Toll-like receptor diversity in 10 threatened bird species: relationship with microsatellite heterozygosity

Abstract

Measuring individual-level heterozygosity in threatened species is one approach to understanding and mitigating losses of genetic diversity and the role of inbreeding depression in those populations. In many conservation contexts, this goal is approached by assaying levels of microsatellite diversity, and inference is often extended to functional genomic regions. Our study quantifies diversity of innate immunity toll-like receptor (TLR) genes in 10 threatened New Zealand birds across four avian orders, with an average of 20.1 individuals and 6.2 TLR loci (sequences averaging 850 bp in length) per species. We provide detailed TLR diversity statistics for these 10 species, which showed more evidence for genetic drift than balancing selection at TLR loci, with two possible exceptions (TLR1LA for hihi and TLR5 for kokako). Our observations also support a possible gene-duplication of TLR7 in rock wren, indicating that a TLR7 duplication previously observed in other passerines may have occurred early in the divergence of this order. In addition to these analyses of population-level TLR sequence diversity, we used an average of 14.6 polymorphic microsatellite loci per species to study, for the first time, the relationship between microsatellite internal relatedness (a measure of individual homozygosity) and TLR heterozygosity. There was no relationship between microsatellite and TLR heterozygosity of individuals within species, suggesting that the predictive power of microsatellites to evaluate functional diversity is poor, and highlighting the value of adding data from putatively functional genomic regions, such as TLRs, in the study of genetic diversity of threatened species. Overall this study provides valuable data for comparison with more widespread species, and facilitates research into the importance of TLR diversity in natural populations of conservation concern.

Appendix

Here we provide species histories and details of study populations for the 10 threatened New Zealand native birds examined in this study. Polynesians were the first settlers to New Zealand from ~1200 AD (Higham et al. 1999), followed by a second colonisation by Europeans in the 1800s. Both immigration events brought about significant environmental changes, and much of New Zealand’s vertebrate fauna has been impacted. For example, New Zealand endemic birds have experienced dramatic population bottlenecks as a result of predation by introduced mammals (such as rats Rattus sp. and mustelids; New Zealand has no native terrestrial mammals except three species of bat), competition from introduced browsers (such as possums Trichosurus vulpecula), and habitat loss and fragmentation due to land conversion (Duncan and Blackburn 2004). These bottlenecks may have resulted in losses of genetic diversity, both at the population level (low numbers of alleles) and at the individual level (low observed heterozygosity). Sites mentioned herein are shown on the map in Fig. 1.

Apterygiformes: North Island brown kiwi Apteryx mantelli

The North Island brown kiwi is the most common of the five species of kiwi, currently numbering ~25,000, spread over four genetically distinct forms (Holzapfel et al. 2009). Despite intensive management, the species is classified under the New Zealand threat classification system as in “serious decline”, primarily as a result of human induced impacts (habitat loss and introduced mammalian predators, especially stoats Mustela erminea), and poor recruitment (Holzapfel et al. 2009; Ziesemann et al. 2011). We used feather samples from a population of brown kiwi near Purua, in the north of the North Island (currently numbers ~8,000; Holzapfel et al. 2009), collected as part of on-going management activities (H. Robertson pers. comm.).

Photo: Colin Miskelly, NZ Birds online (www.nzbirdsonline.org)

Gruiformes: takahe Porphyrio hochstetteri

Takahe were once widespread throughout the South Island of New Zealand, but are thought to have experienced dramatic declines since the arrival of Polynesian settlers in the 1600s and were even thought to be extinct by the 1900s (Lee and Jamieson 2001). A remnant population of birds was discovered in 1948 in the Murchison Mountains, Fiordland and has been subject to intense management since. The current population of <300 comprises birds in the original Murchison Mountains habitat, as well as translocated populations in several predator-free sanctuaries around the country (Wickes et al. 2009). We used samples from the source population, in Fiordland National Park, which were collected for a previous study (Lettink et al. 2002). Previous microsatellite analysis of takahe found very low levels of diversity, both in terms of numbers of polymorphic loci and numbers of alleles at variable loci (Grueber et al. 2008a), probably resulting from a prolonged population bottleneck (Grueber and Jamieson 2011).

Photo: Catherine Grueber

Psittaciformes: kakapo Strigops habroptilus

The kakapo is a large flightless bird, endemic to New Zealand and unique among parrots in its nocturnal, lek-breeding behaviours (Powlesland et al. 2006). The species is severely threatened by the introduction of stoats and rats and were the focus of possibly the earliest-known New Zealand conservation efforts, beginning in 1894. Sadly, only one Fiordland bird (“Richard Henry”) was ultimately rescued and translocated to Codfish Island along with members of a remnant population of birds from Stewart Island (Clout 2006). Very low levels of genetic diversity have been observed in kakapo with microsatellites (Robertson et al. 2000). All samples used in this study were collected as part of on-going management and are from Stewart Island-origin birds, i.e. we did not include samples from “Richard Henry” or his offspring. All extant kakapo (N ~124), except for Richard Henry’s offspring, descend from 62 Stewart Island birds (Powlesland et al. 2006).

