Species recovery programmes are increasingly using genomic data to measure neutral genetic diversity and calculate metrics like relatedness. While these measures can inform conservation management, determining the mechanisms underlying inbreeding depression requires information about functional genes associated with adaptive or maladaptive traits. Of particular interest is the diversity of toll-like receptor (TLR) genes, which play a crucial role in recognition of pathogens and activation of the immune system. To date, these genes have predominantly been identified and characterised using targeted amplification and sequencing.

In this Proof of Concept, I leverage existing short-read reference genomes, whole-genome resequencing data, and bioinformatic tools to develop a novel method to identify TLR genes and characterise TLR gene diversity. I conduct this Proof of Concept in three stages to characterise TLR gene diversity of three nationally critical birds endemic to Aotearoa New Zealand: tūturuatu/shore plover (Thinornis novaeseelandiae), kākāriki karaka/orange-fronted parakeet (Cyanoramphus malherbi), and kakī/black stilt (Himantopus novaezelandiae).

The highest number of TLRs were identified and characterised in tūturuatu, followed by kākāriki karaka, and then kakī, in which could not characterise any of the TLR genes I identified. Consistent with observations in other threatened species and populations, tūturuatu and kākāriki karaka have relatively low TLR gene diversity in comparison to non-threatened species.

Within the captive tūturuatu population, individuals have a high susceptibility to severe avian pox and a low immune response to vaccination. In contrast, anecdotal evidence suggests the wild tūturuatu population on the Chatham Islands have fewer cases of poxvirus, and when a wild bird does contract the virus, the case is usually mild and the bird is able to overcome infection quickly. The low TLR gene diversity within captive tūturuatu may explain the difficulty captive birds have in overcoming poxvirus infection. These findings will ultimately be used to assess the recent

conservation management action to bring wild eggs from the population on Rangatira to supplement the captive population.

While captive kākāriki karaka are not experiencing a current disease outbreak, the threat of known and emerging pathogens is only growing, and disease may be a concern for the captive population because of the low TLR gene diversity I found. The captive kākāriki karaka population has been supplemented with individuals from wild and translocated populations in an attempt to introduce new, or reintroduce lost, founder lineages. Additional sampling is needed to confirm whether there is TLR gene diversity within the source populations used for supplementation that has yet to be captured, and whether bringing in additional eggs from these populations may contribute novel TLR alleles to the captive population.

I had limited success identifying TLR genes within the kakī reference genome, and no success characterising TLR gene diversity within the semi-wild kakī population. I attribute the former to a relatively incomplete kakī genome, due in part to a relatively high percentage of Ns in the draft assembly. I attribute the latter to missingness within several individuals in the population resequencing data. These combined findings suggest this approach may be less effective for threatened species with relatively incomplete genomes and resequencing data.

The novel approach presented in this thesis provides an example of how functional diversity can be characterised using a bioinformatic approach. As more genomic resources become available for non-model threatened species, as a way to measure genome-wide diversity, these data can be leveraged to characterise functional genes. This has broad implications for how TLR gene diversity can be assessed to inform conservation management actions for threatened bird species in Aotearoa New Zealand and beyond.