Christian Lopezguerra (1) and Gavin M. Douglas (2, 3)

(1) Department of Plant and Microbial Biology; (2) Department of Biological Sciences; (3) Bioinformatics Research Center, North Carolina State University, USA

Climate change and anthropogenic stressors are driving rapid biodiversity loss and dynamic shifts in species ranges (Outhwaite et al. 2022). Partially in response to the decline in biodiversity, the European Reference Genome Atlas (ERGA) has been generating high-quality accessible genome resources, allowing for a more collaborative network and assisting conservation efforts.

One recent target was the violet carpenter bee (Xylocopa violacea; Figure 1), one of the many insects that have shown a recent expansion in their range within Europe. This species is a key pollinator and is therefore of great interest for ecological and agricultural purposes. In addition, anticancer research with melittin variants present in the venom of the violet carpenter bee shows potential (Erkoc et al. 2022; von Reumont et al. 2022). However, genetic analyses have been limited by the prior contig-level assembly of the genome (Koludarov et al. 2023). Developing a high-quality, annotated reference genome for the carpenter bee was the goal of Nash and colleagues’ (2024) research, as part of the European Reference Genome Atlas initiative.

Figure 1: Violet carpenter bee (in Margarida, Spain). Copyright Susanne Vogel, a photographer who made this available on iNaturalist.com. Distributed under a CC-BY 4.0 license.

The authors coupled long and short-read sequencing techniques to create an improved assembly. In particular, they used both short-read RNA-seq and long-read Iso-Seq for gene annotation. They also used Hi-C sequencing to capture chromosome conformational information to aid scaffolding. Their final assembly contains 1,300 scaffolds and has a BUSCO completeness level of 99.75% (Manni et al. 2021), aligning with the standards of the European Reference Genome Atlas. The authors generated a 1.02 gigabase assembly, which was much larger than the expected size of 672 megabases based on k-mer content. The authors partially explain the difference by the high repeat content in the genome, particularly specific 109-mer and 217-mer repeats. Due to this high repeat content, the authors could not assemble full chromosomes but instead produced 17 pseudo-chromosomes comprised of 481.4 megabases (in addition to all other unlocalized scaffolds). 

This high-quality reference genome will be valuable for future studies on population and functional genomics of carpenter bees (Xylocopa). Indeed, this is the first high-quality annotated pseudo-chromosomal genome assembly of the genus Xylocopa, which includes hundreds of other species. It will enable improved investigation into genomic signatures associated with shifting populations. More generally, this reference genome will be useful for comparative analyses with other Hymenoptera species.

References

Erkoc P, von Reumont BM, Lüddecke T, Henke M, Ulshöfer T, Vilcinskas A, Fürst R, Schiffmann S (2022) The pharmacological potential of novel melittin variants from the honeybee and solitary bees against inflammation and cancer. Toxins, 14, 818. https://doi.org/10.3390/toxins14120818

Koludarov I, Velasque M, Senoner T, Timm T, Greve C, Hamadou AB, Gupta DK, Lochnit G, Heinzinger M, Vilcinskas A, Gloag R, Harpur BA, Podsiadlowski L, Rost B, Jackson TNW, Dutertre S, Stolle E, von Reumont BM (2023) Prevalent bee venom genes evolved before the aculeate stinger and eusociality. BMC Biology, 21, 229. https://doi.org/10.1186/s12915-023-01656-5

Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM (2021) BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Molecular Biology and Evolution, 38, 4647–4654. https://doi.org/10.1093/molbev/msab199

Nash WJ, Man A, McTaggart S, Baker K, Barker T, Catchpole L, Durrant A, Gharbi K, Irish N, Kaithakottil G, Ku D, Providence A, Shaw F, Swarbreck D, Watkins C, McCartney AM, Formenti G, Mouton A, Vella N, von Reumont BM, Vella A, Haerty W (2024) The genome sequence of the Violet Carpenter Bee, Xylocopa violacea (Linnaeus, 1785): a hymenopteran species undergoing range expansion. Heredity, 133, 381–387. https://doi.org/10.1038/s41437-024-00720-2

Outhwaite CL, McCann P, Newbold T (2022) Agriculture and climate change are reshaping insect biodiversity worldwide. Nature, 605, 97–102. https://doi.org/10.1038/s41586-022-04644-x

von Reumont BM, Dutertre S, Koludarov I (2022) Venom profile of the European carpenter bee Xylocopa violacea: Evolutionary and applied considerations on its toxin components. Toxicon: X, 14, 100117. https://doi.org/10.1016/j.toxcx.2022.100117

The number and prevalence of invasive species have risen in the last decades together with international trade (Hulme 2021). As invasive species, insects have received less attention than plants. In particular, there are fewer reference genomes currently available, although genomic resources can be useful to investigate invasion dynamics and develop more effective management strategies.

