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Despite increasing sequencing efforts, numerous fish families still lack a reference genome, which complicates genetic research. One such understudied family is the sand lances (Ammodytidae, literally: “sand burrower”), a globally distributed clade of over 30 fish species that tend to avoid tidal currents by burrowing into the sand. Here, we present the first annotated chromosome-level genome assembly of the great sand eel (Hyperoplus lanceolatus). The genome assembly was generated using Oxford Nanopore Technologies long sequencing reads and Illumina short reads for polishing. The final assembly has a total length of 808.5 Mbp, of which 97.1% were anchored into 24 chromosome-scale scaffolds using proximity-ligation scaffolding. It is highly contiguous with a scaffold and contig N50 of 33.7 and 31.3 Mbp, respectively, and has a BUSCO completeness score of 96.9%. The presented genome assembly is a valuable resource for future studies of sand lances, as this family is of great ecological and commercial importance and may also contribute to studies aiming to resolve the suprafamiliar taxonomy of bony fishes.

The okapi (Okapia johnstoni), or forest giraffe, is the only species in its genus and the only extant sister group of the giraffe within the family Giraffidae. The species is one of the remaining large vertebrates surrounded by mystery because of its elusive behavior as well as the armed conflicts in the region where it occurs, making it difficult to study. Deforestation puts the okapi under constant anthropogenic pressure, and it is currently listed as “Endangered” on the IUCN Red List. Here, we present the first annotated de novo okapi genome assembly based on PacBio continuous long reads, polished with short reads, and anchored into chromosome-scale scaffolds using Hi-C proximity ligation sequencing. The final assembly (TBG_Okapi_asm_v1) has a length of 2.39 Gbp, of which 98% are represented by 28 scaffolds > 3.9 Mbp. The contig N50 of 61 Mbp and scaffold N50 of 102 Mbp, together with a BUSCO score of 94.7%, and 23 412 annotated genes, underline the high quality of the assembly. This chromosome-scale genome assembly is a valuable resource for future conservation of the species and comparative genomic studies among the giraffids and other ruminants.

Permeable phylogeographic barriers characterize the vast open ocean, boosting gene flow and counteracting population differentiation and speciation of widely distributed and migratory species. However, many widely distributed species consists of distinct populations throughout their distribution, evidencing that our understanding of how the marine environment triggers population and species divergence are insufficient. The sailfish is a circumtropical and highly migratory billfish that inhabits warm and productive areas. Despite its ecological and socioeconomic importance as a predator and fishery resource, the species is threatened by overfishing, requiring innovative approaches to improve their management and conservation status. Thus, we presented a novel high-quality reference genome for the species and applied a seascape genomics approach to understand how marine environmental features may promote local adaptation and how it affects gene flow between populations. We delimit two populations between the Atlantic and Indo-Western Pacific oceans and detect outlier loci correlated with sea surface temperature, salinity, oxygen, and chlorophyll concentrations. However, the most significant explanatory factor that explains the differences between populations was isolation by distance. Despite recent population drops, the sailfish populations are not inbred. For billfishes in general, genome-wide heterozygosity was found to be relatively low compared to other marine fishes, evidencing the need to counteract overfishing effects. In addition, in a climate change scenario, management agencies must implement state-of-the-art sequencing methods, consider our findings in their management plans, and monitor genome-wide heterozygosity over time to improve sustainable fisheries and the long-term viability of its populations.

It is generally recognized that large-scale whaling in the 19th and 20th century led to a substantial reduction of the size of many cetacean populations, particularly those of the baleen whales (Mysticeti). The impact of these operations on genomic diversity of one of the most hunted whales, the fin whale (Balaenoptera physalus), has remained largely unaddressed because of the paucity of adequate samples and the limitation of applicable techniques. Here, we have examined the effect of whaling on the North Atlantic fin whale based on genomes of 51 individuals from Icelandic waters, representing three temporally separated intervals, 1989, 2009 and 2018 and provide a reference genome for the species. Demographic models suggest a noticeable drop of the effective population size of the North Atlantic fin whale around a century ago. The present results suggest that the genome-wide heterozygosity is not markedly reduced and has remained comparable with other baleen whale species. Similarly, there are no signs of apparent inbreeding, as measured by the proportion of long runs of homozygosity, or of a distinctively increased mutational load, as measured by the amount of putative deleterious mutations. Compared with other baleen whales, the North Atlantic fin whale appears to be less affected by anthropogenic influences than other whales such as the North Atlantic right whale, consistent with the presence of long runs of homozygosity and higher levels of mutational load in an otherwise more heterozygous genome. Thus, genome-wide assessments of other species and populations are essential for future, more specific, conservation efforts.

