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Kiwi are a unique and emblematic group of birds endemic to New Zealand. Deep-time evolutionary relationships among the five extant kiwi species have been difficult to resolve, in part due to the absence of pre-Quaternary fossils to inform speciation events. Here, we utilise single representative nuclear genomes of all five extant kiwi species (great spotted kiwi, little spotted kiwi, Okarito brown kiwi, North Island brown kiwi, and southern brown kiwi) and investigate their evolutionary histories with phylogenomic, genetic diversity, and deep-time (past million years) demographic analyses. We uncover relatively low levels of gene-tree phylogenetic discordance across the genomes, suggesting clear distinction between species. However, we also find indications of post-divergence gene flow, concordant with recent reports of interspecific hybrids. The four species for which unbiased levels of genetic diversity could be calculated, due to the availability of reference assemblies (all species except the southern brown kiwi), show relatively low levels of genetic diversity, which we suggest reflects a combination of older environmental as well as more recent anthropogenic influence. In addition, we suggest hypotheses regarding the impact of known past environmental events, such as volcanic eruptions and glacial periods, on the similarities and differences observed in the demographic histories of the five kiwi species over the past million years. [ABSTRACT FROM AUTHOR]

Homeoviscous adaptation (HVA) is a key cellular response by which fish protect their membranes against thermal stress. We investigated evolutionary HVA (long time scale) in Antarctic and non-Antarctic fish. Membrane lipid composition was determined for four Perciformes fish: two closely related Antarctic notothenioid species (Trematomus bernacchii and Pagothenia borchgrevinki); a diversified related notothenioid Antarctic icefish (Chionodraco hamatus); and a New Zealand species (Notolabrus celidotus). The membrane lipid compositions were consistent across the three Antarctic species and these were significantly different from that of the New Zealand species. Furthermore, acclimatory HVA (short time periods with seasonal changes) was investigated to determine whether stenothermal Antarctic fish, which evolved in the cold, stable environment of the Southern Ocean, have lost the acclimatory capacity to modulate their membrane saturation states, making them vulnerable to anthropogenic global warming. We compared liver membrane lipid composition in two closely related Antarctic fish species acclimated at 0 °C (control temperature), 4 °C for a period of 14 days in T. bernacchii and 28 days for P. borchgrevinki, and 6 °C for 7 days in both species. Thermal acclimation at 4 °C did not result in changed membrane saturation states in either Antarctic species. Despite this, membrane functions were not compromised, as indicated by declining serum osmolality, implying positive compensation by enhanced hypo-osmoregulation. Increasing the temperature to 6 °C did not change the membrane lipids of P. borchgrevinki. However, in T. bernacchii, thermal acclimation at 6 °C resulted in an increase of membrane saturated fatty acids and a decline in unsaturated fatty acids. This is the first study to show a homeoviscous response to higher temperatures in an Antarctic fish, although for only one of the two species examined. [ABSTRACT FROM AUTHOR]

Homeoviscous adaptation (HVA) is a key cellular response by which fish protect their membranes against thermal stress. We investigated evolutionary HVA (long time scale) in Antarctic and non-Antarctic fish. Membrane lipid composition was determined for four Perciformes fish: two closely related Antarctic notothenioid species (Trematomus bernacchii and Pagothenia borchgrevinki); a diversified related notothenioid Antarctic icefish (Chionodraco hamatus); and a New Zealand species (Notolabrus celidotus). The membrane lipid compositions were consistent across the three Antarctic species and these were significantly different from that of the New Zealand species. Furthermore, acclimatory HVA (short time periods with seasonal changes) was investigated to determine whether stenothermal Antarctic fish, which evolved in the cold, stable environment of the Southern Ocean, have lost the acclimatory capacity to modulate their membrane saturation states, making them vulnerable to anthropogenic global warming. We compared liver membrane lipid composition in two closely related Antarctic fish species acclimated at 0 °C (control temperature), 4 °C for a period of 14 days in T. bernacchii and 28 days for P. borchgrevinki, and 6 °C for 7 days in both species. Thermal acclimation at 4 °C did not result in changed membrane saturation states in either Antarctic species. Despite this, membrane functions were not compromised, as indicated by declining serum osmolality, implying positive compensation by enhanced hypo-osmoregulation. Increasing the temperature to 6 °C did not change the membrane lipids of P. borchgrevinki. However, in T. bernacchii, thermal acclimation at 6 °C resulted in an increase of membrane saturated fatty acids and a decline in unsaturated fatty acids. This is the first study to show a homeoviscous response to higher temperatures in an Antarctic fish, although for only one of the two species examined. [ABSTRACT FROM AUTHOR]

