Intraskeletal bone growth patterns in the North Island Brown Kiwi (Apteryx mantelli): Growth mark discrepancy and implications for extinct taxa.

Osteohistology, the study of bone microstructure, provides an important avenue for assessing extinct and extant vertebrate growth and life history. Cortical vascularity and collagen fibre organization are direct reflections of growth rate, while bone growth marks are indicative of absolute age. However, each skeletal element has its own ontogenetic trajectory and microstructure of certain bones may not be a true representation of whole body growth. Extensive comparative study of modern taxa is required to resolve intraskeletal discrepancies among age, vascularity and tissue organization in extinct vertebrates. Despite their comparative utility, studies of bone microstructure in modern taxa are severely lacking. Here, we add to a growing comparative osteohistological database by describing (1) bone tissue organization, (2) growth mark count, (3) sexually dimorphic bone (e.g. medullary bone) and (4) secondary cortical reconstruction in the bone microstructure of a 14-year-old male and 5-year-old female North Island Brown Kiwi (Apteryx mantelli). Transverse and longitudinal histological ground sections were processed and described for femora, tibiotarsi, tarsometatarsi, humeri, ulnae and radii in both kiwis. Cortical bone can generally be described as parallel-fibered tissue, interrupted by cyclical growth marks, with vascular canals oriented longitudinally within primary and secondary osteons. Tissue morphologically resembling medullary bone is present in the hindlimbs of the female, and coarse compacted cancellous bone (CCCB) is found sporadically in the male and female hindlimbs. Lines of arrested growth (LAGs) are present in all hindlimb bones of both kiwi, but remodelling has obliterated all LAGs in the male ulnae and radii. LAG count varies intraskeletally, but large weight bearing elements such as femora and tibiotarsi have less remodelling and, thus, higher number of LAGs. LAG count did not match absolute age in any skeletal element; a maximum of seven LAGs are present in the male kiwi and a maximum of seven LAGs in the female kiwi. The tissue organization within the forelimbs and hindlimbs is reflective of the protracted growth strategy of the North Island Brown Kiwi and congruent with previous studies of the kiwi. LAGs were highly variable throughout the skeleton of the kiwi and a decoupling of age and LAG deposition is apparent from the male kiwi samples. Excess LAGs in the 5-year-old female kiwi may be a product of hatching, egg laying or captivity. Regardless, LAG count variation in the kiwi stresses the importance of intraskeletal sampling when assessing growth patterns of extinct taxa. An extensive ontogenetic sampling of kiwi is necessary for future investigations of bone growth patterns, CCCB formation, medullary bone and LAG deposition and obliteration in these elusive birds.

Explosive ice age diversification of kiwi.

Molecular dating largely overturned the paradigm that global cooling during recent Pleistocene glacial cycles resulted in a burst of species diversification although some evidence exists that speciation was commonly promoted in habitats near the expanding and retracting ice sheets. Here, we used a genome-wide dataset of more than half a million base pairs of DNA to test for a glacially induced burst of diversification in kiwi, an avian family distributed within several hundred kilometers of the expanding and retracting glaciers of the Southern Alps of New Zealand. By sampling across the geographic range of the five kiwi species, we discovered many cryptic lineages, bringing the total number of kiwi taxa that currently exist to 11 and the number that existed just before human arrival to 16 or 17. We found that 80% of kiwi diversification events date to the major glacial advances of the Middle and Late Pleistocene. During this period, New Zealand was repeatedly fragmented by glaciers into a series of refugia, with the tiny geographic ranges of many kiwi lineages currently distributed in areas adjacent to these refugia. Estimates of effective population size through time show a dramatic bottleneck during the last glacial cycle in all but one kiwi lineage, as expected if kiwi were isolated in glacially induced refugia. Our results support a fivefold increase in diversification rates during key glacial periods, comparable with levels observed in classic adaptive radiations, and confirm that at least some lineages distributed near glaciated regions underwent rapid ice age diversification.