Feeding exclusively on blood, vampire bats represent the only obligate sanguivorous lineage among mammals. To uncover genomic changes associated with adaptations to this unique dietary specialization, we generated a new haplotype-resolved reference-quality genome of the common vampire bat (Desmodus rotundus) and screened 26 bat species for genes that were specifically lost in the vampire bat lineage. We discovered previously-unknown gene losses that relate to metabolic and physiological changes, such as reduced insulin secretion (FFAR1,SLC30A8), limited glycogen stores (PPP1R3E), and a distinct gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2,CTRL) and distinct pathogen diversity of blood (RNASE7). Interestingly, the loss ofREP15likely helped vampire bats to adapt to high dietary iron levels by enhancing iron excretion and the loss of the 24S-hydroxycholesterol metabolizing enzymeCYP39A1could contribute to their exceptional cognitive abilities. Finally, losses of key cone phototransduction genes (PDE6H,PDE6C) suggest that these strictly-nocturnal bats completely lack cone-based vision. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to sanguivory.

November 2020 marked 2 y since the launch of the Earth BioGenome Project (EBP), which aims to sequence all known eukaryotic species in a 10-y timeframe. Since then, significant progress has been made across all aspects of the EBP roadmap, as outlined in the 2018 article describing the project's goals, strategies, and challenges (1). The launch phase has ended and the clock has started on reaching the EBP's major milestones. This Special Feature explores the many facets of the EBP, including a review of progress, a description of major scientific goals, exemplar projects, ethical legal and social issues, and applications of biodiversity genomics. In this Introduction, we summarize the current status of the EBP, held virtually October 5 to 9, 2020, including recent updates through February 2021. References to the nine Perspective articles included in this Special Feature are cited to guide the reader toward deeper understanding of the goals and challenges facing the EBP.

Striking geographic variation in male advertisement calls was observed in frogs formerly referred to as Limnonectes doriae and L. limborgi, respectively. Subsequent analyses of mtDNA and external morphological data brought supporting evidence for the recognition of these populations as distinct species. We describe two new frog species of the genus Limnonectes (i.e., L. bagoensis sp. nov. and L. bagoyoma sp. nov.) from Myanmar. Limnonectes bagoensis sp. nov. is closely related to L. doriae whereas L. bagoyoma sp. nov. is closely related to L. limborgi. Results of this integrative study provide evidence for the presence of additional undescribed species in these species complexes but due to the lack of bioacoustical data, we consider these additional diverging populations as candidate species that need further study to resolve their respective taxonomic status. Both new species are distributed in Lower Myanmar. Limnonectes doriae is restricted to southern Myanmar along the Malayan Peninsula whereas L. limborgi is known to occur in eastern Myanmar and northwestern Thailand. The remaining populations formerly referred to as either L. doriae or L. limborgi are considered representatives of various candidate species that await further study. We further provide a de novo draft genome of the respective holotypes of L. bagoensis sp. nov. and L. bagoyoma sp. nov. based on short-read sequencing technology to 25-fold coverage.

Background Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non–genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step. Results The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for β-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life. Conclusions It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.

Phylogenetic analyses of nuclear and mitochondrial genomes indicate that polar bears captured the brown bear mitochondrial genome 160,000 years ago, leading to an extinction of the original polar bear mitochondrial genome. However, mitochondrial DNA occasionally integrates into the nuclear genome, forming pseudogenes called numts (nuclear mitochondrial integrations). Screening the polar bear genome identified only 13 numts. Genomic analyses of two additional ursine bears and giant panda indicate that all except one of the discovered numts entered the bear lineage at least 14 million years ago. However, short read genome assemblies might lead to an under-representation of numts or other repetitive sequences. Our findings suggest low integration rates of numts in bears and a loss of the original polar bear mitochondrial genome.