Lombaeart et al. (2025) sequenced and assembled the genomes of three species: Cydalima perspectalis (the box tree moth), Leptoglossus occidentalis (the western conifer seed bug), and Tecia solanivora (the Guatemalan tuber moth), which have in common their rapid spread and severe impact on their respective host plants (boxwoods, conifers and potatoes). The authors generated PacBio HiFi reads, together with Hi-C data to obtain assemblies meeting international quality standards. They also generated short-read RNA-seq data, which they used to provide initial structural annotations of the genes. The resulting reference genomes are still in a draft state because repeats and heterozygosity are notoriously hard to handle. The most challenging genome to assemble was L. occidentalis, with an estimated size of 1.5 Gb, an estimated repeat content of 58%, and an estimated heterozygosity of 1.8%. The raw data produced can still be analysed more in depth to characterise further the repeat content and the heterozygosity of these species.

These reference genomes can readily be used for identifying genetic markers of interest for a variety of applications. In a general context where there is a growing awareness that data production is associated with a significant part of the carbon footprint of research (De Paepe et al. 2024), this dataset has high chances to be extensively reused and analysed by the community.

References

De Paepe M, Jeanneau L, Mariette J, Aumont O, Estevez-Torres A (2024) Purchases dominate the carbon footprint of research laboratories. PLOS Sustainability and Transformation, 3, e0000116. https://doi.org/10.1371/journal.pstr.0000116

Hulme, P E (2021) Unwelcome exchange: international trade as a direct and indirect driver of biological invasions worldwide. One Earth 4, 666–679. https://doi.org/10.1016/j.oneear.2021.04.015

Lombaert E, Klopp C, Blin A, Annonay G, Iampietro C, Lluch J, Sallaberry M, Valière S, Poloni R, Joron M, Deleury E (2025) Draft genome and transcriptomic sequence data of three invasive insect species. bioRxiv, ver. 2 peer-reviewed and recommended by PCI Genomics. https://doi.org/10.1101/2024.12.02.626401

Lampreys are the focus of intense research. Together with hagfishes, they form the Cyclostomata, the sister group of jawed vertebrates, and hence they are a key group for disentangling the early evolution of many vertebrate features (Shimel and Donoghue 2012; McCauley et al. 2015). Ecologically, lamprey species show a diverse array of life modes, including parasitic and non-feeding species, and inhabit freshwater and marine habitats or both (i.e. anadromous species; Docker and Potter 2019). One of these anadromous species, the sea lamprey (Petromyzon marinus), took advantage of man-made canals to invade the North American Great Lakes in the early 20th century, decimating many fish populations. Today, the control of these invasive populations is paramount for the survival of the region’s fishing industry (Ferreira-Martins et al. 2021). All these research avenues will benefit from the generation of new genomic data, an invaluable resource in evolutionary and conservation biology.

In this manuscript, Tørresen‬ et al. (2025) present phased, chromosome-level assemblies from two lamprey species: the European river lamprey (Lampetra fluviatilis) and the brook lamprey (Lampetra planeri). These two genome assemblies are of high quality and will undoubtedly become a key resource in lamprey research. In particular, the authors showcase the potential of such genomes from two perspectives. First, comparing their assemblies to the already published genomes from P. marinus and another specimen of L. fluviatilis, they propose that lamprey genomes are highly conserved and display large syntenic blocks shared among species. Second, phylogenetic analyses and the annotation of SNPs suggest that L. fluviatilis and L. planeri should be considered two ecotypes of the same species complex, instead of two separate species. This might not be new for anyone knowledgeable in lamprey biology (Rougemont et al. 2017), but it is surprising given the distinct ecology of the two lampreys: L. fluviatilis is a parasitic, anadromous species, whereas L. planeri is a non-feeding, freshwater species. 

In addition to the biological significance of this manuscript, I would like to acknowledge the robustness of the analytical approaches. These genomes were assembled and annotated following two pipelines recently developed at EBP-Nor, the Norwegian initiative of the Earth BioGenome Project (EBP). These pipelines are designed to be an easy-to-use, end-to-end solution for genomic analyses and are likely to become a standard for the EBP and European Reference Genome Atlas initiatives. There can be no better evidence of their effectiveness than these two phased, chromosome-level, highly complete genome assemblies.