Wairau Bar, New Zealand, is one of the few prehistoric sites in the world that could lay claim to being a site of first human intrusion into a pristine environment. It is certainly one of the best places to study human impact on a hitherto unoccupied land. Its potential status as a colonization phase settlement for New Zealand's Maori population raises questions that require fine-grained chronological resolution. Unfortunately, the simple stratigraphy of the Wairau Bar site has offered little opportunity for the development of high-resolution chronologies. This situation changed recently when new excavations exposed a complex, midden-rich feature which contained a wide range of dateable material, including hundreds of fragments of eggshell of the extinct megaherbivorous moa (Aves: Dinornithiformes). The thick eggshell, with its minimal inbuilt age and high resistance to contamination, is an ideal material for radiocarbon dating. Its refractory properties also allow high-quality preservation of DNA. The moa eggshell yielded radiocarbon that facilitated reconstruction of the chronology of deposition at a fine resolution. Ancient DNA profiling of eggshell fragments was used to ensure that dated fragments were from different individuals. Bayesian analysis of the dated fragments showed that the midden was laid down over a brief period in the early decades of the 14th century CE. This improved chronology provides a benchmark for understanding the duration of site occupation and revises current interpretations of the timing of Polynesian settlement of New Zealand. [ABSTRACT FROM AUTHOR]

The extinction of New Zealand's moa (Aves: Dinornithiformes) followed the arrival of humans in the late 13th century and was the final event of the prehistoric Late Quaternary megafauna extinctions. Determining the state of the moa populations in the preextinction period is fundamental to understanding the causes of the event. We sampled 281 moa individuals and combined radiocarbon dating with ancient DNA analyses to help resolve the extinction debate and gain insights into moa biology. The samples, which were predominantly from the last 4,000 years preceding the extinction, represent four sympatric moa species excavated from five adjacent fossil deposits. We characterized the moa assemblage using mitochondrial DNA and nuclear microsatellite markers developed specifically for moa. Although genetic diversity differed significantly among the four species, we found that the millennia preceding the extinction were characterized by a remarkable degree of genetic stability in all species, with no loss of heterozygosity and no shifts in allele frequencies over time. The extinction event itself was too rapid to bemanifested in the moa gene pools. Contradicting previous claims of a decline in moa before Polynesian settlement in New Zealand, our findings indicate that the populations were large and stable before suddenly disappearing. This interpretation is supported by approximate Bayesian computation analyses. Our analyses consolidate the disappearance of moa as the most rapid, human-facilitated megafauna extinction documented to date. [ABSTRACT FROM AUTHOR]

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10[-6] per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R[2] = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone. [ABSTRACT FROM AUTHOR]

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means by which the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the 'shotgun' reads, >60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36-70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600-5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research. [ABSTRACT FROM AUTHOR]

Marine nitrogen (N) has been traced into terrestrial and freshwater food chains at petrel (Aves: Procellariiformes) colonies on the South Island of New Zealand, but the effects of N or other marine-derived nutrients on the productivity of mainland forests are unknown. Mean ring widths in matai (Prumnopitys taxifolia; n = 3) and rimu (Dacrydium cupressinum; n = 2) trees growing in a Westland petrel (Procellaria westlandica) colony were 0.9-1.4 mm yr-1, similar to ring widths reported elsewhere. However, series of much wider rings in cores showed that trees in a petrel colony can experience periods of accelerated growth. δ15N values of whole wood were, at 3.2-11.1‰, significantly enriched in comparison to other temperate forest trees. The highest δ15N values were at the cambial and pith ends of different cores, so isotopic fractionation during N remobilisation is unlikely to have been significant. The radial extent of 15N enrichment suggests that petrels have bred at the site since at least the mid 18th century. [ABSTRACT FROM AUTHOR]

Cyclical growth marks in cortical bone, deposited before attainment of adult body size, reflect osteogenetic changes caused by annual rhythms and are a general phenomenon in non-avian ectothermic and endothermic tetrapods. However, the growth periods of ornithurines (the theropod group including all modern birds) are usually apomorphically shortened to less than a year, so annual growth marks are almost unknown in this group. Here we show that cortical growth marks are frequent in long bones of New Zealand's moa (Aves: Dinornithiformes), a recently extinct ratite order. Moa showed the exaggerated K-selected life-history strategy formerly common in the New Zealand avifauna, and in some instances took almost a decade to attain skeletal maturity. This indicates that reproductive maturity in moa was extremely delayed relative to all extant birds. The two presently recognized moa families (Dinornithidae and Emeidae) also showed different postnatal growth rates, which were associated with their relative differences in body size. Both species of giant Dinornis moa attained their massive stature (up to 240 kg live mass) by accelerating their juvenile growth rate compared to the smaller emeid moa species, rather than by extending the skeletal growth period. [ABSTRACT FROM AUTHOR]