We revise the frogs of the genusPhrynoglossusfrom Indochina based on data of external morphology, bioacoustics and molecular genetics. The results of this integrative study provide evidence for the recognition of three distinct species, one of which we describe as new.Phrynoglossus martensiihas a vast geographic distribution from central and southern Thailand across southern China to Vietnam, Laos, and Cambodia.Phrynoglossus myanhesseisp. nov.is distributed in central Myanmar whereasPhrynoglossus magnapustulosusis restricted to the Khorat Plateau, Thailand. These three species occur in allopatry and differ in their mating calls, external morphology, and in genetic distances of the 16S gene of 3.8–5.9%. Finally, we discuss and provide evolutionary evidence for the recognition ofPhrynoglossusas a genus distinct fromOccidozyga. Members of both genera form reciprocal monophyletic groups in our analyses of mtDNA data (16S) and are well differentiated from each other in morphology and ecology. Furthermore, they differ in the amplexus mode withPhrynoglossushaving an inguinal amplexus whereas it is axillary inOccidozyga. We further provide ade novodraft genome of the holotype based on short-read sequencing technology to a coverage of 25-fold. This resource will permanently link the genetic characterization of the species to the name-bearing type specimen.

Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.

Based on two male and two female individuals, we describe a new genus and species of mud snake, Myanophis thanlyinensis gen. nov., sp. nov., from the vicinity of the campus of East Yangon University, Yangon, Thanlyin, Myanmar. This species differs from every other homalopsid species by the following combination of characters: (1) dorsal scales smooth, row formula 21–21–19 or 21–21–17; (2) tail short, ratio tail length/SVL 0.185–0.204 in males, 0.160–0.167 in females; (3) nasal scales separated; (4) 125–126 ventral scales in males, 120–122 in females; (5) 38–39 subcaudal scales in males, 32–34 in females; and (6) hemipenis bilobed. Its matrilineal genealogy (based on analyses of 16S and cytochrome b sequences), associates Myanophis thanlyinensis gen. nov., sp. nov. most closely with species of the genera Myrrophis and Gyiophis. The new taxon differs from the species of Myrrophis and Gyiophis by having a bilobed hemipenis (vs. unilobed). Myanophis thanlyinensis gen. nov., sp. nov. differs further from the species of Myrrophis by having 125–126 ventral scales in males and 120–122 in females (vs. 137–162 and 137–164, respectively), and 38–39 subcaudal scales in males and 32–34 in females (vs. 39–55 and 37–52, respectively). Myanophis thanlyinensis gen. nov., sp. nov. differs further from the species of Gyiophis by lacking dark blotches along flank (vs. present), and by having 21 dorsal scales rows at midbody (vs. 25). We provide an identification key to the homalopsid species known to occur in Myanmar. As a novelty to the classic holotype description and characterization, the individual has been genome sequenced by Illumina short-read technology and its genome has been assembled into a draft nuclear genome and a complete, annotated mitochondrial genome. This innovative approach comprehensively and permanently characterizes the genomic variation of the holotype.