Photo: Ian Jamieson

Psittaciformes: kakariki (red-crowned parakeet) Cyanoramphus novaezelandiae

Kakariki were once abundant on New Zealand’s North and South Islands until the introduction of predatory mammals and the destruction of suitable habitat significantly decreased their range (Higgins 1999). Kakariki are now considered effectively extinct from the mainland and only exist on Stewart Island and a number of offshore islands (BirdLife International 2013). We used samples collected from the wildlife sanctuary of Tiritiri Matangi in 2011 as part of concurrent research (B. Jackson pers. comm.). Since the introduction of approximately 90 birds to Tiritiri Matangi Island between 1974 and 1976 (Dawe 1979) and the subsequent eradication of kiore (Pacific rat Rattus exulans) from the island (Veitch 2002), the abundance of kakariki at the site has increased considerably.

Photo: Emily Weiser

Passeriformes: New Zealand rock wren Xenicus gilviventris

New Zealand rock wren are New Zealand’s only true alpine bird, living above the tree line for its entire life (Michelsen-Heath and Gaze 2007). Rock wren have been reported throughout much of the “main divide” of the Southern Alps, although a distribution study based on records of sightings over the past 100 years indicated that the species is declining (Michelsen-Heath and Gaze 2007). The samples we used here were collected from Fiordland, near the Homer Tunnel, as part of on-going research (Weston and Robertson 2014). This location is considered a stronghold for rock wren (Michelsen-Heath and Gaze 2007).

Photo: Bruce Robertson

Passeriformes: mohua (yellowhead) Mohoua ochrocephala

Mohua were formerly distributed across the entire South Island of New Zealand, but as a result of land conversion the species became fragmented into eight major forest patches (Gaze 1985; O’Donnell et al. 2002; Tracy and Jamieson 2011). Mohua are further threatened today by introduced mammalian predators, especially stoats (O’Donnell et al. 2002). Microsatellite-based studies of population structure and historical diversity (using museum specimens) revealed that the species has lost a significant amount of allelic diversity over the last 100 years and that a pattern of isolation by distance exists among contemporary mohua populations (Tracy and Jamieson 2011). We used samples from the Dart River Valley, Fiordland, for which DNA had been collected for a previous analysis (Tracy and Jamieson 2011); birds from this site are connected with those from other sites within a large forest patch.

Photo: Scott Mouat

Passeriformes: South Island robin Petroica australis australis

The South Island robin is a charismatic forest species endemic to New Zealand, belonging to the widespread Australasian family Petroicidae (Higgins and Peter 2002). Robins were previously distributed throughout the South Island, but the population has been increasingly fragmented as a result of habitat loss. Fragmentation is exacerbated by a reluctance of the birds to cross open water or unforested habitat, further isolating sub-populations of the species. A study of robin microsatellite diversity, in comparison to museum specimens, found little loss of diversity among contemporary birds as a whole, although birds from large mainland populations harboured more diversity than island birds (Boessenkool et al. 2007; Taylor et al. 2007). Here we use samples from one of the largest remnant robin populations, in the Eglinton River Valley, which were collected for a previous study (Boessenkool et al. 2007).

Photo: Ian Jamieson

Passeriformes: hihi (stitchbird) Notiomystis cincta

Once distributed throughout the North Island mainland and on northern offshore islands, hihi were extirpated from the mainland by the 1880s and persisted in a single remnant population on Little Barrier Island (Taylor et al. 2005). The Little Barrier Island population has been used as a source for subsequent translocations to establish populations on additional offshore islands (Taylor et al. 2005; Brekke et al. 2011). Microsatellite data revealed that the Little Barrier Island population has relatively high genetic diversity, possibly as a result of a high degree of extra-pair paternity in the species, reducing male reproductive variance (Brekke et al. 2011). We used samples from Little Barrier Island that were collected as part of on-going research (Brekke et al. 2011).

Photo: Paul Gibson

Passeriformes: North Island kokako Callaeas wilsoni

Kokako were formerly distributed across the North Island, but as a result of land conversion have become restricted to ~15 isolated forest fragments and introduced to several islands (Innes et al. 1999). The birds are particularly vulnerable to ship rats (Rattus rattus) and brushtail possums (Trichosurus vulpecula), so the recovery of kokako depends on the management of these invasive species (Basse et al. 2003). Microsatellite data have shown that the three largest kokako populations show only low levels of genetic differentiation (Hudson et al. 2000). Our samples were collected from the Mapara population, as part of ongoing management (O. Overdyck and T. Thurley, pers. comm.). Although the Mapara population is known to have declined to a small number of breeders in the 1990s, its recovery was rapid (Hudson et al. 2000).

Photo: Emily Weiser

Passeriformes: South Island saddleback Philesturnus carunculatus rufusator

South Island saddleback underwent an extreme population bottleneck when they were extirpated from the South Island of New Zealand at the time of human settlement and as a result of predation by invasive rats. Saddlebacks suffered a further severe bottleneck in the 1960s when the last known population, on Big South Cape Island, was reduced from around 1,000 birds to just 36 after rats arrived on the island. In 1964, these remaining birds were then moved to two rat-free sites, Big and Kaimohu Islands—the first threatened species translocation carried out by the New Zealand government through its Wildlife Service (later renamed the Department of Conservation) (Merton 1975). These populations grew and have been the source for several subsequent saddleback populations; the total population now numbers around 1,200 (Hoosen and Jamieson 2003). We used samples collected from Big Island as part of a previous study (Taylor and Jamieson 2008a, b).

Photo: Scott Mouat