References

Docker MF, Potter IC (2019) Life history evolution in lampreys: Alternative migratory and feeding types. In: Docker M (ed) Lampreys: Biology, Conservation and Control. Fish & Fisheries Series, vol 38. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-1684-8_4

Ferreira-Martins D, Champer J, McCauley DW, Zhang Z, Docker MF (2021) Genetic control of invasive sea lamprey in the Great Lakes. Journal of Great Lakes Research, 47, S764-S775. https://doi.org/10.1016/j.jglr.2021.10.018

McCauley DW, Docker MF, Whyard S, Li W (2015) Lampreys as diverse model organisms in the genomics era. BioScience, 65(11), 1046-1056. https://doi.org/10.1093/biosci/biv139

Rougemont Q, Gagnaire PA, Perrier C, Genthon C, Besnard AL, Launey S, Evanno G (2017) Inferring the demographic history underlying parallel genomic divergence among pairs of parasitic and nonparasitic lamprey ecotypes. Molecular Ecology, 26(1), 142-162. https://doi.org/10.1111/mec.13664

Shimeld SM, Donoghue PC (2012) Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish). Development, 139(12), 2091-2099. https://doi.org/10.1242/dev.074716

Tørresen OK, Garmann-Aarhus B, Hoff SNK, Jentoft S, Svensson M, Schartum E, Tooming-Klunderud A, Skage M, Krabberød‬​ A, ‭Vøllestad‬​ LA, Jakobsen KS (2025) Comparison of whole-genome assemblies of European river lamprey (Lampetra fluviatilis) and brook lamprey (Lampetra planeri). bioRxiv, ver. 5 peer-reviewed and recommended by PCI Genomics https://doi.org/10.1101/2024.12.06.627158​

The study of genomic variability within and between populations, as well as among species, relies on comparative analyses of homologous positions—sites that share a common evolutionary origin. Homology is inferred through sequence similarity (Reeck et al. 1987). However, the ability to detect homologous regions can be compromised when sequence mismatches accumulate due to mutations, especially when analyzing short DNA fragments, as in short-read sequencing (Li et al. 2008). In the genomic era, accurately mapping homologous DNA fragments to a reference genome is essential for obtaining precise estimates of genetic variability and evolutionary inferences (e.g., Li et al. 2008; Ellegren 2014). However, short-read, high-throughput sequencing often introduces mapping bias, disproportionately favoring the reference allele. This bias distorts allele frequency estimates, ancestry proportions, and genotype likelihoods, impacting downstream analyses (e.g., Günther & Nettelblad 2019; Martiniano et al. 2020).

Mapping bias is particularly problematic in ancient DNA studies, where post-mortem damage exacerbates sequencing errors. DNA fragmentation limits read length, while deamination, causing G to A and C to U transitions, increases mismatches and further complicates homology identification (Dabney & Pääbo 2013). These degradation processes contribute to the misidentification of true variants, confounding evolutionary inferences. Various strategies have been developed to mitigate mapping bias, including the commonly used approach, called pseudo-haploid data, that randomly picks a single read at each analyzed position for each  individual, thereby retaining a single allele at each polymorphic site (Günther & Nettelblad 2019; Barlow et al. 2020). 

Günther et al. (2025) introduce a novel method to correct mapping bias using a genotype likelihood-based approach, incorporating a mapping bias ratio to adjust for reference allele overrepresentation. The method specifically targets known single nucleotide polymorphisms (SNPs) because in population genomic analysis of ancient DNA data, low coverage and post-mortem damage often hinder the ability to identify novel SNPs in most individuals. The analysis focuses on DNA fragmentation, assuming that deamination effects are minimal when considering ascertained SNPs. The proposed method was compared against existing approaches, including pseudo-haploid data and standard genotype likelihood-based probabilistic methods. The evaluation was performed using both empirical and simulated data. For empirical data, low-coverage sequencing data from the 1000 Genomes Project (Finnish in Finland, Japanese in Tokyo, Yoruba in Ibadan, Nigeria populations) was analyzed, while for simulated data, ancient DNA-like datasets were generated using ms-prime (Kelleher et al. 2016), modeling different sequencing depths, divergence times, and reference genome choices. The study assesses the impact of mapping bias on the ratio of reference versus non-reference allele mapping, the accuracy of SNP allele frequency estimates relative to true frequencies, the deviation and variance between estimated and true allele frequencies, population differentiation and the estimation of admixture proportions using supervised and unsupervised methods, considering both genotype likelihoods and genotype calls.

Günther et al. (2025) bring to light that all methods analyzed exhibit minor but systematic reference allele bias. The new corrected genotype likelihood method outperforms the standard genotype likelihood approach in correlating with true allele frequencies, although the pseudo-haploid method still provides the most accurate estimates. Mapping bias also affects ancestry estimation, leading to admixture proportion errors of up to 4%, though this effect is smaller than the 10% discrepancy observed across different inference methods.

