PeerJ Preprints: Population Biologyhttps://peerj.com/preprints/index.atom?journal=peerj&subject=7903Population Biology articles published in PeerJ PreprintsLeveraging eDNA to detect and monitor hybrid zoneshttps://peerj.com/preprints/279962019-09-302019-09-30Kathryn StewartScott A. Taylor
Hybrid zones are important windows into evolutionary processes and our understanding of their significance and prevalence in nature has expanded quickly. Yet most hybridization research has restricted temporal and spatial resolution, limiting our ability to draw broad conclusions about evolutionary and conservation related outcomes. Here, we argue rapidly advancing environmental DNA (eDNA) methodology should be adopted for studies of hybrid zones to increase temporal sampling (contemporary and historical), to refine and geographically expand sampling density, and to collect data for taxa that are difficult to directly sample. Genomic data in the environment offer the potential for near real-time biological tracking and eDNA provides broad, as yet untapped potential to address eco-evolutionary questions.
Hybrid zones are important windows into evolutionary processes and our understanding of their significance and prevalence in nature has expanded quickly. Yet most hybridization research has restricted temporal and spatial resolution, limiting our ability to draw broad conclusions about evolutionary and conservation related outcomes. Here, we argue rapidly advancing environmental DNA (eDNA) methodology should be adopted for studies of hybrid zones to increase temporal sampling (contemporary and historical), to refine and geographically expand sampling density, and to collect data for taxa that are difficult to directly sample. Genomic data in the environment offer the potential for near real-time biological tracking and eDNA provides broad, as yet untapped potential to address eco-evolutionary questions.Inbreeding depression in one of the last DFTD-free wild populations of Tasmanian devilshttps://peerj.com/preprints/279852019-09-262019-09-26Rebecca M GooleyCarolyn J HoggSamantha FoxDavid PembertonKatherine BelovCatherine E Grueber
Background. Vulnerable species experiencing inbreeding depression are prone to localised extinctions because of their reduced fitness. For Tasmanian devils, the rapid spread of devil facial tumour disease (DFTD) has led to population declines and fragmentation across the species’ range. Here we show that one of the few remaining DFTD-free populations of Tasmanian devils is experiencing inbreeding depression. Moreover, this population has experienced a significant reduction in reproductive success over recent years.
Methods. We used 32 microsatellite loci to examine changes in genetic diversity and inbreeding in the wild population at Woolnorth, alongside field data on breeding success from females to test for inbreeding depression.
Results. We found that maternal internal relatedness has a negative impact on litter sizes. The results of this study imply that this population has entered an extinction vortex and that to protect the population, genetic rescue may be required. This study provides conservation managers with useful information for managing wild devils and provides support for the “Wild Devil Recovery Program” which is currently augmenting small, isolated populations.
Background. Vulnerable species experiencing inbreeding depression are prone to localised extinctions because of their reduced fitness. For Tasmanian devils, the rapid spread of devil facial tumour disease (DFTD) has led to population declines and fragmentation across the species’ range. Here we show that one of the few remaining DFTD-free populations of Tasmanian devils is experiencing inbreeding depression. Moreover, this population has experienced a significant reduction in reproductive success over recent years.Methods. We used 32 microsatellite loci to examine changes in genetic diversity and inbreeding in the wild population at Woolnorth, alongside field data on breeding success from females to test for inbreeding depression.Results. Wefound that maternal internal relatedness has a negative impact on litter sizes. The results of this study imply that this population has entered an extinction vortex and that to protect the population, genetic rescue may be required. This study provides conservation managers with useful information for managing wild devils and provides support for the “Wild Devil Recovery Program” which is currently augmenting small, isolated populations.A small shift in VSH-gene frequency instead of rapid parallel evolution in bees. A comment on Oddie et al. 2018https://peerj.com/preprints/279382019-09-242019-09-24Jacques J M van AlphenBartJan Fernhout
We refute a recent claim that parallel evolution in four European populations of honeybees has resulted in a not previously reported behavioural defence mechanism of the bees against the parasitic mite Varroa destructor, i.e. the ability of uncapping/recapping to reduce mite reproductive success. There are no data to support this claim, while there is a more plausible alternative interpretation of the reduced mite reproduction, i.e. reduction of mites through Varroa Sensitive Hygiene. We provide evidence why the former mechanism cannot explain resistance against Varroa in honeybees and the latter is instrumental in reducing Varroa populations.
