The impact of sequencing depth and relatedness of the reference genome in population genomic studies: A case study with two caddisfly species (Trichoptera, Rhyacophilidae, Himalopsyche).

Whole genome sequencing for generating SNP data is increasingly used in population genetic studies. However, obtaining genomes for massive numbers of samples is still not within the budgets of many researchers. It is thus imperative to select an appropriate reference genome and sequencing depth to ensure the accuracy of the results for a specific research question, while balancing cost and feasibility. To evaluate the effect of the choice of the reference genome and sequencing depth on downstream analyses, we used five confamilial reference genomes of variable relatedness and three levels of sequencing depth (3.5×, 7.5× and 12×) in a population genomic study on two caddisfly species: Himalopsyche digitata and H. tibetana. Using these 30 datasets (five reference genomes × three depths × two target species), we estimated population genetic indices (inbreeding coefficient, nucleotide diversity, pairwise F ST, and genome-wide distribution of F ST) based on variants and population structure (PCA and admixture) based on genotype likelihood estimates. The results showed that both distantly related reference genomes and lower sequencing depth lead to degradation of resolution. In addition, choosing a more closely related reference genome may significantly remedy the defects caused by low depth. Therefore, we conclude that population genetic studies would benefit from closely related reference genomes, especially as the costs of obtaining a high-quality reference genome continue to decrease. However, to determine a cost-efficient strategy for a specific population genomic study, a trade-off between reference genome relatedness and sequencing depth can be considered.

Plant migration under long-lasting hyperaridity - phylogenomics unravels recent biogeographic history in one of the oldest deserts on Earth.

The post-Miocene climatic histories of arid environments have been identified as key drivers of dispersal and diversification. Here, we investigate how climatic history correlates with the historical biogeography of the Atacama Desert genus Cristaria (Malvaceae). We analyze phylogenetic relationships and historical biogeography by using next-generation sequencing (NGS), molecular clock dating, Dispersal Extinction Cladogenesis and Bayesian sampling approaches. We employ a novel way to identify biogeographically meaningful regions as well as a rarely utilized program permitting the use of dozens of ancestral areas. Partial incongruence between the established taxonomy and our phylogenetic data argue for a complex historical biogeography with repeated introgression and incomplete lineage sorting. Cristaria originated in the central southern part of the Atacama Desert, from there the genus colonized other areas from the late Miocene onwards. The more recently diverged lineages appear to have colonized different habitats in the Atacama Desert during pluvial phases of the Pliocene and early Pleistocene. We show that NGS combined with near-comprehensive sampling can provide an unprecedented degree of phylogenetic resolution and help to correlate the historical biogeography of plant communities with cycles of arid and pluvial phases.

Genome size evolution in the diverse insect order Trichoptera.

BACKGROUND: Genome size is implicated in the form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: polyploidy (i.e., whole-genome duplication) or smaller duplication events and bursts in the activity of repetitive elements. Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages. RESULTS: We detect a ∼14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single-copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments). CONCLUSION: Our findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

Small and slow is safe: On the drought tolerance of tropical tree species.

Understanding how evolutionary history and the coordination between trait trade-off axes shape the drought tolerance of trees is crucial to predict forest dynamics under climate change. Here, we compiled traits related to drought tolerance and the fast-slow and stature-recruitment trade-off axes in 601 tropical woody species to explore their covariations and phylogenetic signals. We found that xylem resistance to embolism (P50) determines the risk of hydraulic failure, while the functional significance of leaf turgor loss point (TLP) relies on its coordination with water use strategies. P50 and TLP exhibit weak phylogenetic signals and substantial variation within genera. TLP is closely associated with the fast-slow trait axis: slow species maintain leaf functioning under higher water stress. P50 is associated with both the fast-slow and stature-recruitment trait axes: slow and small species exhibit more resistant xylem. Lower leaf phosphorus concentration is associated with more resistant xylem, which suggests a (nutrient and drought) stress-tolerance syndrome in the tropics. Overall, our results imply that (1) drought tolerance is under strong selective pressure in tropical forests, and TLP and P50 result from the repeated evolutionary adaptation of closely related taxa, and (2) drought tolerance is coordinated with the ecological strategies governing tropical forest demography. These findings provide a physiological basis to interpret the drought-induced shift toward slow-growing, smaller, denser-wooded trees observed in the tropics, with implications for forest restoration programmes.

