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Habitat destruction and fragmentation are among the major causes of amphibian decline. We investigated to what extent geographic distance and barriers affect the genetic composition of the crested newt, Triturus cristatus in Flanders (North Belgium), causing inbreeding or loss of genetic diversity. Data from seven microsatellite loci and 170 individuals from seven metapopulations up to 180km apart revealed heterozygosities of 0.53 to 0.67 within populations and moderate levels of genetic divergence between populations (FST values from 0.074 to 0.141, harmonic means of Dest between 0.070 and 0.189). In all Flemish meta-populations, more than 90% of the individuals from a given geographic region were assigned to the same genetic cluster indicating little genetic exchange, even in De Panne where the two populations Oosthoek and Westhoek are only a few kilometres apart. Such sub-structuring on a micro-scale has also been described in other amphibians. Unique alleles in most populations further support the probability that genetic drift has already led to some isolation. With the exception of the Oosthoek population, however, we found no significant evidence for bottlenecks. Connectivity within pool clusters seems essential to the maintenance of genetic diversity in crested newts as is indicated by our findings from Tommelen, the population with the largest number of pools in close proximity, which also shows the highest levels of heterozygosity (He and Ho) and the second highest number and richness of alleles. In conclusion, our study indicates that dispersal and migration rates between the Flemish populations of Triturus cristatus are limited at the geographic scale studied here but that habitat fragmentation has not yet led to a significant loss of genetic diversity of the studied Flemish populations, possibly because crested newts are relatively long-lived, fragmentation of their habitat is relatively recent in Flanders, and most investigated pools are still connected at the local scale. [ABSTRACT FROM AUTHOR]

In large parts of Western Europe agricultural intensification after World War II has led to an increased use of fertilisers. The resulting nutrient enrichment (=eutrophication) has a huge impact on the occurrence and distribution of plant species and is one of the main pressures on native plant communities. We used the distribution maps (grid size: 16 km2) of individual plant species, obtained through two consecutive survey projects (1939--1971 and 1972--2004) in Flanders (northern Belgium), to estimate the relative change in their distribution area. The comparison of changes in range size among groups of taxa classified according to habitat preference and Ellenberg indicator values, demonstrated a marked decline in distribution area in species that are characteristic for nutrient-poor habitats. To assess geographic patterns in the change of species assemblages, we calculated the mean Ellenberg N- and R-values for every grid cell during each of both survey periods. Differences between these values were analysed in relation to soil type and estimates of atmospheric nitrogen deposition. The largest shifts in Ellenberg N-values, reflecting a decline of species from nutrient-poor conditions and/or an increase of nitrophilous plants, were observed in areas with nutrient-poor, acid sandy soils and high nitrogen deposition rates. Hence, shifts in species composition were modulated by geographic variation in soil type and levels of nitrogen deposition. As the levels of atmospheric nitrogen deposition are still very high in Flanders, it is likely that species from nutrient-poor habitats such as heathlands, will further decline in the near future. [ABSTRACT FROM AUTHOR]

Squamate reptiles are central for studying phenotypic correlates of evolutionary transitions from oviparity to viviparity because these transitions are numerous, with many of them being recent. Several models of life-history theory predict that viviparity is associated with increased female size, and thus more female-biased sexual size dimorphism (SSD). Yet, the corresponding empirical evidence is overall weak and inconsistent. The lizard Zootoca vivipara, which occupies a major part of Northern Eurasia and includes four viviparous and two non-sister oviparous lineages, represents an excellent model for testing these predictions. We analysed how sex-specific body size and SSD is associated with parity mode, using body length data for nearly 14,000 adult individuals from 97 geographically distinct populations, which cover almost the entire species' range and represent all six lineages. Our analyses controlled for lineage identity, climatic seasonality (the strongest predictor of geographic body size variation in previous studies of this species) and several aspects of data heterogeneity. Parity mode, lineage and seasonality are significantly associated with female size and SSD; the first two predictors accounted for 14%-26% of the total variation each, while seasonality explained 5%-7%. Viviparous populations exhibited a larger female size than oviparous populations, with no concomitant differences in male size. The variation of male size was overall low and poorly explained by our predictors. Albeit fully expected from theory, the strong female bias of the body size differences between oviparous and viviparous populations found in Z. vivipara is not evident from available data on three other lizard systems of closely related lineages differing in parity mode. We confront this pattern with the data on female reproductive traits in the considered systems and the frequencies of evolutionary changes of parity mode in the corresponding lizard families and speculate why the life-history correlates of live-bearing in Z. vivipara are distinct. Comparing conspecific populations, our study provides the most direct evidence for the predicted effect of parity mode on adult body size but also demonstrates that the revealed pattern may not be general. This might explain why across squamates, viviparity is only weakly associated with larger size., (© 2024 The Author(s). Journal of Animal Ecology © 2024 British Ecological Society.)

