Testing the performance of environmental DNA metabarcoding for surveying highly diverse tropical fish communities: A case study from Lake Tanganyika

Background and Aims Environmental DNA (eDNA) metabarcoding provides a highly sensitive method of surveying freshwater fish communities, although studies to date have largely been restricted to temperate ecosystems. Due to limited reference sequence availability and challenges identifying closely related and rare species in diverse tropical ecosystems, the effectiveness of metabarcoding methods for surveying tropical fish communities from eDNA samples remains uncertain. To address this, we applied an eDNA metabarcoding approach to survey Lake Tanganyika's (LT) species‐rich littoral fish communities. Materials and Methods As this system contains many closely related species, particularly cichlid fishes, we used four primer sets including a cichlid‐specific primer set (Cichlid_CR). A reference database was built for the 12s, 16s, and control region for 358 fish species including over 93% of known cichlids. Results and Discussion In silico and in situ results demonstrated wide variability in the taxonomic resolution of assignments by each primer with the cichlid‐specific marker (Cichlid_CR) enabling greater species‐level assignments for this highly diverse family. A greater number of non‐cichlid teleost species were detected at sites compared to the visual survey data. For cichlid species however, sequencing depth substantially influenced species richness estimates obtained from eDNA samples, with increased depths producing estimates comparable to that obtained from the visual survey data. Conclusions Our study highlights the importance of sequencing depth and local reference databases when undertaking metabarcoding studies within diverse ecosystems, as well as demonstrating the potential of eDNA metabarcoding for surveying diverse tropical fish communities, even those containing closely related species within evolutionary radiations

A diet rich in C3 plants reveals the sensitivity of an alpine mammal to climate change

Plant-herbivore interactions provide critical insights into the mechanisms that govern the spatiotemporal distributions of organisms. These interactions are crucial to understanding the impacts of climate change, which are likely to have an effect on the population dynamics of alpine herbivores. The Royle's pika (Ochotona roylei, hereafter pika) is a lagomorph found in the western Himalaya and is dependent on alpine plants that are at risk from climate change. As the main prey of many carnivores in the region, the pika plays a crucial role in trophic interactions. We examined topographical features, plant genera presence and seasonal dynamics as drivers of the plant richness in the pika's diet across an elevational gradient (2,600-4,450 m). We identified 79 plant genera in the faecal pellets of pikas, of which 89% were forbs, >60% were endemic to the Himalaya, and 97.5% of the diet plant genera identified followed the C3 photosynthetic pathway. We found that, during the premonsoon season, the number of genera in the pika's diet decreased with increasing elevation. We demonstrate that a large area of talus supports greater plant diversity and, not surprisingly, results in higher species richness in the pika's diet. However, in talus habitat with deep crevices, pikas consumed fewer plant genera suggesting they may be foraging suboptimally due to predation risk. The continued increase in global temperature is expected to have an effect on the distribution dynamics of C3 plants and consequently influence pika diet and distribution, resulting in a significant negative cascading effect on the Himalayan ecosystem

Negative effects of urbanisation on diurnal and nocturnal pollen‐transport networks

Pollinating insects are declining due to habitat loss and climate change, and cities with limited habitat and floral resources may be particularly vulnerable. The effects of urban landscapes on pollination networks remain poorly understood, and comparative studies of taxa with divergent niches are lacking. Here, for the first time, we simultaneously compare nocturnal moth and diurnal bee pollen-transport networks using DNA metabarcoding and ask how pollination networks are affected by increasing urbanisation. Bees and moths exhibited substantial divergence in the communities of plants they interact with. Increasing urbanisation had comparable negative effects on pollen-transport networks of both taxa, with significant declines in pollen species richness. We show that moths are an important, but overlooked, component of urban pollen-transport networks for wild flowering plants, horticultural crops, and trees. Our findings highlight the need to include both bee and non-bee taxa when assessing the status of critical plant-insect interactions in urbanised landscapes