SNP data sets can be used to infer a wealth of information about natural populations, including information about their structure, genetic diversity, and the presence of loci under selection. However, SNP data analysis can be a time-consuming and challenging process, not in the least because at present many different software packages are needed to execute and depict the wide variety of mainstream population-genetic analyses. Here, we present SambaR, an integrative and user-friendly R package which automates and simplifies quality control and population-genetic analyses of biallelic SNP data sets. SambaR allows users to perform mainstream population-genetic analyses and to generate a wide variety of ready to publish graphs with a minimum number of commands (less than 10). These wrapper commands call functions of existing packages (including adegenet, ape, LEA, poppr, pcadapt and StAMPP) as well as new tools uniquely implemented in SambaR. We tested SambaR on online available SNP data sets and found that SambaR can process data sets of over 100,000 SNPs and hundreds of individuals within hours, given sufficient computing power. Newly developed tools implemented in SambaR facilitate optimization of filter settings, objective interpretation of ordination analyses, enhance comparability of diversity estimates from reduced representation library SNP data sets, and generate reduced SNP panels and structure-like plots with Bayesian population assignment probabilities. SambaR facilitates rapid population genetic analyses on biallelic SNP data sets by removing three major time sinks: file handling, software learning, and data plotting. In addition, SambaR provides a convenient platform for SNP data storage and management, as well as several new utilities, including guidance in setting appropriate data filters. The SambaR source script, manual and example data set are distributed through GitHub: https://github.com/mennodejong1986/SambaR.

BackgroundThe common dragonet, Callionymus lyra, is one of three Callionymus species inhabiting the North Sea. All three species show strong sexual dimorphism. The males show strong morphological differentiation, e.g., species-specific colouration and size relations, while the females of different species have few distinguishing characters. Callionymus belongs to the ‘benthic associated clade’ of the order Syngnathiformes. The ‘benthic associated clade’ so far is not represented by genome data and serves as an important outgroup to understand the morphological transformation in ‘long-snouted’ syngnatiforms such as seahorses and pipefishes.FindingsHere, we present the chromosome-level genome assembly of C. lyra. We applied Oxford Nanopore Technologies’ long-read sequencing, short-read DNBseq, and proximity-ligation-based scaffolding to generate a high-quality genome assembly. The resulting assembly has a contig N50 of 2.2 Mbp, a scaffold N50 of 26.7 Mbp. The total assembly length is 568.7 Mbp, of which over 538 Mbp were scaffolded into 19 chromosome-length scaffolds. The identification of 94.5% of complete BUSCO genes indicates high assembly completeness. Additionally, we sequenced and assembled a multi-tissue transcriptome with a total length of 255.5 Mbp that was used to aid the annotation of the genome assembly. The annotation resulted in 19,849 annotated transcripts and identified a repeat content of 27.66%.ConclusionsThe chromosome-level assembly of C. lyra provides a high-quality reference genome for future population genomic, phylogenomic, and phylogeographic analyses.

Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in evolutionary sciences, ecology, systematics and in biodiversity-related applied fields such as environmental management and natural product research. Advances in DNA sequencing technologies make genome sequencing feasible for many non-genetic model species. However, genome sequencing today relies on large quantities of high quality, high molecular weight (HMW) DNA which is mostly obtained from fresh tissues. This is problematic for biodiversity genomics of Metazoa as most species are small and yield minute amounts of DNA. Furthermore, briging living specimens to the lab bench not realistic for the majority of species.Here we overcome those difficulties by sequencing two species of springtails (Collembola) from single specimens preserved in ethanol. We used a newly developed, genome-wide amplification-based protocol to generate PacBio libraries for HiFi long-read sequencing.The assembled genomes were highly continuous. They can be considered complete as we recovered over 95% of BUSCOs. Genome-wide amplification does not seem to bias genome recovery. Presence of almost complete copies of the mitochondrial genome in the nuclear genome were pitfalls for automatic assemblers. The genomes fit well into an existing phylogeny of springtails. A neotype is designated for one of the species, blending genome sequencing and creation of taxonomic references.Our study shows that it is possible to obtain high quality genomes from small, field-preserved sub-millimeter metazoans, thus making their vast diversity accessible to the fields of genomics.