Apparent lack of efficacy of toltrazuril against Eimeria species affecting brown kiwi (Apteryx mantelli) at a captive rearing facility.

AIM: To assess the efficacy of toltrazuril against the Eimeria spp. affecting brown kiwi (Apteryx mantelli). METHODS: Droppings were collected from three brown kiwi, aged <6 months old, at a captive rearing facility in the North Island of New Zealand, between 22 February and 20 April 2017, on 14 sampling dates. Only droppings (n=30) that were excreted between 03:00 and 07:00, as determined using video surveillance, were included for analysis, reflecting the peak time for shedding of coccidial oocysts for brown kiwi. Oocysts were quantified in each sample and Eimeria species identified on the basis of oocyst morphology. All samples were collected between 2 and 10 days after the birds had been treated with 25 mg/kg toltrazuril. RESULTS: Eimeria spp. oocysts were identified in 28/30 individual samples and on 14/14 sampling dates. Oocyst counts varied from 0 to 328,080 oocysts per gram (opg), and at least one oocyst count >10,000 opg was measured on 12/14 sampling dates. Three species of Eimeria were observed, with Eimeria apteryxii and E. kiwii most commonly encountered, whereas only one sample contained E. paraurii. CONCLUSIONS AND CLINICAL RELEVANCE: In the three birds monitored at this research site, there was a high abundance of E. apteryxii and E. kiwii oocysts in droppings despite recent administration of toltrazuril. These results suggest that the populations of Eimeria spp. affecting brown kiwi at this location appear to possess an ability to survive exposure to toltrazuril. Toltrazuril is widely used at captive rearing facilities to limit the effects of coccidiosis in juvenile kiwi. If a lack of efficacy is confirmed, it will be necessary to investigate alternative treatment regimens alongside broader environmental management strategies.

Independent pseudogenization of CYP2J19 in penguins, owls and kiwis implicates gene in red carotenoid synthesis.

Carotenoids have important roles in bird behavior, including pigmentation for sexual signaling and improving color vision via retinal oil droplets. Yellow carotenoids are diet-derived, but red carotenoids (ketocarotenoids) are typically synthesized from yellow precursors via a carotenoid ketolase. Recent research on passerines has provided evidence that a cytochrome p450 enzyme, CYP2J19, is responsible for this reaction, though it is unclear if this function is phylogenetically restricted. Here I provide evidence that CYP2J19 is the carotenoid ketolase common to Aves using the genomes of 65 birds and the retinal transcriptomes of 15 avian taxa. CYP2J19 is functionally intact and robustly transcribed in all taxa except for several species adapted to foraging in dim light conditions. Two penguins, an owl and a kiwi show evidence of genetic lesions and relaxed selection in their genomic copy of CYP2J19, and six owls show evidence of marked reduction in CYP2J19 retinal transcription compared to nine diurnal avian taxa. Furthermore, one of the owls appears to transcribe a CYP2J19 pseudogene. Notably, none of these taxa are known to use red carotenoids for sexual signaling and several species of owls and penguins represent the only birds known to completely lack red retinal oil droplets. The remaining avian taxa belong to groups known to possess red oil droplets, are known or expected to deposit red carotenoids in skin and/or plumage, and/or frequently forage in bright light. The loss and reduced expression of CYP2J19 is likely an adaptation to maximize retinal sensitivity, given that oil droplets reduce the amount of light available to the retina.

The circadian variation of oocyst shedding of Eimeria spp. affecting brown kiwi (Apteryx mantelli).

