DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera-recorded diets.

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free-living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole-nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size-adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera-recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R = .85 (95CI:0.68-0.94) at order level and at R = .75 (CI:0.67-0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.

Growth, maturity, and diet of the pearl whipray (Fontitrygon margaritella) from the Bijagós Archipelago, Guinea-Bissau.

The pearl whipray Fontitrygon margaritella (Compagno & Roberts, 1984) is a common elasmobranch in coastal western African waters. However, knowledge on their life-history and trophic ecology remains limited. Therefore, we aimed to determine the growth, maturity and diet of F. margaritella from the Bijagós Archipelago in Guinea-Bissau. Growth was modelled with: von Bertalanffy, Gompertz and logistic functions. Model selection revealed no model significantly outperformed another. The sampled age ranged from less than 1 to 7 years (1.8 ± 1.9 cm, mean ± standard deviation) and size (disc width) ranged from 12.2 to 30.6 cm (18.7 ± 5.2 cm). Size-at-maturity was estimated at 20.3 cm (95% CI [18.8-21.8 cm]) for males and 24.3 cm for females (95% CI [21.9-26.5 cm]), corresponding ages of 2.2 and 3.9 years. The diet differed significantly among young-of-the-year (YOY), juveniles and adults (p = 0.001). Diet of all life stages consisted mainly of crustaceans (27.4%, 28.5%, 33.3%) and polychaetes (12.5%, 26.7%, 20.3%), for YOY, juveniles and adults respectively. This study shows that F. margaritella is relatively fast-growing, matures early and experiences ontogenetic diet shifts. These results contribute to status assessments and conservation efforts of F. margaritella and closely related species.

Global flyway evolution in red knots Calidris canutus and genetic evidence for a Nearctic refugium.

Present-day ecology and population structure are the legacies of past climate and habitat perturbations, and this is particularly true for species that are widely distributed at high latitudes. The red knot, Calidris canutus, is an arctic-breeding, long-distance migratory shorebird with six recognized subspecies defined by differences in morphology, migration behavior, and annual cycle phenology, in a global distribution thought to have arisen just since the last glacial maximum (LGM). We used nextRAD sequencing of 10,881 single-nucleotide polymorphisms (SNPs) to assess the neutral genetic structure and phylogeographic history of 172 red knots representing all known global breeding populations. Using population genetics approaches, including model-based scenario-testing in an approximate Bayesian computation (ABC) framework, we infer that red knots derive from two main lineages that diverged ca. 34,000 years ago, and thus most probably persisted at the LGM in both Palearctic and Nearctic refugia, followed by at least two instances of secondary contact and admixture. Within two Beringian subspecies (C. c. roselaari and rogersi), we detected previously unknown genetic structure among sub-populations sharing a migratory flyway, reflecting additional complexity in the phylogeographic history of the region. Conversely, we found very weak genetic differentiation between two Nearctic populations (rufa and islandica) with clearly divergent migratory phenotypes and little or no apparent contact throughout the annual cycle. Together, these results suggest that relative gene flow among migratory populations reflects a complex interplay of historical, geographical, and ecological factors.

Prenatal Transfer of Gut Bacteria in Rock Pigeon.

Vertebrates evolved in concert with bacteria and have developed essential mutualistic relationships. Gut bacteria are vital for the postnatal development of most organs and the immune and metabolic systems and may likewise play a role during prenatal development. Prenatal transfer of gut bacteria is shown in four mammalian species, including humans. For the 92% of the vertebrates that are oviparous, prenatal transfer is debated, but it has been demonstrated in domestic chicken. We hypothesize that also non-domestic birds can prenatally transmit gut bacteria. We investigated this in medium-sized Rock pigeon (Columba livia), ensuring neonates producing fair-sized first faeces. The first faeces of 21 neonate rock pigeons hatched in an incubator, contained a microbiome (bacterial community) the composition of which resembled the cloacal microbiome of females sampled from the same population (N = 5) as indicated by multiple shared phyla, orders, families, and genera. Neonates and females shared 16.1% of the total number of OTUs present (2881), and neonates shared 45.5% of their core microbiome with females. In contrast, the five females shared only 0.3% of the 1030 female OTUs present. These findings suggest that prenatal gut bacterial transfer may occur in birds. Our results support the hypothesis that gut bacteria may be important for prenatal development and present a heritability pathway of gut bacteria in vertebrates.