Ecological speciation in sympatric palms: 2. Pre- and post-zygotic isolation

We evaluated reproductive isolation in two species of palms (Howea) that have evolved sympatrically on Lord Howe Island (LHI, Australia). We estimated the strength of some pre- and post-zygotic mechanisms in maintaining current species boundaries. We found that flowering time displacement between species is consistent across in and ex situ common gardens and is thus partly genetically determined. On LHI, pre-zygotic isolation due solely to flowering displacement was 97% for Howea belmoreana and 80% for H. forsteriana; this asymmetry results from H. forsteriana flowering earlier than H. belmoreana and being protandrous. As expected, only a few hybrids (here confirmed by genotyping) at both juvenile and adult stages could be detected in two sites on LHI, in which the two species grow intermingled (the Far Flats) or adjacently (Transit Hill). Yet, the distribution of hybrids was different between sites. At Transit Hill, we found no hybrid adult trees, but 13.5% of younger palms examined there were of late hybrid classes. In contrast, we found four hybrid adult trees, mostly of late hybrid classes, and only one juvenile F1 hybrid in the Far Flats. This pattern indicates that selection acts against hybrids between the juvenile and adult stages. An in situ reciprocal seed transplant between volcanic and calcareous soils also shows that early fitness components (up to 36 months) were affected by species and soil. These results are indicative of divergent selection in reproductive isolation, although it does not solely explain the current distribution of the two species on LHI

Whole‐genome sequencing and genome regions of special interest : lessons from major histocompatibility complex, sex determination, and plant self‐incompatibility

Whole-genome sequencing of non-model organisms is now widely accessible and has allowed a range of questions in the field of molecular ecology to be investigated with greater power. However, some genomic regions that are of high biological interest remain problematic for assembly and data-handling. Three such regions are the major histocompatibility complex (MHC), sex-determining regions (SDRs) and the plant self-incompatibility locus (S-locus). Using these as examples, we illustrate the challenges of both assembling and resequencing these highly polymorphic regions and how bioinformatic and technological developments are enabling new approaches to their study. Mapping short-read sequences against multiple alternative references improves genotyping comprehensiveness at the S-locus thereby contributing to more accurate assessments of allelic frequencies. Long-read sequencing, producing reads of several tens to hundreds of kilobase pairs in length, facilitates the assembly of such regions as single sequences can span the multiple duplicated gene copies of the MHC region, and sequence through repetitive stretches and translocations in SDRs and S-locus haplotypes. These advances are adding value to short-read genome resequencing approaches by allowing, for example, more accurate haplotype phasing across longer regions. Finally, we assessed further technical improvements, such as nanopore adaptive sequencing and bioinformatic tools using pangenomes, which have the potential to further expand our knowledge of a number of genomic regions that remain challenging to study with classical resequencing approaches

The diet of red-throated divers (Gavia stellata) overwintering in the German Bight (North Sea) analysed using molecular diagnostics

In Europe, the German Bight is one of the most important non-breeding areas for protected red-throated divers (Gavia stellata). It is unclear what attracts the birds to this area, especially as the food composition of seabirds outside the breeding season is notoriously difficult to study. To obtain information on prey species composition of red-throated divers in this area, faecal samples from 34 birds caught alive were analysed using DNA metabarcoding. Prey DNA was detected in 85% of the samples with a mean number of 4.2 ± 0.7 taxa per sample (n = 29). Altogether, we found a broad prey spectrum with 19 fish taxa from 13 families dominated by five groups: clupeids, mackerel, gadoids, flatfish and sand lances with clupeids being the most frequently detected prey. Our results indicate that red-throated divers are generalist opportunistic feeders in the German Bight, but pelagic schooling fish that aggregate at frontal zones and have a high energetic value might be favoured. Atlantic mackerel appears to be a more important prey for red-throated divers in this area than previously thought. The precision achievable using metabarcoding has revealed a number of prey species that are consumed by red-throated divers in the German Bight, which helps to explain the selection of this area by divers in winter and spring

Ecological speciation in sympatric palms: 1. Gene expression, selection and pleiotropy

Ecological speciation requires divergent selection, reproductive isolation and a genetic mechanism to link the two. We examined the role of gene expression and coding sequence evolution in this process using two species of Howea palms that have diverged sympatrically on Lord Howe Island, Australia. These palms are associated with distinct soil types and have displaced flowering times, representing an ideal candidate for ecological speciation. We generated large amounts of RNA-Seq data from multiple individuals and tissue types collected on the island from each of the two species. We found that differentially expressed loci as well as those with divergent coding sequences between Howea species were associated with known ecological and phenotypic differences, including response to salinity, drought, pH and flowering time. From these loci, we identified potential 'ecological speciation genes' and further validate their effect on flowering time by knocking out orthologous loci in a model plant species. Finally, we put forward six plausible ecological speciation loci, providing support for the hypothesis that pleiotropy could help to overcome the antagonism between selection and recombination during speciation with gene flow