BackgroundSNP datasets can be used to infer a wealth of information about natural populations, including information about their structure, genetic diversity, and the presence of loci under selection. However, SNP data analysis can be a time-consuming and challenging process, not in the least because at present many different software packages are needed to execute and depict the wide variety of mainstream population-genetic analyses. Here we present SambaR, an integrative and user-friendly R package which automates and simplifies quality control and population-genetic analyses of biallelic SNP datasets. SambaR allows users to perform mainstream population-genetic analyses and to generate a wide variety of ready to publish graphs with a minimum number of commands (less than ten). These wrapper commands call functions of existing packages (including adegenet, ape, LEA, poppr, pcadapt and StAMPP) as well as new tools uniquely implemented in SambaR.ResultsWe tested SambaR on online available SNP datasets and found that SambaR can process datasets of millions of SNPs and hundreds of individuals within hours, given sufficient computing power. Newly developed tools implemented in SambaR facilitate optimization of filter settings, objective interpretation of ordination analyses, enhance comparability of diversity estimates from reduced representation library SNP datasets, and generate reduced SNP panels and structure-like plots with Bayesian population assignment probabilities.ConclusionSambaR facilitates rapid population genetic analyses on biallelic SNP datasets by removing three major time sinks: file handling, software learning, and data plotting. In addition, SambaR provides a convenient platform for SNP data storage and management, as well as several new utilities, including guidance in setting appropriate data filters.Availability and implementationThe SambaR source script, manual and example datasets are distributed through GitHub:https://github.com/mennodejong1986/SambaR

Summary Species is the fundamental taxonomic unit in biology and its delimitation has implications for conservation. In giraffe (Giraffa spp.), multiple taxonomic classifications have been proposed since the early 1900s. 1 However, one species with nine subspecies has been generally accepted, 2 likely due to limited in-depth assessments, subspecies hybridizing in captivity, 3 , 4 and anecdotal reports of hybrids in the wild. 5 Giraffe taxonomy received new attention after population genetic studies using traditional genetic markers suggested at least four species. 6 , 7 This view has been met with controversy, 8 setting the stage for debate. 9 , 10 Genomics is significantly enhancing our understanding of biodiversity and speciation relative to traditional genetic approaches and thus has important implications for species delineation and conservation. 11 We present a high-quality de novo genome assembly of the critically endangered Kordofan giraffe (G. camelopardalis antiquorum) 12 and a comprehensive whole-genome analysis of 50 giraffe representing all traditionally recognized subspecies. Population structure and phylogenomic analyses support four separately evolving giraffe lineages, which diverged 230–370 ka ago. These lineages underwent distinct demographic histories and show different levels of heterozygosity and inbreeding. Our results strengthen previous findings of limited gene flow and admixture among putative giraffe species 6 , 7 , 9 and establish a genomic foundation for recognizing four species and seven subspecies, the latter of which should be considered as evolutionary significant units. Achieving a consensus over the number of species and subspecies in giraffe is essential for adequately assessing their threat level and will improve conservation efforts for these iconic taxa.

Historically, giraffe have been translocated across Africa to supplement extant populations, reintroduce extinct populations or to establish new populations, often for conservation and tourism. Such faunal relocations were often carried out disregarding taxonomic affiliation. Today, the small giraffe populations in the Republic of Malawi are assumed to consist of South African giraffe (Giraffa giraffa giraffa), which have likely descended from five individuals translocated from Imire Game Park (Zimbabwe) to Nyala Game Park (Malawi) in 1993. However, during the last 25 years, unknown additional translocations, migrations or unrecognized local populations of potential Masai giraffe (Giraffa tippelskirchi) in Malawi may have resulted in introgressive hybridization. Thus, the current taxonomic affiliation for Malawi’s giraffe is uncertain, calling for a genetic assessment to implement further management. We analyzed mitochondrial sequences and nuclear introns for 14 individuals, representing approximately half of the known Malawian population, to genetically determine the (sub)species of giraffe that occur in the Republic of Malawi by comparison with a comprehensive Giraffa dataset. Additionally, we genotyped individuals at ten microsatellite loci to determine the level of inbreeding and potential introgression. All data identify individuals unambiguously as South African giraffe, although two individuals shared a single nuclear allele with Masai giraffe. The low microsatellite genetic variability suggests high inbreeding in the current population. Thus, supplementing Malawi’s giraffe populations with G. g. giraffa will prevent further loss of their genetic diversity and avoid inbreeding depression.