We refute a recent claim that parallel evolution in four European populations of honeybees has resulted in a not previously reported behavioural defence mechanism of the bees against the parasitic mite Varroa destructor, i.e. the ability of uncapping/recapping to reduce mite reproductive success. There are no data to support this claim, while there is a more plausible alternative interpretation of the reduced mite reproduction, i.e. reduction of mites through Varroa Sensitive Hygiene. We provide evidence why the former mechanism cannot explain resistance against Varroa in honeybees and the latter is instrumental in reducing Varroa populations.Barnacle recruit density and size increase from high to middle intertidal elevations in wave-exposed habitats on the Atlantic coast of Nova Scotiahttps://peerj.com/preprints/279662019-09-172019-09-17Ricardo A Scrosati
Barnacle recruitment is often studied in rocky intertidal habitats due to the relevant role that barnacles can play in intertidal communities. In 2014, barnacle (Semibalanus balanoides) recruitment was measured at high elevations in wave-exposed intertidal habitats on the NW Atlantic coast in Nova Scotia, Canada. Values were considerably lower than previously reported for middle elevations in wave-exposed intertidal habitats on the NE Atlantic and NE Pacific coasts. To determine if such differences in recruitment may have resulted from elevation influences, I did a field experiment in 2019 in wave-exposed intertidal habitats in Nova Scotia to test the hypothesis that recruitment is higher at middle than at high elevations, based on known environmental differences between both elevation zones. Based on data from three locations spanning 158 km of the Nova Scotia coast, barnacle recruitment was, on average, nearly 200 % higher (and recruits were larger) at middle than at high elevations. However, even with this increase, barnacle recruitment on this NW Atlantic coast is still lower than for comparable habitats on the NE Atlantic and NE Pacific coasts, and also lower than previously reported for wave-exposed locations farther south on the NW Atlantic coast, in Maine, USA. Therefore, barnacle recruitment in wave-exposed intertidal environments in Nova Scotia appears to be only moderate relative to other shores. This difference in the supply of barnacle recruits might influence the intensity of interspecific interactions involving barnacles.
Barnacle recruitment is often studied in rocky intertidal habitats due to the relevant role that barnacles can play in intertidal communities. In 2014, barnacle (Semibalanus balanoides) recruitment was measured at high elevations in wave-exposed intertidal habitats on the NW Atlantic coast in Nova Scotia, Canada. Values were considerably lower than previously reported for middle elevations in wave-exposed intertidal habitats on the NE Atlantic and NE Pacific coasts. To determine if such differences in recruitment may have resulted from elevation influences, I did a field experiment in 2019 in wave-exposed intertidal habitats in Nova Scotia to test the hypothesis that recruitment is higher at middle than at high elevations, based on known environmental differences between both elevation zones. Based on data from three locations spanning 158 km of the Nova Scotia coast, barnacle recruitment was, on average, nearly 200 % higher (and recruits were larger) at middle than at high elevations. However, even with this increase, barnacle recruitment on this NW Atlantic coast is still lower than for comparable habitats on the NE Atlantic and NE Pacific coasts, and also lower than previously reported for wave-exposed locations farther south on the NW Atlantic coast, in Maine, USA. Therefore, barnacle recruitment in wave-exposed intertidal environments in Nova Scotia appears to be only moderate relative to other shores. This difference in the supply of barnacle recruits might influence the intensity of interspecific interactions involving barnacles.Call overlapping signals sexual status in Darwin’s frogshttps://peerj.com/preprints/279432019-09-072019-09-07Jose M SerranoNoé GuzmánMario PennaMarco A MéndezClaudio Soto-Azat
Background. In animal reproductive contexts, calling behaviour is mostly performed by males but in species in which females call, it is not known how vocal interaction occurs between sexes, particularly when sexual dimorphism in signals is low, as in cases in which call repertoire is identical but acoustic properties differ. In Darwin’s frog (Rhinoderma darwinii), a species in which males brood larvae inside their vocal sacs, females have higher dominant frequency and shorter calls and notes than males. Since in this species males persist calling after getting pregnant with larvae, different vocal interaction patterns are expected to occur among animals having dissimilar reproductive status.
