Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ’s natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses.

The recently extinct New Zealand adzebills (Aptornithidae, Aptornis spp.) were an enigmatic group of large flightless birds that have long eluded precise taxonomic assignment as they do not closely resemble any extant birds. Adzebills were nearly wingless, weighed approximately 16–19 kg, and possessed massive adze-like reinforced bills whose function remains unknown. Using hybridisation enrichment and high-throughput sequencing of DNA extracted from subfossil bone and eggshell, near-complete mitochondrial genomes were successfully assembled from the two Quaternary adzebill species: the North Island Adzebill (Aptornis otidiformis) and South Island Adzebill (A. defossor). Molecular phylogenetic analyses confirm that adzebills are members of the Ralloidea (rails and allies) and are sister-taxon to the Sarothruridae, which our results suggest comprises the Madagascan wood rails (Mentocrex, two likely sp.) in addition to the tiny (<50 gram) rail-like Afro-Madagascan flufftails (Sarothrura, 9 spp.). Node age estimates indicate that the split between adzebills and Sarothruridae occurred ~39.6 Ma, suggesting that the ancestors of the adzebills arrived in New Zealand by long-distance dispersal rather than continental vicariance. This newly identified biogeographic link between physically distant New Zealand and Afro-Madagascar, echoed by the relationship between the New Zealand kiwi (Apterygiformes) and Madagascan elephant-birds (Aepyornithiformes), suggests that the adzebill’s near relatives were formerly more widespread. In addition, our estimate for the divergence time between the two Quaternary adzebill species (0.2–2.3 Ma) coincides with the emergence of a land-bridge between the North and South islands of New Zealand (ca. 1.5–2 Ma). This relatively recent divergence suggests that North Island adzebills are the result of a relatively recent dispersal from the South Island, from which the earliest (Miocene) adzebill fossil has been described.

Mycovores (animals that consume fungi) are important for fungal spore dispersal, including ectomycorrhizal (ECM) fungi symbiotic with forest-forming trees. As such, fungi and their symbionts may be impacted by mycovore extinction. New Zealand (NZ) has a diversity of unusual, colourful, endemic sequestrate (truffle-like) fungi, most of which are ECM. As NZ lacks native land mammals (except bats), and sequestrate fungi are typically drab and mammal-dispersed, NZ’s sequestrate fungi are hypothesized to be adapted for bird dispersal. However, there is little direct evidence for this hypothesis, as 41% of NZ’s native land bird species became extinct since initial human settlement in the thirteenth century. Here, we report ancient DNA and spores from the inside of two coprolites of NZ’s extinct, endemic upland moa ( Megalapteryx didinus ) that reveal consumption and likely dispersal of ECM fungi, including at least one colourful sequestrate species. Contemporary data from NZ show that birds rarely consume fungi and that the introduced mammals preferentially consume exotic fungi. NZ’s endemic sequestrate fungi could therefore be dispersal limited compared with fungi that co-evolved with mammalian dispersers. NZ’s fungal communities may thus be undergoing a gradual species turnover following avian mycovore extinction and the establishment of mammalian mycovores, potentially affecting forest resilience and facilitating invasion by exotic tree taxa.

The Chathamiidae are an interesting family of caddisflies, unusual as all of the five known species are believed to breed entirely within the marine intertidal, comprising one of very few known marine insect groups. Additionally the family approaches almost complete endemicity status in New Zealand, and may represent an ancient lineage representative of ancient vicariance from Gondwana. However one species, the common and widespread Philanisus plebeius is also known to have a disjunct population in New South Wales Australia, hypothesised to represent a recent anthropogenic dispersal. This thesis, using DNA information, examined the Chathamiidae at varying phylogenetic levels. Firstly the species Philanisus plebeius was incorporated into a thorough intraspecific phylogeography, including samples from both New Zealand and Australia. The population as a whole was genetically diverse, with the population divisible into two major haplogroups, each restricted to discrete geographic areas with no overlap being observed. One of these groups was restricted to just two localities in the central eastern North Island, whereas the remainder included most remaining samples from both Islands of New Zealand, and also Australia. All Australian samples were found to comprise a single haplotype, differing by a single base pair from the most common haplotype in New Zealand. It was decided that the Australian population therefore represents a recent dispersal event from New Zealand, although unless the Australian haplotype remains undiscovered in New Zealand the level of divergence found is not congruent with a human introduction. One sequence intermediate between the two major haplogroups was identified from a single haplotype from Tauranga. It seemed that much of the population of Philanisus plebeius has been affected by recent demographic expansion, likely due to the effects of the last glacial maximum (LGM). The five species of the Chathamiidae were then analysed in a phylogeny. It was found that the genus Chathamia was polyphyletic, with the species C. integripennis nested within the genus Philanisus. The remaining species, C. brevipennis from the Chatham Islands, was basal to all the remaining members of the family. A strict molecular clock found a recent Pleistocene age (roughly 0.5 Ma) for divergence of the Kermadec Island species Philanisus fasciatus, and a Pliocene-Pleistocene age (roughly 3 Ma) for the Chatham Island species Chathamia brevipennis. For a comparison with the species C. brevipennis, the other Chatham Island caddisfly taxa Oecetis chathamensis, and Hydrobiosis lindsayi were compared with New Zealand relatives; indicated to have late and early Pleistocene ages respectively. A short sequence of the gene COI was amplified for the species Philanisus mataua, however this was found to contain two sequences reflecting either heteroplasmy or sample contamination, inhibiting confident phylogenetic placement. Additionally a larval sample from Sydney was demonstrated to represent C. integripennis, recorded outside of Northern New Zealand for the first time. Finally the Chathamiidae was included in a higher level phylogeny with related families, and was show to comprise a monophyletic group, sister to the Australasian family of the Conoesucidae. A relaxed molecular clock estimated a Cretaceous (roughly 90 Ma) age for the Chathamiidae, congruent with a vicariant age in New Zealand.