1. Stable isotope ratios of aquatic invertebrates, aquatic mosses and leaves of riparian plants were used to determine whether marine-derived nutrients from breeding colonies of the Westland petrel (Procellaria westlandica) were incorporated into the food webs of small streams in New Zealand. 2. The δ15N signatures of all plants and animals examined were higher by 3.6-4.6‰ in small streams draining catchments with petrel colonies than in nearby streams where petrels were absent. δ13C values of leaves from terrestrial plants were also enriched by about 2‰ where petrels were present, but the carbon ratios of aquatic species were depleted in 13C, rather than enriched, suggesting that any marine signal was over-ridden by isotopic shifts related to photosynthetic fractionation. 3. A high marine-nitrogen signal was maintained along the 3 km length of Scotchman Creek with the δ15N values of leptophlebiid mayflies and predatory insects ranging from 7.4-9.5 and 9.2-11.9‰, respectively. 4. Most nutrients derived from petrels are likely to be translocated to streams via the soil, which they enter in the form of excreta, spilled food, feathers, dead chicks, and abandoned eggs. However, because changes in δ15N values are brought about by soil processes such as volatilisation of ammonia, nitrification and denitrification, it is difficult to predict the exact isotope signature of nitrogen entering a stream. Tentative estimates of the proportion of marine-derived nitrogen in stream biota, calculated using a mass-balance approach, ranged from 28-38%. 5. Our findings indicate that marine nutrients transported inland by seabirds can be incorporated into the food webs of streams. In pre-human times when there were many more seabird colonies on mainland New Zealand than exist today, marine-derived nutrients introduced by birds may have had significant effects on nitrogen cycling and the productivity of New Zealand streams. [ABSTRACT FROM AUTHOR]

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals—apart from three species of bats—instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10–15 kg; 2–3 m wingspan) was 30%–40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems. A phylogenetic analysis reveals the rapid increase in size of a New Zealand eagle, demonstrating the speed at which evolution can act on islands. [ABSTRACT FROM AUTHOR]

Tenacibaculum maritimum is a cosmopolitan bacterial pathogen with the potential to cause significant losses in a broad range of farmed and wild marine fish species. This study investigated the antigenic diversity of T. maritimum isolated in culture from farmed Chinook (king) salmon (Oncorhynchus tshawytscha) from the Marlborough Sounds in New Zealand. A total of 36 isolates were examined using antibody serotyping and rapid molecular serotyping via multiplex PCR (mPCR) targeting genes encoding O-antigen biosynthesis enzymes. Serological analysis using three different polyclonal antisera developed against T. maritimum isolated from farmed Atlantic salmon (Salmo salar) in Tasmania, Australia revealed that there are three putative serotypes of T. maritimum that occur in New Zealand. The predominant serotype was defined by a positive reaction to all three Tasmanian antisera (antisera A, B and C), designated as serotype ABC. This serotype was found at all nine farm locations tested and represented 81% of all isolates examined. The same library of isolates was evaluated by mPCR serotyping and found three O-AGC types among tested isolates. O-AGC Type 3 was not only the predominant type (72%) present, but it also had a wide distribution, having been isolated at eight of the nine farms. Two other O-AGC types (O-AGC Type 2–1 and O-AGC Type 3–2) were identified, providing evidence of genetic variation. However, there was only partial concordance between the two serotyping techniques, which is likely linked to differences in the way serotypes are defined in the two approaches that were used. Nevertheless, in broad terms there is good evidence of intraspecific antigenic variation within our library of isolates, and collectively these data will be of crucial importance for assessing the pathogenicity of the isolates and the subsequent development of a vaccine for this emerging disease in New Zealand marine salmon farms. • Three putative serogroups of Tenacibaculum maritimum occur in New Zealand fish farms. • The predominant serotype occurred at all farm sites tested and represented 81% of all isolates examined. • There was partial concordance between two serotyping techniques. • Subsequent pathogenicity testing of the isolates will guide vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: The human colonisation of New Zealand in the late thirteenth century ad led to catastrophic impacts on the local biota and is among the most compelling examples of human over-exploitation of native fauna, including megafauna. Nearly half of the species in New Zealand'' s pre-human avifauna are now extinct, including all nine species of large, flightless moa (Aves: Dinornithiformes). The abundance of moa in early archaeological sites demonstrates the significance of these megaherbivores in the diet of the first New Zealanders. Combining moa assemblage data, based on DNA identification of eggshell and bone, with morphological identification of bone (literature and museum catalogued specimens), we present the most comprehensive audit of moa to date from several significant 13th–15th century ad archaeological deposits across the east coast of the South Island. Mitochondrial DNA (mtDNA) was amplified from 251 of 323 (78%) eggshell fragments and 22 of 27 (88%) bone samples, and the analyses revealed the presence of four moa species: Anomalopteryx didiformis; Dinornis robustus; Emeus crassus and Euryapteryx curtus. The mtDNA, along with polymorphic microsatellite markers, enabled an estimate of the minimum number of individual eggs consumed at each site. Remarkably, in one deposit over 50 individual eggs were identified – a number that likely represents a considerable proportion of the total reproductive output of moa in the area and emphasises that human predation of all life stages of moa was intense. Molecular sexing was conducted on bones (n = 11). Contrary to previous ancient DNA studies from natural sites that consistently report an excess of female moa, we observed an excess of males (2.7:1), suggestive that males were preferential targets. This could be related to different behaviour between the two highly size-dimorphic sexes in moa. Lastly, we investigated the moa species from recovered skeletal and eggshell remains from seven Wairau Bar burials, and identified the presence of only the larger species of moa, E. curtus and D. robustus. [Copyright &y& Elsevier]

