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

Microsatellite characterisation and sex-typing in two invasive parakeet species, the monk parakeet Myiopsitta monachus and ring-necked parakeet Psittacula krameri

Invasive species can have wide-ranging negative impacts, and an understanding of the process and success of invasions can be vital to determine management strategies, mitigate impacts and predict range expansions of such species. Monk parakeets (Myiopsitta monachus) and ring-necked parakeets (Psittacula krameri) are both widespread invasive species, but there has been little research into the genetic and social structure of these two species despite the potential links with invasion success. The aim of this study was to isolate novel microsatellite loci from the monk parakeet and characterise them in both monk and ring-necked parakeets in order to facilitate future investigations into their behaviour and population ecology. Sex-typing markers were also tested in both species. Of the 20 microsatellite loci assessed in 24 unrelated monk parakeets, 16 successfully amplified and were polymorphic displaying between 2 and 14 alleles (mean = 8.06). Expected heterozygosity ranged from 0.43 to 0.93 and observed heterozygosity ranged from 0.23 to 0.96. Nine of the 20 loci also successfully amplified and were polymorphic in the ring-necked parakeet, displaying between 2 and 10 alleles. Suitable markers to sex both species and a Z-linked microsatellite locus were identified. A multiplex marker set was validated for monk parakeets. These novel microsatellite loci will facilitate fine and broad-scale population genetic analyses of these two widespread invasive species

Quantitative Trait Locus Analysis of Mating Behavior and Male Sex Pheromones in Nasonia Wasps.

A major focus in speciation genetics is to identify the chromosomal regions and genes that reduce hybridization and gene flow. We investigated the genetic architecture of mating behavior in the parasitoid wasp species pair Nasonia giraulti and Nasonia oneida that exhibit strong prezygotic isolation. Behavioral analysis showed that N. oneida females had consistently higher latency times and broke off the mating sequence more often in the mounting stage when confronted with N. giraulti males compared with males of their own species. N. oneida males produce a lower quantity of the long-range male sex pheromone, (4R,5S)-5-hydroxy-4-decanolide (RS-HDL). Crosses between the two species yielded hybrid males with various pheromone quantities and these males were used in mating trials with females of either species to measure female mate discrimination rates. A quantitative trait locus (QTL) analysis involving 475 recombinant hybrid males (F2), 2148 reciprocally backcrossed females (F3), and a linkage map of 52 equally spaced neutral single nucleotide polymorphism (SNP) markers plus SNPs in 40 candidate mating behavior genes revealed four QTL for male pheromone amount depending on partner species. Our results demonstrate that the RS-HDL pheromone plays a role in the mating system of N. giraulti and N. oneida, but also that additional communication cues are involved in mate choice. No QTL were found for female mate discrimination which points at a polygenic architecture of female choice with strong environmental influences

A multiplex microsatellite set for non-invasive genotyping and sexing of the osprey (Pandion haliaetus)

During the 1950s and 1970s the osprey (Pandion haliaetus) experienced a dramatic population crash and remains of conservation concern in several parts of the world. We isolated 37 microsatellite loci and assessed these in ospreys sampled in the UK and Norway (using mouth swabs/feathers). From 26 loci variable in four ospreys, we selected 13, combined these into two multiplex-PCR sets and included a sex-typing marker. Additional markers confirmed sexes. In 17 ospreys, feather-sampled in central Norway, we found 3–10 alleles per locus. The 13 loci are autosomal (heterozygotes were present in both sexes) and observed heterozygosities ranged from 0.24 to 0.94. The combined probability of identity for the 13 loci was 8.0 × 10−12. These microsatellite loci will be useful for genetic monitoring, parentage analysis and population genetic studies of the osprey

Admixture and reproductive skew shape the conservation value of ex situ populations of the Critically Endangered eastern black rhino

Small populations of endangered species risk losing already eroded genetic diversity, important for adaptive potential, through the effects of genetic drift. The magnitude of drift can be mitigated by maximising the effective population size, as is the goal of genetic management strategies. Different mating systems, specifically those leading to reproductive skew, exacerbate genetic drift by distorting contributions. In the absence of an active management strategy, reproductive skew will have long-term effects on the genetic composition of a population, particularly where admixture is present. Here we examine the contrasting effects of conservation management strategies in two ex situ populations of the Critically Endangered eastern black rhino (Diceros bicornis michaeli), one managed as a semi-wild population in South Africa (SAx), and one managed under a mean-kinship breeding strategy in European zoos. We use molecular data to reconstruct pedigrees for both populations and validate the method using the zoo studbook. Using the reconstructed pedigree and studbook we show there is male sex-specific skew in both populations. However, the zoo’s mean-kinship breeding strategy effectively reduces reproductive skew in comparison to a semi-wild population with little genetic management. We also show that strong male reproductive skew in SAx has resulted in extensive admixture, which may require a re-evaluation of the population’s original intended role in the black rhino meta-population. With a high potential for admixture in many ex situ populations of endangered species, molecular and pedigree data remain vital tools for populations needing to balance drift and selection

Assessing rates of parasite coinfection and spatiotemporal strain variation via metabarcoding: insights for the conservation of European turtle doves Streptopelia turtur

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe's most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intrastrain variation and four novel subtypes revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89%–100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations

Conservation by trans-border cooperation: population genetic structure and diversity of geoffroy’s bat (Myotis emarginatus) at its north-western european range edge

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy’s bat (Myotis emarginatus) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species’ genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented

Similar yet different: co-analysis of the genetic diversity and structure of an invasive nematode parasite and its invasive mammalian host

Animal parasitic nematodes can cause serious diseases and their emergence in new areas can be an issue of major concern for biodiversity conservation and human health. Their ability to adapt to new environments and hosts is likely to be affected by their degree of genetic diversity, with gene flow between distinct populations counteracting genetic drift and increasing effective population size. The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal parasite of the raccoon (Procyon lotor), has increased its global geographic range after being translocated with its host. The raccoon has been introduced multiple times to Germany, but not all its populations are infected with the parasite. While fewer introduced individuals may have led to reduced diversity in the parasite, admixture between different founder populations may have counteracted genetic drift and bottlenecks. Here, we analyse the population genetic structure of the roundworm and its raccoon host at the intersection of distinct raccoon populations infected with B. procyonis. We found evidence for two parasite clusters resulting from independent introductions. Both clusters exhibited an extremely low genetic diversity, suggesting small founding populations subjected to inbreeding and genetic drift with no, or very limited, genetic influx from population admixture. Comparison of the population genetic structures of both host and parasite suggested that the parasite spread to an uninfected raccoon founder population. On the other hand, an almost perfect match between cluster boundaries also suggested that the population genetic structure of B. procyonis has remained stable since its introduction, mirroring that of its raccoon host

High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species

Background Microsatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility. Results We selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species. Conclusions When combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available