A study on the genetic population structure and the tetrodotoxin content of rough-skinned newts, Taricha granulosa (Salamandridae), from their northern range of distribution.

Rough-skinned newts, Taricha granulosa, which contain tetrodotoxin (TTX), a potent neurotoxin, are widely distributed along the west-coast of North America up to British Columbia (BC), Canada, and Southeast Alaska. Their genetic population structure using DNA-microsatellites and the TTX-content of specimens from British Columbia (Prince Rupert area) and Alaska (Revillagigedo Island, Shelter Island, and Juneau) were analysed. TTX-concentrations were low in newts from BC and Revillagigedo Island, but high in specimens from mainland Juneau, which had been deliberately introduced from Shelter Island, where TTX was not detectable in the individuals sampled. No significant genetic differences were detected between these populations, which may correlate with the high intraspecies variability of TTX. It is still an open question, which factors favour or induce the toxin production in the newts.

Joining forces in Ochnaceae phylogenomics: a tale of two targeted sequencing probe kits.

PREMISE: Both universal and family-specific targeted sequencing probe kits are becoming widely used for reconstruction of phylogenetic relationships in angiosperms. Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits. Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships. METHODS: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support. RESULTS: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree. CONCLUSIONS: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.

Resolving Recalcitrant Clades in the Pantropical Ochnaceae: Insights From Comparative Phylogenomics of Plastome and Nuclear Genomic Data Derived From Targeted Sequencing.

Plastid DNA sequence data have been traditionally widely used in plant phylogenetics because of the high copy number of plastids, their uniparental inheritance, and the blend of coding and non-coding regions with divergent substitution rates that allow the reconstruction of phylogenetic relationships at different taxonomic ranks. In the present study, we evaluate the utility of the plastome for the reconstruction of phylogenetic relationships in the pantropical plant family Ochnaceae (Malpighiales). We used the off-target sequence read fraction of a targeted sequencing study (targeting nuclear loci only) to recover more than 100 kb of the plastid genome from the majority of the more than 200 species of Ochnaceae and all but two genera using de novo and reference-based assembly strategies. Most of the recalcitrant nodes in the family's backbone were resolved by our plastome-based phylogenetic inference, corroborating the most recent classification system of Ochnaceae and findings from a phylogenomic study based on nuclear loci. Nonetheless, the phylogenetic relationships within the major clades of tribe Ochnineae, which comprise about two thirds of the family's species diversity, received mostly low support. Generally, the phylogenetic resolution was lowest at the infrageneric level. Overall there was little phylogenetic conflict compared to a recent analysis of nuclear loci. Effects of taxon sampling were invoked as the most likely reason for some of the few well-supported discords. Our study demonstrates the utility of the off-target fraction of a target enrichment study for assembling near-complete plastid genomes for a large proportion of samples.

Annotated Draft Genomes of Two Caddisfly Species Plectrocnemia conspersa CURTIS and Hydropsyche tenuis NAVAS (Insecta: Trichoptera).

Members of the speciose insect order Trichoptera (caddisflies) provide important ecosystem services, for example, nutrient cycling through breaking down of organic matter. They are also of industrial interest due to their larval silk secretions. These form the basis for their diverse case-making behavior that allows them to exploit a wide range of ecological niches. Only five genomes of this order have been published thus far, with variable qualities regarding contiguity and completeness. A low-cost sequencing strategy, that is, using a single Oxford Nanopore flow cell per individual along with Illumina sequence reads was successfully used to generate high-quality genomes of two Trichoptera species, Plectrocnemia conspersa and Hydropsyche tenuis. Of the de novo assembly methods compared, assembly of low coverage Nanopore reads (∼18×) and subsequent polishing with long reads followed by Illumina short reads (∼80-170× coverage) yielded the highest genome quality both in terms of contiguity and BUSCO completeness. The presented genomes are the shortest to date and extend our knowledge of genome size across caddisfly families. The genomic region that encodes for light (L)-chain fibroin, a protein component of larval caddisfly silk was identified and compared with existing L-fibroin gene clusters. The new genomic resources presented in this paper are among the highest quality Trichoptera genomes and will increase the knowledge of this important insect order by serving as the basis for phylogenomic and comparative genomic studies.

