Nuclear microsatellite DNA markers for New Zealand kiwi (Apteryx spp.)

Kiwi (Apterygidae) is an endemic New Zealand avian family comprising five species whose conservation is actively managed. We present five polymorphic microsatellite DNA loci isolated from North Island brown kiwi (Apteryx mantelli). In addition, we demonstrate cross‐amplification, and in some cases, polymorphism, of these microsatellite DNA loci in four other kiwi species. Therefore, these markers may be broadly applicable to conservation genetic studies within this family.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the North Island brown kiwi, Apteryx mantelli

We report the isolation and characterization of eight polymorphic and five monomorphic microsatellites in North Island brown kiwi (NIBK, Apteryx mantelli), using two polymerase chain reaction (PCR) techniques employing either short-tandem repeat primers (STR method) or random PCR-based isolation of microsatellite arrays (PIMA method). Microsatellite polymorphism was subsequently determined using 65 individuals. There were two to seven alleles for each polymorphic locus with heterozygozity ranging between 0.04 and 0.86. These primers will be used in future studies to determine the level of extra-pair copulation, dispersal patterns, and genetic diversity within and between wild populations of NIBK.     

Two sibling species sympatrically breeding: a new conservation concern for the critically endangered Balearic shearwater

Two Puffinus shearwater species are endemic to the Mediterranean region: the Yelkouan shearwater P.␣yelkouan breeding in the central and eastern, and the critically endangered Balearic shearwater P.␣mauretanicus in the western, exclusively breeding at the Balearic archipelago. All individuals caught in Minorca, a Balearic breeding site, showed phenotypic traits of Yelkouan shearwaters. Ten birds from that colony were sampled and genetically analysed. A fragment of 857 base pairs of the cytochrome b was sequenced and compared to published sequences of both species obtained from GenBank. A Neighbor-Joining (NJ) reconstruction distinguished two clades with high bootstrap support, showing that both species bred sympatrically in that colony. This result was unexpected since shearwaters do not usually form mixed colonies. New conservation concerns arise also from our study: at first, breeding population of Yelkouan shearwaters is even smaller than previously thought; additionally, other potential threats emerge, such as hybridization and ecological competition.     

Reconstructing the evolutionary history of China: a caveat about inferences drawn from ancient DNA

The decipherment of the meager information provided by short fragments of ancient mitochondrial DNA (mtDNA) is notoriously difficult but is regarded as a most promising way toward reconstructing the past from the genetic perspective. By haplogroup-specific hypervariable segment (HVS) motif search and matching or near-matching with available modern data sets, most of the ancient mtDNAs can be tentatively assigned to haplogroups, which are often subcontinent specific. Further typing for mtDNA haplogroup-diagnostic coding region polymorphisms, however, is indispensable for establishing the geographic/genetic affinities of ancient samples with less ambiguity. In the present study, we sequenced a fragment (approximately 982 bp) of the mtDNA control region in 76 Han individuals from Taian, Shandong, China, and we combined these data with previously reported samples from Zibo and Qingdao, Shandong. The reanalysis of two previously published ancient mtDNA population data sets from Linzi (same province) then indicates that the ancient populations had features in common with the modern populations from south China rather than any specific affinity to the European mtDNA pool. Our results highlight that ancient mtDNA data obtained under different sampling schemes and subject to potential contamination can easily create the impression of drastic spatiotemporal changes in the genetic structure of a regional population during the past few thousand years if inappropriate methods of data analysis are employed.     