The work performed by Günther et al. (2025) provides a rigorous and innovative evaluation of mapping bias in the context of ascertained SNPs, introducing a probabilistic approach that improves bias correction. Unlike non-probabilistic methods such as pseudo-haploid data, the genotype likelihood framework leverages all sequencing reads for each analyzed SNP, and can incorporate additional bias corrections, enhancing its applicability across different sequencing conditions. While probabilistic approaches offer clear advantages in bias correction, they can be less intuitive to interpret compared to traditional genotype calling methods. This study highlights that mapping bias is pervasive across all methods, influencing evolutionary inferences such as selection signals and population differentiation. Although the improvements in allele frequency recovery may seem modest, the genome-wide impact of mapping bias is significant, especially in ancient DNA studies, making bias correction essential for robust evolutionary analyses.

References
 
Barlow A, Hartmann S, Gonzalez J, Hofreiter M, Paijmans JLA. (2020) Consensify: A method for generating pseudohaploid genome sequences from palaeogenomic datasets with reduced error rates. Genes;11(1):50. https://doi.org/10.3390/genes11010050 
 
Dabney J, Meyer M, Pääbo S. (2013) Ancient DNA damage. Cold Spring Harb Perspect Biol. 5(7):a012567. https://doi.org/10.1101/cshperspect.a012567 

Ellegren H. (2014) Genome sequencing and population genomics in non-model organisms. Trends Ecol Evol. 29(1):51-63. https://doi.org/10.1016/j.tree.2013.09.008 

Günther T, Nettelblad C. (2019) The presence and impact of reference bias on population genomic studies of prehistoric human populations. PLoS Genet.15(7):e1008302. https://doi.org/10.1371/journal.pgen.1008302 

Günther T., Goldberg A., Schraiber J. G.  (2025) Estimating allele frequencies, ancestry proportions and genotype likelihoods in the presence of mapping bias. bioRxiv, ver. 5 peer-reviewed and recommended by PCI Genomics https://doi.org/10.1101/2024.07.01.601500 

Kelleher J., Etheridge A. M., McVean G. (2016) Efficient coalescent simulation and genealogical analysis for large sample sizes. PLoS computational biology, 12(5):e1004842. https://doi.org/10.1371/journal.pcbi.1004842

Li H, Ruan J, Durbin R. (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18(11):1851-8. https://doi.org/10.1101/gr.078212.108 

Reeck GR, de Haën C, Teller DC, Doolittle RF, Fitch WM, Dickerson RE, et al. (1987) "Homology" in proteins and nucleic acids: a terminology muddle and a way out of it. Cell. 50 (5): 667. https://doi.org/10.1016/0092-8674(87)90322-9 

The first time I heard of ”onagres” in French was when I was a teenager, through the books of Pierre Bordage as fantastic monsters, or through historical games as Roman siege weapons (onagers). At this time, I was far from imagining that “onagre” also refers to a very large flowering plant family, as it is the French term for evening primroses.

In this family, the genus Ludwigia comprises species that are invasive (resembling in that way the ancient armies using onagers to invade cities) in aquatic environments, degrading ecosystems already fragilized by human activities. To counteract this phenomenon, it is of high importance to understand their propagation of these species. However, the knowledge about their genetics and diversity is very scarce, and thus tracking their dispersal using genetic information is complicated, and in fact almost impossible.

Barloy-Hubler et al. (2024) proposed in the present manuscript a new set of chloroplastic genomes from two of these species, Ludwigia grandiflora subsp. hexapetala and Ludwigia peploides subsp. montevidensis, and compared them to the published chloroplastic genome of Ludwigia octovalis. They explored the possibility of assembling these genomes relying solely on short reads and showed that long reads were necessary to obtain an almost complete assembly for these plastid genomes. In addition, through this approach, they detected two haplotypes in Ludwigia grandiflora subsp. hexapetala as compared to one in a short-read assembly. This highlights the need for long reads data to assess the structure and diversity of chloroplastic genomes. The authors were also able to clarify the phylogeny of the genus Ludwigia. Finally, they identified multiple potential single nucleotide polymorphisms and simple sequence repeats for future evaluation of diversity and dispersal of those invasive species.

This analysis, while appearing more technical than biological at first glance, is in fact of high importance for the understanding of ecology and preservation of fragile ecosystems, such as the European watersheds. Indeed, new scientific results and insights are generally linked to a reevaluation of previously analyzed data or samples through new technologies, and this paper is a quite clever example of that matter.

References

Barloy-Hubler F, Gac A-LL, Boury C, Guichoux E, Barloy D (2024) Sequencing, de novo assembly of Ludwigia plastomes, and comparative analysis within the Onagraceae family. bioRxiv, ver. 5 peer-reviewed and recommended by PCI Genomics. https://doi.org/10.1101/2023.10.20.563230

Bordage, P (1993) Les Guerriers du Silence, L'Atalante, ISBN 9782905158697