Captive rearing of wild brown kiwi (Apteryx mantelli) is widely carried out to assist in the recovery of this declining species. As a consequence, high densities of immunologically naïve kiwi are commonly housed in semi-captive conditions, with the potential to result in substantial morbidity and mortality from coccidiosis caused by multiple species of Eimeria. Previous research has described circadian variation in oocyst shedding across multiple avian host species. The aim of this research was to describe any circadian variation in oocyst shedding in brown kiwi. Droppings were collected from brown kiwi (n = 4) at a single captive rearing facility using video surveillance to determine the time of excretion, and oocyst counts were undertaken. Results show that two of the Eimeria spp. affecting brown kiwi exhibit a peak in oocyst shedding between 03.00 and 07.00 with few or no oocysts shed between 08.00 and midnight. These results are not able to be explained by the current hypotheses theorising the evolutionary forces behind the development of this adaptive trait. Our findings increase the current understanding of the biology of the Eimeria spp. affecting brown kiwi and have important implications for the management of captive-reared kiwi, in particular for the accurate interpretation of faecal oocyst counts.

Sixteen kiwi (Apteryx spp) transcriptomes provide a wealth of genetic markers and insight into sex chromosome evolution in birds.

BACKGROUND: Kiwi represent the most basal extant avian lineage (paleognaths) and exhibit biological attributes that are unusual or extreme among living birds, such as large egg size, strong olfaction, nocturnality, flightlessness and long lifespan. Despite intense interest in their evolution and their threatened status, genomic resources for kiwi were virtually non-existent until the recent publication of a single genome. Here we present the most comprehensive kiwi transcriptomes to date, obtained via Illumina sequencing of whole blood and de novo assembly of mRNA sequences of eight individuals from each of the two rarest kiwi species, little spotted kiwi (LSK; Apteryx owenii) and rowi (A. rowi). RESULTS: Sequences obtained were orthologous with a wide diversity of functional genes despite the sequencing of a single tissue type. Individual and composite assemblies contain more than 7900 unique protein coding transcripts in each of LSK and rowi that show strong homology with chicken (Gallus gallus), including those associated with growth, development, disease resistance, reproduction and behavior. The assemblies also contain 66,909 SNPs that distinguish between LSK and rowi, 12,384 SNPs among LSK (associated with 3088 genes), and 29,313 SNPs among rowi (associated with 4953 genes). We found 3084 transcripts differentially expressed between LSK and rowi and 150 transcripts differentially expressed between the sexes. Of the latter, 83 could be mapped to chicken chromosomes with 95% syntenic with chromosome Z. CONCLUSIONS: Our study has simultaneously sequenced multiple species, sexes, and individual kiwi at thousands of genes, and thus represents a significant leap forward in genomic resources available for kiwi. The expression pattern we observed among chromosome Z related genes in kiwi is similar to that observed in ostriches and emu, suggesting a common and ancestral pattern of sex chromosome homomorphy, recombination, and gene dosage among living paleognaths. The transcriptome assemblies described here will provide a rich resource for polymorphic marker development and studies of adaptation of these highly unusual and endangered birds.

Genetic consequences of a century of protection: serial founder events and survival of the little spotted kiwi (Apteryx owenii).

We present the outcome of a century of post-bottleneck isolation of a long-lived species, the little spotted kiwi (Apteryx owenii, LSK) and demonstrate that profound genetic consequences can result from protecting few individuals in isolation. LSK were saved from extinction by translocation of five birds from South Island, New Zealand to Kapiti Island 100 years ago. The Kapiti population now numbers some 1200 birds and provides founders for new populations. We used 15 microsatellite loci to compare genetic variation among Kapiti LSK and the populations of Red Mercury, Tiritiri Matangi and Long Islands that were founded with birds from Kapiti. Two LSK native to D'Urville Island were also placed on Long Island. We found extremely low genetic variation and signatures of acute and recent genetic bottleneck effects in all four populations, indicating that LSK have survived multiple genetic bottlenecks. The Long Island population appears to have arisen from a single mating pair from Kapiti, suggesting there is no genetic contribution from D'Urville birds among extant LSK. The Ne/NC ratio of Kapiti Island LSK (0.03) is exceptionally low for terrestrial vertebrates and suggests that genetic diversity might still be eroding in this population, despite its large census size.