Mating system and genetic variation in the endangered New Zealand takahe

The takahe (Porphyrio hochstetteri) is ahighly endangered flightless rail that isendemic to New Zealand. Only one remnantpopulation of takahe (120 adults) is left inthe wild in Fiordland, which has been thesource for introductions to four predator-freeislands. The objective of the present studywas to determine the mating system andamount of genetic variation in takahe usingmultilocus DNA profiling, in order to assist inthe management of the island populations. There was no evidence of extra-pair paternityfor the 27 (73%, n = 37) offspring towhich paternity could be resolved. Thepaternity of the remaining 10 offspring couldnot be resolved due to low levels ofminisatellite DNA variation, but in none wasthe resident male excluded. Overall, the DNAresults along with behavioral and life historyinformation indicate that extra-pairfertilizations should be rare or absent, andtakahe join a small but growing list oflong-lived species of birds that have beenshown to exhibit genetic monogamy. Inaddition, the levels of minisatellite DNAvariation detected in takahe are low relativeto those reported for most other known outbredavian populations, and are consistent with theevidence of the takahe's persistence as asmall, isolated population in Fiordland over atleast the last 100 years. The low geneticvariation is discussed in relation to possibleevidence of environment depended inbreedingdepression in translocated island populationsof takahe.     

Focusing and accommodation in the brown kiwi (Apteryx australis)

Summary Brown kiwis are an endangered species of nocturnal, flightless birds which are native to New Zealand. The resting focus of two specimens has previously been studied by retinoscopy in a zoo while the birds were restrained by their keeper (Sivak and Howland 1987). Those birds appeared to be hyperopic (farsighted) by 2–7 D. In this study, examination with infrared photorefraction of the focusing of two unrestrained, feeding birds showed that they could focus objects at infinity and objects in their immediate environment and that they had modest powers of accommodation. Measurements on two 6 month old kiwi chicks showed their corneal radius of curvature to be between 2.90 and 3.00 mm (117 D and 101 D in power).     

Ancient human mtDNA genotypes from England reveal lost variation over the last millennium

We analysed the historical genetic diversity of human populations in Europe at the mtDNA control region for 48 ancient Britons who lived between ca AD 300 and 1000, and compared these with 6320 modern mtDNA genotypes from England and across Europe and the Middle East. We found that the historical sample shows greater genetic diversity than for modern England and other modern populations, indicating the loss of diversity over the last millennium. The pattern of haplotypic diversity was clearly European in the ancient sample, representing each of the modern haplogroups. There was also increased representation of one of the ancient haplotypes in modern populations. We consider these results in the context of possible selection or stochastic processes.     

Genetic variation in populations of the endangered fish Ladigesocypris ghigii and its implications for conservation

1. The genetic variation of the endangered freshwater fish Ladigesocypris ghigii, endemic to the island of Rhodes (Greece), was investigated for nine populations, originating from seven different stream systems and a reservoir, both at the mtDNA and nuclear level, in order to suggest conservation actions. 2. Both restriction fragment length polymorphism analysis of five segments of mitochondrial DNA (ND‐5/6, COI and 12S‐16S rRNA) amplified by polymerase chain reaction, and random amplified polymorphic DNA analysis, revealed extremely low levels of intra‐population polymorphism. It is highly likely that the low intra‐population variability is the result of successive bottleneck events evident in shrinkage and expansion of the populations year after year, which may have led to a complete loss of several genotypes and haplotypes, and an increased degree of inbreeding. 3. Inter‐population genetic structuring was high, with fixation of haplotypes within six of the nine populations and fixation of alleles within populations originating from different waterbodies. It is probable that all haplotypes and/or alleles found were initially represented in all populations. However, because of the long time of isolation coupled with successive bottleneck and subsequent genetic drift, common mtDNA haplotypes and alleles among the populations may have become rare or extinct through stochastic lineage loss. 4. Although nucleotide divergence among haplotypes was very shallow, half of the haplotypes recorded (three of six), resulted from nucleotide changes on the 12S–16S rRNA segments, which are the most conserved part of the mitochondrial genome. This fact may indicate that the observed genetic variation did not necessarily result only from the retention of ancestral polymorphism, but may have arisen through mutation and complete lineage sorting over a relatively small number of generations, once the populations had become isolated from one another. 5. Our data suggest that two of the L. ghigii populations may be on independent evolutionary trajectories. Considering that each population appears so far well adapted within each site, all populations should be managed and conserved separately.     