Allelic diversity and patterns of selection at the major histocompatibility complex class I and II loci in a threatened shorebird, the Snowy Plover (Charadrius nivosus)

Background: Understanding the structure and variability of adaptive loci such as the major histocompatibility complex (MHC) genes is a primary research goal for evolutionary and conservation genetics. Typically, classical MHC genes show high polymorphism and are under strong balancing selection, as their products trigger the adaptive immune response in vertebrates. Here, we assess the allelic diversity and patterns of selection for MHC class I and class II loci in a threatened shorebird with highly flexible mating and parental care behaviour, the Snowy Plover (Charadrius nivosus) across its broad geographic range. Results: We determined the allelic and nucleotide diversity for MHC class I and class II genes using samples of 250 individuals from eight breeding population of Snowy Plovers. We found 40 alleles at MHC class I and six alleles at MHC class II, with individuals carrying two to seven different alleles (mean 3.70) at MHC class I and up to two alleles (mean 1.45) at MHC class II. Diversity was higher in the peptide-binding region, which suggests balancing selection. The MHC class I locus showed stronger signatures of both positive and negative selection than the MHC class II locus. Most alleles were present in more than one population. If present, private alleles generally occurred at very low frequencies in each population, except for the private alleles of MHC class I in one island population (Puerto Rico, lineage tenuirostris). Conclusion: Snowy Plovers exhibited an intermediate level of diversity at the MHC, similar to that reported in other Charadriiformes. The differences found in the patterns of selection between the class I and II loci are consistent with the hypothesis that different mechanisms shape the sequence evolution of MHC class I and class II genes. The rarity of private alleles across populations is consistent with high natal and breeding dispersal and the low genetic structure previously observed at neutral genetic markers in this species

Evidence for strain-specific virulence of <i>Trichomonas gallinae</i> in African columbiformes

Infection by parasites or pathogens can have marked physiological impacts on individuals. In birds, infection may affect moult and feather growth, which is an energetically demanding time in the annual cycle. Previous work has suggested a potential link between clinically visible Trichomonas gallinae infection and wing length in turtle doves Streptopelia turtur arriving on breeding grounds. First, T. gallinae infection was characterized in 149 columbids from 5 species, sampled on turtle dove wintering grounds in Senegal during the moulting period, testing whether infection by T. gallinae is linked to moult. Trichomonas gallinae prevalence was 100%, so rather than testing for differences between infected and uninfected birds, we tested for differences in moult progression between birds infected by different T. gallinae strains. Twelve strains of T. gallinae were characterized at the internal transcribed spacer 1 (ITS1)/5.8S/ITS2 region, of which 6 were newly identified within this study. In turtle doves only, evidence for differences in wing length by strain was found, with birds infected by strain Tcl-1 having wings nearly 6 mm longer than those infected with strain GEO. No evidence was found for an effect of strain identity within species on moult progression, but comparisons between infected and uninfected birds should be further investigated in species where prevalence is lower

Evidence for strain-specific virulence of Trichomonas gallinae in African columbiformes

Infection by parasites or pathogens can have marked physiological impacts on individuals. In birds, infection may affect moult and feather growth, which is an energetically demanding time in the annual cycle. Previous work has suggested a potential link between clinically visible Trichomonas gallinae infection and wing length in turtle doves Streptopelia turtur arriving on breeding grounds. First, T. gallinae infection was characterized in 149 columbids from 5 species, sampled on turtle dove wintering grounds in Senegal during the moulting period, testing whether infection by T. gallinae is linked to moult. Trichomonas gallinae prevalence was 100%, so rather than testing for differences between infected and uninfected birds, we tested for differences in moult progression between birds infected by different T. gallinae strains. Twelve strains of T. gallinae were characterized at the internal transcribed spacer 1 (ITS1)/5.8S/ITS2 region, of which 6 were newly identified within this study. In turtle doves only, evidence for differences in wing length by strain was found, with birds infected by strain Tcl-1 having wings nearly 6 mm longer than those infected with strain GEO. No evidence was found for an effect of strain identity within species on moult progression, but comparisons between infected and uninfected birds should be further investigated in species where prevalence is lower