Methodology. To explore the mechanisms underlying vocal recognition among the different sexual status of R. darwinii, we recorded natural duets between non-pregnant males (NPM), pregnant males (PM) and females (F) and evaluated their evoked vocal response to natural playback stimuli of each sexual status from November to February 2015-2016 in Chiloé island, Chile. Call rate, phase angles, sound pressure level (SPL), number of overlapping calls and delay of overlapping calls were measured to determine differential responses between natural duets and in response to stimuli consisting of natural calls of individuals of different sexual status.
Results. Spontaneous duet interactions occurred mainly between males and no clear differences between duets were detected. In playbacks, call ratios in response to calls of different sexual status were similar. Females decreased their SPL in response to F calls, while F and PM had longer call delays and lower call overlaps between each other. Major differences were observed in call overlap, as the occurrence of this phenomenon was larger in playback experiments than during natural duets. The number of calls overlapped during natural duets was fewer (10.9 %) than during playback experiments (36.8 %).
Conclusions. Our results suggest that in R. darwinii, PM and F signalize their sexual status by decreasing their call overlap and that NPM respond indistinctly to the other sexual status. In general, these differences in selective call overlap between Darwin's frogs arise as a novel mechanism for signal recognition between animal vocal interactions.
Background. In animal reproductive contexts, calling behaviour is mostly performed by males but in species in which females call, it is not known how vocal interaction occurs between sexes, particularly when sexual dimorphism in signals is low, as in cases in which call repertoire is identical but acoustic properties differ. In Darwin’s frog (Rhinoderma darwinii), a species in which males brood larvae inside their vocal sacs, females have higher dominant frequency and shorter calls and notes than males. Since in this species males persist calling after getting pregnant with larvae, different vocal interaction patterns are expected to occur among animals having dissimilar reproductive status.Methodology. To explore the mechanisms underlying vocal recognition among the different sexual status of R. darwinii, we recorded natural duets between non-pregnant males (NPM), pregnant males (PM) and females (F) and evaluated their evoked vocal response to natural playback stimuli of each sexual status from November to February 2015-2016 in Chiloé island, Chile. Call rate, phase angles, sound pressure level (SPL), number of overlapping calls and delay of overlapping calls were measured to determine differential responses between natural duets and in response to stimuli consisting of natural calls of individuals of different sexual status.Results. Spontaneous duet interactions occurred mainly between males and no clear differences between duets were detected. In playbacks, call ratios in response to calls of different sexual status were similar. Females decreased their SPL in response to F calls, while F and PM had longer call delays and lower call overlaps between each other. Major differences were observed in call overlap, as the occurrence of this phenomenon was larger in playback experiments than during natural duets. The number of calls overlapped during natural duets was fewer (10.9 %) than during playback experiments (36.8 %).Conclusions. Our results suggest that in R. darwinii, PM and F signalize their sexual status by decreasing their call overlap and that NPM respond indistinctly to the other sexual status. In general, these differences in selective call overlap between Darwin's frogs arise as a novel mechanism for signal recognition between animal vocal interactions.The interplay between movement, dispersal and morphology in Tetrahymena ciliateshttps://peerj.com/preprints/265402019-09-032019-09-03Frank PennekampJean ClobertNicolas Schtickzelle
Understanding how and why individual movement translates into dispersal between populations is a long-term goal in ecology. Movement is broadly defined as “any change in the spatial location of an individual”, whereas dispersal is more narrowly defined as a movement that may lead to gene flow. Because the former may create the condition for the latter, behavioural decisions that lead to dispersal may be detectable in underlying movement behaviour. In addition, dispersing individuals also have specific sets of morphological and behavioural traits that help them coping with the costs of movement and dispersal, and traits that mitigate costs should be under selection and evolve if they have a genetic basis. Here we experimentally study the relationships between movement behaviour, morphology and dispersal across 44 genotypes of the actively dispersing unicellular, aquatic model organism Tetrahymena thermophila. We used two-patch populations to quantify individual movement trajectories, as well as activity, morphology