Toxoplasma gondii Exposure Prevalence in Little Spotted Kiwi (Apteryx owenii).

Toxoplasma gondii has been reported as a cause of morbidity and mortality in New Zealand's native avifauna, including the ground-dwelling Kiwi (Apteryx spp.). To better understand the extent of T. gondii infection in Little Spotted Kiwi (Apteryx owenii), a prevalence survey of kiwi living inside a 200-ha predator-proof mainland ecosanctuary (Zealandia Te Mara a Tane, Wellington, New Zealand) was undertaken. Antibodies to T. gondii were detected by a latex agglutination test (LAT) with a cutoff positive titer of ≥1:64, and T. gondii DNA was detected by PCR. In total, 16/19 (84.2%) birds tested were positive for T. gondii by LAT (10/11), PCR (10/19), or both (4/11). Antibody titers ranged from 1:32 to ≥1:2,048. These results suggest widespread exposure of T. gondii in this population of Little Spotted Kiwi and, in conjunction with earlier reports of toxoplasmosis causing mortality in kiwi, raise important questions as to the effect this parasite may be having on this rare endemic species. Further information on the epidemiology of T. gondii infections within free-living and managed kiwi populations is urgently needed.

Legacy of supervolcanic eruptions on population genetic structure of brown kiwi.

Supervolcanoes are volcanoes capable of mega-colossal eruptions that emit more than 1,000 km3 of ash and other particles.1 The earth's most recent mega-colossal eruption was the Oruanui eruption of the Taupo supervolcano 25,580 years before present (YBP) on the central North Island of New Zealand.2 This eruption blanketed major swaths of the North Island in thick layers of ash and igneous rock,2,3 devastating habitats and likely causing widespread population extinctions.4-7 An additional devastating super-colossal eruption (>100 km3) of the Taupo supervolcano occurred approximately 1,690 YBP.8 The impacts of such massive but ephemeral natural disasters on contemporary population genetic structure remain underexplored. Here, we combined data for 4,951 SNPs with spatially explicit demographic and coalescent models within an approximate Bayesian computation framework to test the drivers of genetic structure in brown kiwi (Apteryx mantelli). Our results strongly support the importance of eruptions of the Taupo supervolcano in restructuring pre-existing geographic patterns of population differentiation and genetic diversity. Range shifts due to climatic oscillations-a frequent explanation for genetic structure9-are insufficient to fully explain the empirical data. Meanwhile, recent range contraction and fragmentation due to historically documented anthropogenic habitat alteration adds no explanatory power to our models. Our results support a major role for cycles of destruction and post-volcanic recolonization in restructuring the population genomic landscape of brown kiwi and highlight how ancient and ephemeral mega-disasters may leave a lasting legacy on patterns of intraspecific genetic variation.

The eggshell structure in apteryx; form, function, and adaptation.

Apteryx is a genus of flightless birds endemic to New Zealand known to lay very large eggs in proportion to body weight. The eggshell of Apteryx is unusually thin and less porous than allometrically expected possibly as a compensation for a very long incubation period. Past studies have been carried out on Apteryx australis, a species which once comprised all kiwi with brown plumage, now separated into three distinct species. These species use different habitats and live at different latitudes and altitudes, therefore generating a need to revise our knowledge of the attributes of their eggshells. In this study, we measured the physical characteristics and water conductance on eggshell fragments of these three species and Great-spotted Kiwi and relate them to the environmental conditions of their respective environments; we also measured the water vapor conductance of Brown Kiwi eggs of late stages of incubation. We found that several trade-offs exist between incubation behavior, environmental conditions, and eggshell structure. We found differences between species in eggshell water vapor conductance seemingly related to altitude; Brown Kiwi and Rowi generally inhabiting lower altitudes had the highest conductance and Tokoeka, generally living in montane environments, the lowest. This is achieved by an increased eggshell thickness rather than a pore area reduction. Finally, the water vapor conductance late in incubation was 58% higher than infertile unincubated eggs, suggesting a drastic increase in conductance throughout the long incubation period. Using the values previously reported, we calculated the embryonic eggshell thinning to be 32.5% at the equatorial region of the eggshell. We describe several new features, such as triangular mineral particles in the cuticle, reported for the extinct Trigonoolithus amoei, and confirmed the existence of plugged pores. We suggest that these structures provide microbial protection needed by a burrow nesting species with a long incubation period.