Recent advances in genome sequencing technologies have simplified the generation of genome data and reduced the costs for genome assemblies, even for complex genomes like those of vertebrates. More practically oriented genomic courses can prepare university students for the increasing importance of genomic data used in biological and medical research. Low-cost third-generation sequencing technology, along with publicly available data, can be used to teach students how to process genomic data, assemble full chromosome-level genomes, and publish the results in peer-reviewed journals, or preprint servers. Here we outline experiences gained from 2 master's-level courses and discuss practical considerations for teaching hands-on genome assembly courses.

The distribution maps of giraffe (Giraffa) subspecies in southern Africa are based on historical assumptions, yet some populations have likely been misidentified, hindering effective conservation efforts. Particularly, the populations in Zimbabwe are poorly studied, and translocations, such as the 1991 movement of giraffe from Namibia's Etosha National Park to mitigate the human-induced decline of Namibia's desert-dwelling giraffe in the lower Hoanib and Hoarusib Rivers, was concluded without consideration of genotype. Mitochondrial DNA sequence analyses from cytochrome b and control region revealed that instead of the South African giraffe subspecies (G. giraffa giraffa), the Angolan giraffe (G. g. angolensis) occurs in southern Zimbabwe's Bubye Valley Conservancy. Furthermore, Namibia's desert-dwelling giraffe and those from Etosha National Park form distinct matrilineal lineages within the Angolan giraffe subspecies clade. Thus, despite the translocation and proximity of the Etosha National Park, the Etosha giraffe genotype has not spread amongst the desert-dwelling giraffe. Use of mutation rate estimates indicates that there has been no matrilineal exchange between Etosha and the desert-dwelling giraffe for ca. 40,000 years. While philopatry could produce distinct mitochondrial lineages, the short geographical distance and long time involved, makes it more likely that the Etosha giraffe cannot successfully compete with a putatively better adapted desert-dwelling giraffe. The analyses also show that the distribution of the Angolan giraffe extends further eastwards than expected. These findings provide important implications for giraffe conservation, and translocation in particular, which may not always be an effective means of improving genetic diversity.

Three of the four species of giraffe are threatened, particularly the northern giraffe (Giraffa camelopardalis), which collectively have the smallest known wild population estimates. Among the three subspecies of the northern giraffe, the West African giraffe (Giraffa camelopardalis peralta) had declined to 49 individuals by 1996 and only recovered due to conservation efforts undertaken in the past 25 years, while the Kordofan giraffe (Giraffa camelopardalis antiquorum) remains at

Wastewater-based monitoring of the spread of the new SARS-CoV-2 virus, also referred to as wastewater-based epidemiology (WBE), has been suggested as a tool to support epidemiology. An extensive sampling campaign, including nine municipal wastewater treatment plants, has been conducted in different cities of the Federal State of North Rhine-Westphalia (Germany) on the same day in April 2020, close to the first peak of the corona crisis. Samples were processed and analysed for a set of SARS-CoV-2-specific genes, as well as pan-genotypic gene sequences also covering other coronavirus types, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, a comprehensive set of chemical reference parameters and bioindicators was analysed to characterize the wastewater quality and composition. Results of the RT-qPCR based gene analysis indicate the presence of SARS-CoV-2 genetic traces in different raw wastewaters. Furthermore, selected samples have been sequenced using Sanger technology to confirm the specificity of the RT-qPCR and the origin of the coronavirus. A comparison of the particle-bound and the dissolved portion of SARS-CoV-2 virus genes shows that quantifications must not neglect the solid-phase reservoir. The infectivity of the raw wastewater has also been assessed by viral outgrowth assay with a potential SARS-CoV-2 host cell line in vitro, which were not infected when exposed to the samples. This first evidence suggests that wastewater might be no major route for transmission to humans. Our findings draw attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater., Graphical abstract Unlabelled Image, Highlights • The first study that reports the detection of SARS-CoV-2 in wastewater in Germany using RT-qPCR. • The presence of SARS-CoV-2 was confirmed by sequencing, but also the risk of false-positive results has been elucidated. • In raw wastewater, 3.0 to 20 gene equivalents/mL was found in raw wastewater. • The replication potential tests were negative for wastewater samples. • Sanger sequencing was required to differentiate the genetic pattern clearly.