and dispersal rate. First, we studied variation in movement behaviour among and within genotypes (i.e. between dispersers and residents) and tested whether this variation can be explained by morphology. Then, we address how much the dispersal rate is driven by differences in the underlying movement behaviour. Genotypes revealed clear differences in terms of movement speed and linearity. We also detected marked movement differences between resident and dispersing individuals, mediated by the genotype. Movement variation was partly explained by morphological properties such as cell size and shape, with larger cells consistently showing higher movement speed and higher linearity. Genetic differences in activity and diffusion rates were positively related to the observed dispersal and jointly explained 47% of the variation in dispersal rate. Our study shows that a detailed understanding of the interplay between morphology, movement and dispersal may have potential to improve dispersal predictions over broader spatio-temporal scales.
Understanding how and why individual movement translates into dispersal between populations is a long-term goal in ecology. Movement is broadly defined as “any change in the spatial location of an individual”, whereas dispersal is more narrowly defined as a movement that may lead to gene flow. Because the former may create the condition for the latter, behavioural decisions that lead to dispersal may be detectable in underlying movement behaviour. In addition, dispersing individuals also have specific sets of morphological and behavioural traits that help them coping with the costs of movement and dispersal, and traits that mitigate costs should be under selection and evolve if they have a genetic basis. Here we experimentally study the relationships between movement behaviour, morphology and dispersal across 44 genotypes of the actively dispersing unicellular, aquatic model organism Tetrahymena thermophila. We used two-patch populations to quantify individual movement trajectories, as well as activity, morphology and dispersal rate. First, we studied variation in movement behaviour among and within genotypes (i.e. between dispersers and residents) and tested whether this variation can be explained by morphology. Then, we address how much the dispersal rate is driven by differences in the underlying movement behaviour. Genotypes revealed clear differences in terms of movement speed and linearity. We also detected marked movement differences between resident and dispersing individuals, mediated by the genotype. Movement variation was partly explained by morphological properties such as cell size and shape, with larger cells consistently showing higher movement speed and higher linearity. Genetic differences in activity and diffusion rates were positively related to the observed dispersal and jointly explained 47% of the variation in dispersal rate. Our study shows that a detailed understanding of the interplay between morphology, movement and dispersal may have potential to improve dispersal predictions over broader spatio-temporal scales.Impact of hybridisation in two Cossypha robin-chat species in southern Africahttps://peerj.com/preprints/279122019-08-212019-08-21Jean MollettNaadhirah MunshiCraig Symes
Chorister Robin-Chat Cossypha dichroa, a South African forest endemic, and Red-capped Robin-Chat C. natalensis, a widely distributed species in African forest and woodland, are inferred to hybridise in areas of sympatry. DNA was extracted from blood samples of C. dichroa (n = 18), C. natalensis (n = 47), and two phenotypic hybrids. The mitochondrial cytochrome c oxidase I (COI) gene was amplified by PCR and sequenced. Phylogenetic analysis was performed on the sequence data to investigate taxonomic status and putative interspecific hybridisation. Phenotypic hybrids grouped with C. natalensis, suggesting maternal parentage from that species. Intra- and interspecific genetic and geographic distances were compared between C. dichroa and C. natalensis to assess genetic introgression. Seven of the thirteen microsatellite primer pairs developed for C. natalensis cross amplified in C. dichroa. These seven markers were then used for further analysis. STRUCTURE v2.3.4 was used to assign individuals to a particular genetic cluster and determine any admixture. NEWHYBRIDS v1.1 was used to assign hybrid status to samples beyond the F1 generation. Despite the hybridisation events recorded between C. dichroa and C. natalensis they still form two separate clusters as expected, and two genetic clusters (K=2) were identified using STRUCTURE. These two species are proficient vocal mimics and it is likely that reproductive isolation mechanisms are overcome through vocalisations. Genotypic hybrids are evident in the sampled population and hybridisation and backcrossing across a zone of sympatry is occurring. However, hybridisation is expected to have very little evolutionary influence on the integrity of recently diverged species which retain reproductive isolation across a wide region of sympatry through call distinctness.