Ventral dermatitis in rowi (Apteryx rowi) caused by cutaneous capillariasis.

In 2013 there was an outbreak of crusting ventral dermatitis among a group of juvenile rowi (Apteryx rowi), a species of the endangered New Zealand kiwi, that were being raised on an off-shore island sanctuary. Biopsies taken at the time found nematodes migrating within the epidermis of affected skin but the specific identity and origin of the organisms was not established, and sporadic cases of similar skin disease continue to occur on the island. On examination of additional sections from the original skin biopsies, adult nematodes and eggs were identified, the histomorphology of which was consistent with Capillaria sensu lato. PCR was performed on DNA extracted from archived formalin-fixed, paraffin-embedded tissue blocks of skin from eight affected rowi, using primers targeting the 18S region of nuclear ribosomal DNA and the COI gene of mitochondrial DNA of capillarid nematodes. The 18S sequences from all rowi samples were identical and matched sequences from members of the genus Eucoleus. In contrast, two distinct capillarid COI sequences were obtained, in one case both from the same rowi skin biopsy. While there were no close matches, both COI sequences also aligned nearest to sequences identified as Eucoleus spp. It is considered unlikely that two different nematode species are involved in the rowi skin lesions and the possible amplification of a COI pseudogene or "numt" is discussed. A species-level identification of the capillarid nematodes causing skin disease in rowi was not obtained, however based on histological evaluation the infections include reproductively-active adult nematodes. This finding indicates the possibility of perpetuation of the skin disease in the absence of the original source, as well as raising potential for the transfer of infection from the island when the juvenile rowi are translocated to their new habitats.

Nematode larva migrans caused by Toxocara cati in the North Island brown kiwi (Apteryx mantelli).

Sporadic cases of visceral and neural nematode larva migrans have been diagnosed at necropsy in the endangered New Zealand kiwi (Apteryx spp.), but the causative organisms have not yet been definitively identified. From an initial group of five affected kiwi, PCR was performed on DNA extracted from archival formalin-fixed paraffin-embedded tissue sections in which larval nematodes had been histologically identified. Sequencing of positive results from four out of the five kiwi aligned with sequences from Toxocara cati, a nematode parasite whose definitive host is the domestic cat. PCR was then performed on a second group of 12 kiwi that had histologic inflammatory lesions consistent with larva migrans, but variable larval presence. Repeatable positive PCR results were only achieved in one tissue, in which larval organisms were histologically confirmed. This study supports the use of PCR as an alternative or adjunct to the morphological identification of nematode larvae in formalin-fixed histopathological samples, as well as showing that in investigation of larva migrans, PCR has greatest chance of success from sections where nematode larvae are evident histologically. The identification of Toxocara cati from lesions of larva migrans in kiwi reflects an indirect, parasite-mediated effect of an invasive mammalian species on a native species.

Extraction of DNA from captive-sourced feces and molted feathers provides a novel method for conservation management of New Zealand kiwi (Apteryx spp.).