Conservation genetic theory suggests that small and isolated populations should be subject to reduced genetic diversity i.e., heterozygosity and allelic diversity. Our 34 years study of an isolated island population of adders (Vipera berus) in southern Sweden challenges this notion. Despite a lack of gene flow and a yearly mean estimated reproductive adult population size of only 65 adult adders (range 12-171), the population maintains high levels of heterozygosity and allelic diversity similar to that observed in two mainland populations. Even a 14-year major "bottleneck" i.e., a reduction in adult adder numbers, encompassing at least four adder generations, did not result in any reduction in the island adders' heterozygosity and allelic diversity. Female adders are polyandrous, and fertilisation is non-random, which our empirical data and modelling suggest are underpinning the maintenance of the population's high level of heterozygosity. Our empirical results and subsequent modelling suggest that the positive genetic effects of polyandry in combination with non-random fertilisation, often overlooked in conservation genetic analyses, deserve greater consideration when predicting long-term survival of small and isolated populations., (© 2022. The Author(s), under exclusive licence to The Genetics Society.)

Color pattern polymorphism occurs when more than one form is found within the same population. It is widespread in a variety of taxa, leading us to ask what maintains this variation. One stabilizing mechanism is negative frequency-dependent selection, also known as apostatic selection, in which the fitness of a phenotype decreases with its frequency. 1-6 Negative frequency-dependent selection has been proposed as one of the most powerful selective forces in maintaining phenotypic and genetic diversity in both plant and animal populations. 1 , 5 Despite its importance and experimental evidence, 1 , 2 , 6 no study has documented that natural selection due to predation may result in negative frequency-dependent selection in a wild undisturbed vertebrate population. Here, we report the results of a long-term study, spanning 37 years from 1984 to 2020, of two distinct color morphs, zigzag and melanistic, within a population of adult adders (Vipera berus) on the island of Hallands Väderö in southern Sweden (Figure 1). Our results strongly suggest that the color pattern polymorphism is maintained by negative frequency-dependent natural selection in both males and females., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microorganisms possess a plethora of regulatory mechanisms to tightly control the flux through their metabolic network, allowing optimal behaviour in response to environmental conditions. However, these mechanisms typically counteract metabolic engineering efforts to rewire the metabolism with a view to overproduction. Hence, overcoming flux control is key in the development of microbial cell factories, illustrated in this contribution using the strictly controlled hexosamine biosynthesis pathway. The hexosamine biosynthesis pathway has recently garnered attention as gateway for the industrial biotechnological production of numerous mono-, oligo- and polysaccharidic compounds, composed of, i.a., glucosamine, N-acetylglucosamine, and neuraminic acid and with a vast application potential in the health, comsetics, and agricultural sector. First, the various alternative pathways in eukaryotes and prokaryotes are discussed. Second, the main regulatory mechanisms on transcriptional, translational and post-translational control, and the strategies to circumvent these pathway bottlenecks are highlighted. These efforts can serve as an inspiration to tackle regulatory control when optimizing any microbial cell factory., (Copyright © 2020. Published by Elsevier Inc.)

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology., Competing Interests: The authors have declared that no competing interests exist.