Chorister Robin-Chat Cossypha dichroa, a South African forest endemic, and Red-capped Robin-Chat C. natalensis, a widely distributed species in African forest and woodland, are inferred to hybridise in areas of sympatry. DNA was extracted from blood samples of C. dichroa (n = 18), C. natalensis (n = 47), and two phenotypic hybrids. The mitochondrial cytochrome c oxidase I (COI) gene was amplified by PCR and sequenced. Phylogenetic analysis was performed on the sequence data to investigate taxonomic status and putative interspecific hybridisation. Phenotypic hybrids grouped with C. natalensis, suggesting maternal parentage from that species. Intra- and interspecific genetic and geographic distances were compared between C. dichroa and C. natalensis to assess genetic introgression. Seven of the thirteen microsatellite primer pairs developed for C. natalensis cross amplified in C. dichroa. These seven markers were then used for further analysis. STRUCTURE v2.3.4 was used to assign individuals to a particular genetic cluster and determine any admixture. NEWHYBRIDS v1.1 was used to assign hybrid status to samples beyond the F1 generation. Despite the hybridisation events recorded between C. dichroa and C. natalensis they still form two separate clusters as expected, and two genetic clusters (K=2) were identified using STRUCTURE. These two species are proficient vocal mimics and it is likely that reproductive isolation mechanisms are overcome through vocalisations. Genotypic hybrids are evident in the sampled population and hybridisation and backcrossing across a zone of sympatry is occurring. However, hybridisation is expected to have very little evolutionary influence on the integrity of recently diverged species which retain reproductive isolation across a wide region of sympatry through call distinctness.Twisted tale of the tiger: the case of inappropriate data and deficient sciencehttps://peerj.com/preprints/273492019-07-312019-07-31Qamar QureshiRajesh GopalYadvendradev V Jhala
Publications in peer reviewed journals are often looked upon as tenets on which future scientific thought is built. Published information is not always flawless and errors in published research should be expediently reported, preferably by a peer review process. We review a recent publication by Gopalaswamy et al (doi:10.1111/2041-210X.12351) that challenges the use of “double sampling” in large scale animal surveys. Double sampling is often resorted to as an established economical and practical approach for large scale surveys since it calibrates abundance indices against absolute abundance, thereby potentially addressing the statistical shortfalls of indices. Empirical data used by Gopalaswamy et al. to test their theoretical model, relate to tiger sign and tiger abundance referred to as an Index Calibration experiment (IC-Karanth). These data on tiger abundance and signs should be paired in time and space to qualify as a calibration experiment for double sampling, but original data of IC-Karanth show lags of (up to) several years. Further, data points used in the paper do not match the original sources. We show that by use of inappropriate and incorrect data collected through a faulty experimental design, poor parameterization of their theoretical model, and selectively-picked estimates from literature on detection probability, the inferences of this paper are highly questionable. We highlight how the results of Gopalaswamy et al. were further distorted in popular media. If left unaddressed, Gopalaswamy et al. paper could have serious implications on statistical design of large-scale animal surveys by propagating unreliable inferences.