Abstract: Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) – one of New Zealand’s most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts. [Copyright &y& Elsevier]

Abstract: Ancient DNA was isolated from the bones of 267 individuals of the extinct New Zealand moa (Aves: Dinornithiformes) from two late Holocene deposits [Pyramid Valley (PV) and Bell Hill Vineyard (BHV)] located 5.7km apart in North Canterbury, South Island. The two sites’ combined fossil record cover the last 3000years of pre-human New Zealand and mitochondrial DNA confirmed that four species (Dinornis robustus, Euryapteryx curtus, Emeus crassus, and Pachyornis elephantopus) were sympatric in the region. However, the relative species compositions in the two deposits differed significantly with D. robustus and E. crassus being most abundant at PV while E. curtus outnumbered the other three moa taxa combined at BHV. A subsample of 227 individuals had sufficient nuclear DNA preservation to warrant the use of molecular sexing techniques, and the analyses uncovered a remarkable excess of females in both deposits with an overall male to female ratio of 1:5.1. Among juveniles of E. curtus, the only species which was represented by a substantial fraction of juveniles, the sex ratio was not skewed (10 ♂, 10 ♀), suggesting that the observed imbalance arose as a result of differential mortality during maturation. Surprisingly, sex ratios proved significantly different between sites with a 1:2.2 ratio at BHV (n =90) and 1:14.2 at PV (n =137). Given the mobility of large ratites, and the proximity of the two fossil assemblages in space and time, these differences in taxonomic and gender composition indicate that moa biology and the local environment have affected the fossil representation dramatically and several possible explanations are offered. Apart from adding to our understanding of moa biology, these discoveries reinforce the need for caution when basing interpretation of the fossil record on material from a single site. [Copyright &y& Elsevier]

New Zealand moa (Aves: Dinornithiformes) are the only late Quaternary megafauna whose extinction was clearly caused by humans. New Zealand offers the best opportunity to estimate the number of people involved in a megafaunal extinction event because, uniquely, both the Polynesian settlement of New Zealand and moa extinction are recent enough to be dated with a high degree of precision. In addition, the founding human population can be estimated from genetic evidence. Here we show that the Polynesian population of New Zealand would not have exceeded 2,000 individuals before extinction of moa populations in the habitable areas of the eastern South Island. During a brief (<150 years) period and at population densities that never exceeded ~0.01 km(-2), Polynesians exterminated viable populations of moa by hunting and removal of habitat. High human population densities are not required in models of megafaunal extinction.

The extinct moa of New Zealand included three families (Megalapterygidae; Dinornithidae; Emeidae) of flightless palaeognath bird, ranging in mass from <15 kg to >200 kg. They are perceived to have evolved extremely robust leg bones, yet current estimates of body mass have very wide confidence intervals. Without reliable estimators of mass, the extent to which dinornithid and emeid hindlimbs were more robust than modern species remains unclear. Using the convex hull volumetric-based method on CT-scanned skeletons, we estimate the mass of a female Dinornis robustus (Dinornithidae) at 196 kg (range 155-245 kg) and of a female Pachyornis australis (Emeidae) as 50 kg (range 33-68 kg). Finite element analysis of CT-scanned femora and tibiotarsi of two moa and six species of modern palaeognath showed that P. australis experienced the lowest values for stress under all loading conditions, confirming it to be highly robust. In contrast, stress values in the femur of D. robustus were similar to those of modern flightless birds, whereas the tibiotarsus experienced the highest level of stress of any palaeognath. We consider that these two families of Dinornithiformes diverged in their biomechanical responses to selection for robustness and mobility, and exaggerated hindlimb strength was not the only successful evolutionary pathway.

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10(-6) per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R(2) = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.