Female adders ( Vipera berus ) are "capital breeders" that exhibit delayed maturity and intermittent reproductive frequency. We studied the attainment of sexual maturity, the initiation of annual breeding in mature females, the energy and mortality costs associated with breeding, the length of the reproductive cycle and female lifetime reproductive frequency. We use longitudinal data obtained during an 18-year (2000-2017) mark-recapture study in a large population of adders in northern Belgium. A minority (15%) of the females gave birth to their first litter when they attained the actual age of 3 years, upon surpassing a minimum snout-vent-length of 38 cm. However, most females reproduced for the first time when they were 4 years or older. In mature females, breeding in a given year depends to a large extent on their body condition at the onset of the active season, indicating that a threshold level of energy reserves is necessary to start a reproductive cycle. During breeding years females stop growing, lose about one-third of their initial body mass and are very emaciated after parturition. The decrease in relative body mass was most notable initially in the largest and fattiest females. During the non-breeding years, females forage intensely to rebuild their fat reserves; their abilities to do so will strongly affect whether and when they engage in subsequent reproductive bouts. We used a multistate mark-recapture analytical method to simultaneously estimate the capture and survival rates of breeding and non-breeding females, a necessary procedure to obtain accurate estimates of survival probabilities. The analysis indicated much higher capture rates during the breeding years, but did not reveal a substantial effect of reproductive state on annual survival rates. Although some females were observed to breed in successive years, the reproductive cycle was most often biennial or triennial. However, most females (ca. 70%) that attained sexual maturity reproduced only once during their lifetime, while a minority of the females (ca. 5%) were observed to breed on 3-5 annual occasions. On average, females produced only ca. 1.3 litters during their reproductive lifetime. The short reproductive lifetime is a consequence not only of mortality directly related to the reproductive activities, but also of mortality associated with recovering from the weakened post-parturient body condition during the long intervals (1-2 years) between reproductive bouts., Competing Interests: The authors declare there are no competing interests.

We analyzed seasonal variation in mortality rates in adult males and females of the European adder ( Vipera berus ), using data collected during a 13-year capture-recapture study (2005-2017) in a large population. We concurrently obtained quantitative information on the seasonal variation in the detectability and body condition of adders. Our results show strong seasonality in body condition, encounter, and capture rates of adult adders, and these patterns differ markedly between sexes and between breeding and nonbreeding females. Seasonal variation in mortality rates was however virtually nonexistent in males and moderately low in both breeding and nonbreeding females. In addition, we found no evidence for among-year differences in the seasonal mortality schedules of males and females. During periods of intensive basking, both males and pregnant females are highly visible for humans, but are not subject to strong natural mortality. This low susceptibility to predation is presumably induced by various factors, including the limitation of overt exposure to short periods of time and specific microhabitats, the dorsal coloration pattern that provides cryptic protection and possibly also an aposematic warning signal, and presumed seasonal differences in the foraging behavior and food requirements of natural predators. Our data provide some evidence that female adders, but not males, are relatively vulnerable to predation during the seasonal migrations between the hibernation and feeding habitats. Mortality in the females was not much elevated during their breeding years, but was notably highest in the spring of the ensuing nonbreeding year. After giving birth, reproductive females are extremely emaciated and have a weakened general condition. They then run the risk of dying from starvation either before, during, or after hibernation. The higher mortality after giving birth, that is sustained over a period of ca. 9 months, should be considered as an indirect and delayed survival cost of reproduction., Competing Interests: None declared.

Biotechnological production routes for fine and bulk chemicals are progressively explored and developed. Yet this development is hampered by the many constraints determining the metabolic sweet spot, such as optimal expression levels, metabolic stress, feedback regulation, etc.In this regard, we introduce a novel, highly reliable, and rapid single-strand assembly (SSA) methods for combinatorial pathway engineering. In this contribution, SSA is elucidated which enables one to modulate the expression via promoter and/or RBS randomization. Moreover, a new combinatorial multigene pathway assembly scheme based on single-strand assembly (SSA) methods and Golden Gate Assembly is introduced, exploiting the strengths of both assembly techniques.

Capture-mark-recapture procedures are a basic tool in population studies and require that individual animals are correctly identified throughout their lifetime. A method that has become more and more popular uses photographic records of natural markings, such as pigmentation pattern and scalation configuration. As with any other marking tool, the validity of the photographic identification technique should be evaluated thoroughly. Here, we report on a large-scale double-marking study in which European adders ( Vipera berus ) were identified by both microsatellite genetic markers and by the pattern of head scalation. Samples that were successfully genotyped for all nine loci yielded 624 unique genotypes, which matched on a one-to-one basis with the individual assignments based on the head scalation pattern. Thus, adders considered as different individuals by their genotypes were also identified as different individuals by their head scalation pattern, and vice versa. Overall, variation in the numbers, shape, and arrangement of the head scales enabled us to distinguish among 3200+ photographed individual snakes. Adders that were repeatedly sequenced genetically over intervals of 2-3 years showed no indication whatsoever for a change in the head scale pattern. Photographic records of 900+ adders that were recaptured over periods of up to 12 years showed a very detailed and precise match of the head scale characteristics. These natural marks are thus robust over time and do not change during an individual's lifetime. With very low frequency (0.3%), we detected small changes in scalation that were readily discernible and could be attributed to physical injury or infection. Our study provides a conclusive validation for the use of photo-identification by head scale patterns in the European adder.