Although some taxa are increasing in number due to active management and predator control, the overall number of kiwi (Apteryx spp.) is declining. Kiwi are cryptic and rare, meaning current monitoring tools, such as call counts, radio telemetry, and surveys using detection dogs are labor-intensive, yield small datasets, and require substantial resources or provide inaccurate estimates of population sizes. A noninvasive genetic approach could help the conservation effort. We optimized a panel of 23 genetic markers (22 autosomal microsatellite loci and an allosomal marker) to discriminate between all species of kiwi and major lineages within species, while simultaneously determining sex. Markers successfully amplified from both fecal and shed feather DNA samples collected in captivity. We found that DNA extraction was more efficient from shed feathers, but DNA quality was greater with feces, although this was sampling dependent. Our microsatellite panel was able to distinguish between contemporary kiwi populations and lineages and provided PI values in the range of 4.3 × 10-5 to 2.0 × 10-19, which in some cases were sufficient for individualization and mark-recapture studies. As such, we have tested a wide-reaching, noninvasive molecular approach that will improve conservation management by providing better parameter estimates associated with population ecology and demographics such as abundance, growth rates, and genetic diversity.

The complete mitochondrial genome of North Island brown kiwi (Apteryx mantelli).

Here, we report the complete mitochondrial genome sequence of North Island brown kiwi(Apteryx mantelli). The genome is found to be 16,694 bp in length and has a base composition of A (30.74%), G (13.46%), C (26.50%), and T (29.30%). Similar to other Apteryx species, it contains a typically conserved structure including 13 protein-coding genes, 2 rRNA genes, 1 control region (D-loop), and 22tRNA genes. The proportion of coding sequences with a total length of 11,431 bp is 68.47%, which encodes 3776 amino acids. All protein-coding genes started with Met, and ND2, COX2, and COX3 ended by TAA as a stop codon. The lengths of 12S ribosomal RNA and 16S ribosomal RNA are 973 bp and 1596 bp, respectively. The length of control region is 1112 bp, ranging from 15,583 bp to 16,694 bp. The complete mitochondrial genome sequence provided here would be useful for further understanding the evolution of ratite and conservation genetics of A. mantelli.

Prevalence of and risk factors for coccidiosis in kiwi between 1977 and 2011.

AIMS: To identify risk factors associated with coccidiosis in kiwi (Apteryx spp.) using a retrospective analysis of historical data from kiwi examined post-mortem, and to determine the prevalence of coccidial oocysts in the droppings of wild and captive kiwi. METHODS: Necropsy reports were examined from kiwi submitted to the National Wildlife Mortality Database of New Zealand (Huia) between February 1977 and May 2011. All cases that reported histological examination of one or more hepatic, intestinal or renal tissues were included in the study (n=372). Data collated for analysis included the presence or absence of coccidiosis in one or more tissues, age, host species, habitat, and season of submission. Fisher's exact test was used to determine the association between each independent variable and the prevalence of coccidiosis. Droppings opportunistically collected from wild and captive kiwi between January 2008 and June 2010 were also examined for the presence of coccidial oocysts. RESULTS: Coccidiosis was evident in 47/372 (12.6%) kiwi examined post-mortem and was considered the primary cause of death in 12/47 (26%) infected cases. Examination of 412 droppings from wild and captive kiwi collected over a 29-month period revealed that 98 (23.8%) samples contained coccidia at the time of sampling. Enteric (n=27) coccidiosis was the most common form diagnosed using histology, followed by renal (n=19) and hepatic (n=11), with splenic (n=2) and pulmonary (n=1) infections infrequently seen. Many kiwi demonstrated infections in multiple tissues. The prevalence of coccidiosis was greater in juvenile kiwi (36/148, 24.3%) than adults (8/133, 6.0%) or chicks (2/83, 2.4%) (p<0.001), although there was no difference in mortality between age groups. Season of year was also associated with overall prevalence (p=0.05), with most cases being diagnosed in the autumn and winter. Coccidiosis was histologically evident in four of five species of kiwi examined, and in all host species upon analysis of droppings. Host species or habitat (captive vs. wild) did not influence the prevalence of disease detected histologically. CONCLUSIONS: Age and season were the only factors that influenced the prevalence of coccidiosis in kiwi in this study. Coccidiosis was present in all species of kiwi, and this is the first report of coccidiosis in rowi (Apteryx rowi).