Ancient DNA sequences point to a large loss of mitochondrial genetic diversity in the saiga antelope (Saiga tatarica) since the Pleistocene

Prior to the Holocene, the range of the saiga antelope (Saiga tatarica) spanned from France to the Northwest Territories of Canada. Although its distribution subsequently contracted to the steppes of Central Asia, historical records indicate that it remained extremely abundant until the end of the Soviet Union, after which its populations were reduced by over 95%. We have analysed the mitochondrial control region sequence variation of 27 ancient and 38 modern specimens, to assay how the species’ genetic diversity has changed since the Pleistocene. Phylogenetic analyses reveal the existence of two well‐supported, and clearly distinct, clades of saiga. The first, spanning a time range from >49 500 ¹⁴C ybp to the present, comprises all the modern specimens and ancient samples from the Northern Urals, Middle Urals and Northeast Yakutia. The second clade is exclusive to the Northern Urals and includes samples dating from between 40 400 to 10 250 ¹⁴C ybp. Current genetic diversity is much lower than that present during the Pleistocene, an observation that data modelling using serial coalescent indicates cannot be explained by genetic drift in a population of constant size. Approximate Bayesian Computation analyses show the observed data is more compatible with a drastic population size reduction (c. 66–77%) following either a demographic bottleneck in the course of the Holocene or late Pleistocene, or a geographic fragmentation (followed by local extinction of one subpopulation) at the Holocene/Pleistocene transition.     

Identity of an endangered grasshopper (Acrididae: Brachaspis): Taxonomy, molecules and conservation

Brachaspis robustus is an endangeredgrasshopper endemic to South Island, NewZealand. It is both rare and localised;occupying low altitude floodplain terraces andbraided riverbeds of the Mackenzie Basin. Thisis in stark contrast to the two other speciesin this genus (B. nivalis and B.collinus) which occupy montane habitats.Mitochondrial and nuclear sequence data wereemployed to explore genetic diversity andphylogenetic relationships of populations ofBrachaspis with a view to establishingthe status of B. robustus. Molecularevidence indicates that Brachaspisprobably radiated during the Pliocene and thatdivisions within the genus relate more tospatial distribution developed during thePleistocene than to ecology. The mitochondrial(Cytochrome oxidase I) and nuclear (ITS)sequence data indicate that Brachaspisnivalis is divided into northern and southernpopulations. The northern clade is furthersubdivided geographically. The southern cladecomprises alpine populations of B.nivalis and includes the lowland B.robustus. Additionally, it is observed thatsome morphological features previously thoughtto be specific to B. robustus also occurin members of the southern B. nivalisclade. It is suggested that the taxon B.robustus should include all of the southernBrachaspis populations. But it is arguedthat the absence of genetic evidencedistinguishing the endangered population doesnot preclude it from conservation effort. Acombination of morphological and habitatpeculiarities indicate that the survival ofB. robustus (sensu lato) isimportant to the maintenance of diversity.     

Ancient DNA evidence for the loss of a highly divergent brown bear clade during historical times

The genetic diversity of present-day brown bears (Ursus arctos) has been extensively studied over the years and appears to be geographically structured into five main clades. The question of the past diversity of the species has been recently addressed by ancient DNA studies that concluded to a relative genetic stability over the last 35,000 years. However, the post-last glacial maximum genetic diversity of the species still remains poorly documented, notably in the Old World. Here, we analyse Atlas brown bears, which became extinct during the Holocene period. A divergent brown bear mitochondrial DNA lineage not present in any of the previously studied modern or ancient bear samples was uncovered, suggesting that the diversity of U. arctos was larger in the past than it is now. Specifically, a significant portion (with respect to sequence divergence) of the intraspecific diversity of the brown bear was lost with the extinction of the Atlas brown bear after the Pleistocene/Holocene transition.     