Publications in peer reviewed journals are often looked upon as tenets on which future scientific thought is built. Published information is not always flawless and errors in published research should be expediently reported, preferably by a peer review process. We review a recent publication by Gopalaswamy et al (doi:10.1111/2041-210X.12351) that challenges the use of “double sampling” in large scale animal surveys. Double sampling is often resorted to as an established economical and practical approach for large scale surveys since it calibrates abundance indices against absolute abundance, thereby potentially addressing the statistical shortfalls of indices. Empirical data used by Gopalaswamy et al. to test their theoretical model, relate to tiger sign and tiger abundance referred to as an Index Calibration experiment (IC-Karanth). These data on tiger abundance and signs should be paired in time and space to qualify as a calibration experiment for double sampling, but original data of IC-Karanth show lags of (up to) several years. Further, data points used in the paper do not match the original sources. We show that by use of inappropriate and incorrect data collected through a faulty experimental design, poor parameterization of their theoretical model, and selectively-picked estimates from literature on detection probability, the inferences of this paper are highly questionable. We highlight how the results of Gopalaswamy et al. were further distorted in popular media. If left unaddressed, Gopalaswamy et al. paper could have serious implications on statistical design of large-scale animal surveys by propagating unreliable inferences.Almost faithful: SNP markers reveal low levels of extra-pair paternity in the Eurasian beavershttps://peerj.com/preprints/278662019-07-172019-07-17Priyank Sharad NimjeHelga Veronica TinnesandChristina BueschingMona SæbøHelen SennAndreas ZedrosserFrank Rosell
Mating systems largely affect individual reproductive strategies which further drives evolution. Monogamy, where males and females form exclusive pairs for more than one breeding season, is particularly intriguing in this context, as there are real and potential costs of genetic monogamy to both sexes. However, molecular studies in a variety of species have revealed that social monogamy does not necessarily imply genetic monogamy due to occurrence of extra-pair copulations resulting in extra-pair offspring. Although common in birds, <10% of mammals are monogamous. Here we use single nucleotide polymorphism (SNP) to investigate the genetic mating system of the Eurasian beaver (Castor fiber), a species traditionally considered to be not only socially but also genetically monogamous. We found evidence for low frequency of extra-pair paternity (EPP) and multiple paternity within litter. Only 5.4% young were produced by EPP and only 7% of litter contain at least one extra-pair young. Moreover, we found indications that only pairings of old individuals engaged in EPP. None of these pairs produced more than one litter as a result of EPP and none of the EPP events resulted in mate change. Our findings suggest that EPP in beavers might be the consequence of a lapse in mate guarding ability of old males.
Mating systems largely affect individual reproductive strategies which further drives evolution. Monogamy, where males and females form exclusive pairs for more than one breeding season, is particularly intriguing in this context, as there are real and potential costs of genetic monogamy to both sexes. However, molecular studies in a variety of species have revealed that social monogamy does not necessarily imply genetic monogamy due to occurrence of extra-pair copulations resulting in extra-pair offspring. Although common in birds, <10% of mammals are monogamous. Here we use single nucleotide polymorphism (SNP) to investigate the genetic mating system of the Eurasian beaver (Castor fiber), a species traditionally considered to be not only socially but also genetically monogamous. We found evidence for low frequency of extra-pair paternity (EPP) and multiple paternity within litter. Only 5.4% young were produced by EPP and only 7% of litter contain at least one extra-pair young. Moreover, we found indications that only pairings of old individuals engaged in EPP. None of these pairs produced more than one litter as a result of EPP and none of the EPP events resulted in mate change. Our findings suggest that EPP in beavers might be the consequence of a lapse in mate guarding ability of old males.The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNAhttps://peerj.com/preprints/277802019-07-152019-07-15Adam ŠťovíčekSmadar Cohen-ChalamishOsnat Gillor
It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.
It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.