All giraffe (Giraffa) were previously assigned to a single species (G. camelopardalis) and nine subspecies. However, multi-locus analyses of all subspecies have shown that there are four genetically distinct clades and suggest four giraffe species. This conclusion might not be fully accepted due to limited data and lack of explicit gene flow analyses. Here we present an extended study based on 21 independent nuclear loci from 137 individuals. Explicit gene flow analyses identify less than one migrant per generation, including between the closely related northern and reticulated giraffe. Thus, gene flow analyses and population genetics of the extended dataset confirm four genetically distinct giraffe clades and support four independent giraffe species. The new findings call for a revision of the IUCN classification of giraffe taxonomy. Three of the four species are threatened with extinction, mostly occurring in politically unstable regions, and as such, require the highest conservation support possible.

In recent articles published in Molecular Phylogenetics and Evolution, Mark Springer and John Gatesy (S&G) present numerous criticisms of recent implementations and testing of the multispecies coalescent (MSC) model in phylogenomics, popularly known as "species tree" methods. After pointing out errors in alignments and gene tree rooting in recent phylogenomic data sets, particularly in Song et al. (2012) on mammals and Xi et al. (2014) on plants, they suggest that these errors seriously compromise the conclusions of these studies. Additionally, S&G enumerate numerous perceived violated assumptions and deficiencies in the application of the MSC model in phylogenomics, such as its assumption of neutrality and in particular the use of transcriptomes, which are deemed inappropriate for the MSC because the constituent exons often subtend large regions of chromosomes within which recombination is substantial. We acknowledge these previously reported errors in recent phylogenomic data sets, but disapprove of S&G's excessively combative and taunting tone. We show that these errors, as well as two nucleotide sorting methods used in the analysis of Amborella, have little impact on the conclusions of those papers. Moreover, several concepts introduced by S&G and an appeal to "first principles" of phylogenetics in an attempt to discredit MSC models are invalid and reveal numerous misunderstandings of the MSC. Contrary to the claims of S&G we show that recent computer simulations used to test the robustness of MSC models are not circular and do not unfairly favor MSC models over concatenation. In fact, although both concatenation and MSC models clearly perform well in regions of tree space with long branches and little incomplete lineage sorting (ILS), simulations reveal the erratic behavior of concatenation when subjected to data subsampling and its tendency to produce spuriously confident yet conflicting results in regions of parameter space where MSC models still perform well. S&G's claims that MSC models explain little or none (0-15%) of the observed gene tree heterogeneity observed in a mammal data set and that MSC models assume ILS as the only source of gene tree variation are flawed. Overall many of their criticisms of MSC models are invalidated when concatenation is appropriately viewed as a special case of the MSC, which in turn is a special case of emerging network models in phylogenomics. We reiterate that there is enormous promise and value in recent implementations and tests of the MSC and look forward to its increased use and refinement in phylogenomics.

High-resolution, male-inherited Y-chromosomal markers are a useful tool for population genetic analyses of wildlife species, but to date have only been applied in this context to relatively few species besides humans. Using nine Y-chromosomal STRs and three Y-chromosomal single nucleotide polymorphism markers (Y-SNPs), we studied whether male gene flow was important for the recent recovery of the brown bear (Ursus arctos) in Northern Europe, where the species declined dramatically in numbers and geographical distribution during the last centuries but is expanding now. We found 36 haplotypes in 443 male extant brown bears from Sweden, Norway, Finland and northwestern Russia. In 14 individuals from southern Norway from 1780 to 1920, we found two Y chromosome haplotypes present in the extant population as well as four Y chromosome haplotypes not present among the modern samples. Our results suggested major differences in genetic connectivity, diversity and structure between the eastern and the western populations in Northern Europe. In the west, our results indicated that the recovered population originated from only four male lineages, displaying pronounced spatial structuring suggestive of large-scale population size increase under limited male gene flow within the western subpopulation. In the east, we found a contrasting pattern, with high haplotype diversity and admixture. This first population genetic analysis of male brown bears shows conclusively that male gene flow was not the main force of population recovery.