A supergene determines highly divergent male reproductive morphs in the ruff.

Three strikingly different alternative male mating morphs (aggressive 'independents', semicooperative 'satellites' and female-mimic 'faeders') coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

Genetic variation in nuclear and mitochondrial markers supports a large sex difference in lifetime reproductive skew in a lekking species.

Sex differences in skews of vertebrate lifetime reproductive success are difficult to measure directly. Evolutionary histories of differential skew should be detectable in the genome. For example, male-biased skew should reduce variation in the biparentally inherited genome relative to the maternally inherited genome. We tested this approach in lek-breeding ruff (Class Aves, Philomachus pugnax) by comparing genetic variation of nuclear microsatellites (θ n ; biparental) versus mitochondrial D-loop sequences (θ m ; maternal), and conversion to comparable nuclear (N e ) and female (N ef ) effective population size using published ranges of mutation rates for each marker (µ). We provide a Bayesian method to calculate N e (θ n = 4N e µ n ) and N ef (θ m = 2N ef µ m ) using 95% credible intervals (CI) of θ n and θ m as informative priors, and accounting for uncertainty in µ. In 96 male ruffs from one population, N e was 97% (79-100%) lower than expected under random mating in an ideal population, where N e :N ef = 2. This substantially lower autosomal variation represents the first genomic support of strong male reproductive skew in a lekking species.

Characterization of MHC class I in a long-distance migrant shorebird suggests multiple transcribed genes and intergenic recombination.

The major histocompatibility complex (MHC) includes highly polymorphic gene families encoding proteins crucial to the vertebrate acquired immune system. Classical MHC class I (MHCI) genes code for molecules expressed on the surfaces of most nucleated cells and are associated with defense against intracellular pathogens, such as viruses. These genes have been studied in a few wild bird species, but have not been studied in long-distance migrating shorebirds. Red Knots Calidris canutus are medium-sized, monogamous sandpipers with migratory routes that span the globe. Understanding how such long-distance migrants protect themselves from disease has gained new relevance since the emergence of avian-borne diseases, including intracellular pathogens recognized by MHCI molecules, such as avian influenza. In this study, we characterized MHCI genes in knots and found 36 alleles in eight individuals and evidence for six putatively functional and expressed MHCI genes in a single bird. We also found evidence for recombination and for positive selection at putative peptide binding sites in exons 2 and 3. These results suggest surprisingly high MHC diversity in knots, given their demographic history. This may be a result of selection from diverse pathogens encountered by shorebirds throughout their annual migrations.

A novel mitochondrial gene order in shorebirds (Scolopacidae, Charadriiformes).

Although the mitochondrial genome in birds has highly conserved features, with protein genes similar to mammals and amphibians, several variations in gene order around the hypervariable control region have been found. Here we report a novel gene arrangement around the control region in shorebirds (Charadriiformes). In ruffs Philomachus pugnax, the mitochondrial genome between cytochrome b and 12SrRNA was over 1.5 kb longer than reported for other Charadriiformes and contained a duplication of the control region together with NADH dehydrogenase subunit 6 (ND6) and the adjacent transfer RNAs: tRNA(Pro) and tRNA(Glu). Sequence data from 68 individuals from several stopover and breeding populations show that the duplication is widespread in ruffs. Similar gene re-arrangements have been found independently in unrelated tube-nosed seabirds and spoonbills.

Characterization of polymorphic microsatellite DNA markers in the black-tailed godwit (Limosa limosa: Aves).

We isolated and tested 16 microsatellite loci in black-tailed godwits from the Netherlands (Limosa limosa limosa), and from Australasia (subspecies melanuroides). One locus was monomorphic, two loci had null-alleles and one was significantly heterozygote deficient. The remaining 12 polymorphic loci had on average 7.9 alleles (range 5-11) and the mean expected heterozygosity was 0.69. No significant linkage disequilibrium between the loci was observed and all loci were autosomal. Fourteen loci were successfully cross-amplified in bar-tailed godwit (Limosa lapponica).