Improved non-destructive 2D and 3D X-ray imaging of leaf venation.

BACKGROUND: Leaf venation traits are important for many research fields such as systematics and evolutionary biology, plant physiology, climate change, and paleoecology. In spite of an increasing demand for vein trait data, studies are often still data-limited because the development of methods that allow rapid generation of large sets of vein data has lagged behind. Recently, non-destructive X-ray technology has proven useful as an alternative to traditional slow and destructive chemical-based methods. Non-destructive techniques more readily allow the use of herbarium specimens, which provide an invaluable but underexploited resource of vein data and related environmental information. The utility of 2D X-ray technology and microfocus X-ray computed tomography, however, has been compromised by insufficient image resolution. Here, we advanced X-ray technology by increasing image resolution and throughput without the application of contrast agents. RESULTS: For 2D contact microradiography, we developed a method which allowed us to achieve image resolutions of up to 7 µm, i.e. a 3.6-fold increase compared to the industrial standard (25 µm resolution). Vein tracing was further optimized with our image processing standards that were specifically adjusted for different types of leaf structure and the needs of higher imaging throughput. Based on a test dataset, in 91% of the samples the 7 µm approach led to a significant improvement in estimations of minor vein density compared to the industrial standard. Using microfocus X-ray computed tomography, very high-resolution images were obtained from a virtual 3D-2D transformation process, which was superior to that of 3D images. CONCLUSIONS: Our 2D X-ray method with a significantly improved resolution advances rapid non-destructive bulk scanning at a quality that in many cases is sufficient to determine key venation traits. Together with our high-resolution microfocus X-ray computed tomography method, both non-destructive approaches will help in vein trait data mining from museum collections, which provide an underexploited resource of historical and recent data on environmental and evolutionary change. In spite of the significant increase in effective image resolution, a combination of high-throughput and full visibility of the vein network (including the smallest veins and their connectivity) remains challenging, however.

Water supply and demand remain coordinated during breakdown of the global scaling relationship between leaf size and major vein density.

Vein networks that disobey the global scaling of major vein density with leaf size shed light on functional constraints of vein network formation in dicotyledons. Understanding their evolution, distribution and impact on vein-stomata-climate associations is an important contribution to our global view of vein network organization. Based on vein traits of 55 species of pantropical Ochnaceae, stomata and climatic niche data, and a dated molecular phylogeny, we unveil major structural shifts in vein networks through deep time, relationships between leaf size, vein and stomata traits, and their interplay with climate. Dense 2° veins, reduction of minor veins and the associated breakdown of vein-leaf size scaling evolved multiple times independently in Ochnaceae. In spite of the drastic changes in vein architecture in this venation type, vein and stomatal densities remain correlated. Our study demonstrates that shortening the major vein-stomata distance is economically not less advantageous than by increasing minor vein density, as illustrated by the same degree of coordination between vein and stomatal densities and the similar construction costs across networks with dense 2° veins and those with 'normally' spaced 2° veins.

Phylogenetics, ancestral state reconstruction, and a new infrafamilial classification of the pantropical Ochnaceae (Medusagynaceae, Ochnaceae s.str., Quiinaceae) based on five DNA regions.

Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.

Molecular evidence for hybridization and introgression in the neotropical coastal desert-endemic Palaua (Malveae, Malvaceae).

Recent years have seen an increasing awareness of the importance of hybridization and introgression in plant evolution. Both processes were also invoked to have had a strong impact in the evolution of Palaua, based on morphological intermediacy. Here, we used nuclear ITS and cellulose synthase DNA sequences to assess previous phylogenetic hypotheses and to uncover previously undescribed reticulate evolution in Palaua. Both nuclear regions recovered largely the same reticulate pattern, showing that interspecific crosses occurred between distantly related species of the Dissecta and Integrifolia clade. Hybridization between species occurred only in a narrow contact zone. Moreover, many sequences of Palaua dissecta were placed in the strongly divergent Integrifolia clade, a pattern that is best explained by introgression. These results support our view that the strong interannual fluctuations of humidity and habitat expansion provide opportunities for the isolation of taxa, establishment of hybrid species, and for introgression between incompletely reproductively isolated taxa following recurrent contact.