Background

Long-read shotgun metagenomic sequencing is gaining in popularity and offers many advantages over short-read sequencing. The higher information content in long reads is useful for a variety of metagenomics analyses, including taxonomic classification and profiling. The development of long-read specific tools for taxonomic classification is accelerating, yet there is a lack of information regarding their relative performance. Here, we perform a critical benchmarking study using 11 methods, including five methods designed specifically for long reads. We applied these tools to several mock community datasets generated using Pacific Biosciences (PacBio) HiFi or Oxford Nanopore Technology sequencing, and evaluated their performance based on read utilization, detection metrics, and relative abundance estimates.

Results

Our results show that long-read classifiers generally performed best. Several short-read classification and profiling methods produced many false positives (particularly at lower abundances), required heavy filtering to achieve acceptable precision (at the cost of reduced recall), and produced inaccurate abundance estimates. By contrast, two long-read methods (BugSeq, MEGAN-LR & DIAMOND) and one generalized method (sourmash) displayed high precision and recall without any filtering required. Furthermore, in the PacBio HiFi datasets these methods detected all species down to the 0.1% abundance level with high precision. Some long-read methods, such as MetaMaps and MMseqs2, required moderate filtering to reduce false positives to resemble the precision and recall of the top-performing methods. We found read quality affected performance for methods relying on protein prediction or exact k-mer matching, and these methods performed better with PacBio HiFi datasets. We also found that long-read datasets with a large proportion of shorter reads (< 2 kb length) resulted in lower precision and worse abundance estimates, relative to length-filtered datasets. Finally, for classification methods, we found that the long-read datasets produced significantly better results than short-read datasets, demonstrating clear advantages for long-read metagenomic sequencing.

Conclusions

Our critical assessment of available methods provides best-practice recommendations for current research using long reads and establishes a baseline for future benchmarking studies.

Sexual size dimorphism (SSD) is shaped by multiple selective forces that drive the evolution of sex-specific body size, resulting in male or female-biased SSD. Stronger selection on one sex can result in an allometric body size scaling relationship consistent with Rensch's rule or its converse. Anurans (frogs and toads) generally display female-biased SSD, but there is variation across clades and the mechanisms driving the evolution of SSD remain poorly understood. We investigated these topics in a diverse family of African treefrogs (Hyperoliidae). Hyperoliids display traits considered rare among amphibians, including sexual dichromatism and protogynous sex change. Using phylogenetic comparative methods, we tested if adult ecology, sexual dichromatism, and sex change were predictors of body size or SSD. We also tested whether hyperoliids displayed allometric interspecific body size scaling relationships. We found a majority of hyperoliid taxa display female-biased SSD, but that adult ecology and sexual dichromatism are poor predictors of sex-specific body size and SSD. Regardless of the groupings analyzed (partitioned by clades or traits), we found support for isometric body size scaling. However, we found that sex change is a significant predictor of SSD variation. Species in the Hyperolius viridiflavus complex, which putatively display this trait, show a significant reduction in SSD and are frequently sexually monomorphic in size. Although protogynous sex change needs to be validated for several of these species, we tentatively propose this trait is a novel mechanism influencing anuran body size evolution. Beyond this association, additional factors that shape the evolution of anuran body size and SSD remain elusive.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Theory predicts that sexually dimorphic traits under strong sexual selection, particularly those involved with intersexual signaling, can accelerate speciation and produce bursts of diversification. Sexual dichromatism (sexual dimorphism in color) is widely used as a proxy for sexual selection and is associated with rapid diversification in several animal groups, yet studies using phylogenetic comparative methods to explicitly test for an association between sexual dichromatism and diversification have produced conflicting results. Sexual dichromatism is rare in frogs, but it is both striking and prevalent in African reed frogs, a major component of the diverse frog radiation termed Afrobatrachia. In contrast to most other vertebrates, reed frogs display female-biased dichromatism in which females undergo color transformation, often resulting in more ornate coloration in females than in males. We produce a robust phylogeny of Afrobatrachia to investigate the evolutionary origins of sexual dichromatism in this radiation and examine whether the presence of dichromatism is associated with increased rates of net diversification. We find that sexual dichromatism evolved once within hyperoliids and was followed by numerous independent reversals to monochromatism. We detect significant diversification rate heterogeneity in Afrobatrachia and find that sexually dichromatic lineages have double the average net diversification rate of monochromatic lineages. By conducting trait simulations on our empirical phylogeny, we demonstrate that our inference of trait-dependent diversification is robust. Although sexual dichromatism in hyperoliid frogs is linked to their rapid diversification and supports macroevolutionary predictions of speciation by sexual selection, the function of dichromatism in reed frogs remains unclear. We propose that reed frogs are a compelling system for studying the roles of natural and sexual selection on the evolution of sexual dichromatism across micro- and macroevolutionary timescales.

Theory predicts that sexually dimorphic traits under strong sexual selection, particularly those involved with intersexual signaling, can accelerate speciation and produce bursts of diversification. Sexual dichromatism (sexual dimorphism in color) is widely used as a proxy for sexual selection and is associated with rapid diversification in several animal groups, yet studies using phylogenetic comparative methods to explicitly test for an association between sexual dichromatism and diversification have produced conflicting results. Sexual dichromatism is rare in frogs, but it is both striking and prevalent in African reed frogs, a major component of the diverse frog radiation termed Afrobatrachia. In contrast to most other vertebrates, reed frogs display female-biased dichromatism in which females undergo color transformation, often resulting in more ornate coloration in females than in males. We produce a robust phylogeny of Afrobatrachia to investigate the evolutionary origins of sexual dichromatism in this radiation and examine whether the presence of dichromatism is associated with increased rates of net diversification. We find that sexual dichromatism evolved once within hyperoliids and was followed by numerous independent reversals to monochromatism. We detect significant diversification rate heterogeneity in Afrobatrachia and find that sexually dichromatic lineages have double the average net diversification rate of monochromatic lineages. By conducting trait simulations on our empirical phylogeny, we demonstrate that our inference of trait-dependent diversification is robust. Although sexual dichromatism in hyperoliid frogs is linked to their rapid diversification and supports macroevolutionary predictions of speciation by sexual selection, the function of dichromatism in reed frogs remains unclear. We propose that reed frogs are a compelling system for studying the roles of natural and sexual selection on the evolution of sexual dichromatism across micro- and macroevolutionary timescales.