Ancient mtDNA haplogroups: a new insight into the genetic history of European populations

In the present work, DNA was extracted from 63 skeletal samples recovered at the Neolithic site of San Juan ante Portam Latinam (SJAPL) (Araba, Basque Country). These samples have proved useful as genetic material for the performance of population studies. To achieve this it was necessary to overcome the methodological problems arising when working with damaged DNA molecules. We succeeded in performing an amplification and restriction analysis of the polymorphisms present in the mtDNA. Ninety seven percent of the samples were classified as belonging to one of the nine mtDNA haplogroups described in Caucasians. This work shows that restriction analysis is a useful methodological tool to perform reliable population genetic analysis on archaeological remains. Tha analysis of ancient and modern haplogroup distribution can shed more light on the genetic evolution of human populations. Moreover, a more exhaustive data on prehistoric populations will allow to build stronger hypothesis on the genetic relationships among human populations.     

Ancient mitochondrial DNA from the northern fringe of the Neolithic farming expansion in Europe sheds light on the dispersion process

The European Neolithization process started around 12 000 years ago in the Near East. The introduction of agriculture spread north and west throughout Europe and a key question has been if this was brought about by migrating individuals, by an exchange of ideas or a by a mixture of these. The earliest farming evidence in Scandinavia is found within the Funnel Beaker Culture complex (Trichterbecherkultur, TRB) which represents the northernmost extension of Neolithic farmers in Europe. The TRB coexisted for almost a millennium with hunter–gatherers of the Pitted Ware Cultural complex (PWC). If migration was a substantial part of the Neolithization, even the northerly TRB community would display a closer genetic affinity to other farmer populations than to hunter–gatherer populations. We deep-sequenced the mitochondrial hypervariable region 1 from seven farmers (six TRB and one Battle Axe complex, BAC) and 13 hunter–gatherers (PWC) and authenticated the sequences using postmortem DNA damage patterns. A comparison with 124 previously published sequences from prehistoric Europe shows that the TRB individuals share a close affinity to Central European farmer populations, and that they are distinct from hunter–gatherer groups, including the geographically close and partially contemporary PWC that show a close affinity to the European Mesolithic hunter–gatherers.     

Deeply divergent lineages of the widespread New Zealand amphipod Paracalliope fluviatilis revealed using allozyme and mitochondrial DNA analyses

1. We evaluated the population genetic structure of the common New Zealand amphipod Paracalliope fluviatilis using eight allozyme loci, and the mitochondrial cytochrome oxidase c subunit I (COI) gene locus. Morphological analyses were also conducted to evaluate any phenotypic differences. Individuals belonging to P. fluviatilis were collected from a total of 14 freshwater fluvial habitats on the North and South Islands, New Zealand. 2. We found evidence for strong genetic differentiation among locations (Wright's F_ST > 0.25), and fixed differences (non‐shared alleles) at two of the eight allozyme loci indicating the possibility of previously unknown species. Analysis of a 545‐bp fragment of the COI locus was mostly congruent with the allozyme data and revealed the same deeply divergent lineages (sequence divergences up to 26%). 3. Clear genetic breaks were identified between North Island and South Island populations. North Island populations separated by <100 km also showed genetic differences between east and west draining watersheds (sequence divergence >12%). Accordingly, present‐day dispersal among hydrologically isolated habitats appears minimal for this taxon. 4. Although population differences were clearly shown by allozyme and mtDNA analyses, individuals were morphologically indistinguishable. This suggests that, as in North American and European taxa (e.g. Hyalella and Gammarus), morphological conservatism may be prevalent among New Zealand's freshwater amphipods. We conclude that molecular techniques, particularly the COI gene locus, may be powerful tools for resolving species that show no distinctive morphological differences.     

Panbiogeography and conservation science in New Zealand

Abstract

New Zealand’s official conservation science framework, the Protected Natural Areas Programme (PNA), is evaluated in relation to current biogeographic/systematic methods and principles with reference to philosophy, taxonomy, information content, and historical geological/biological relations. The PNA Programme and its parent International Union for Conservation of Nature and Natural Resources (IUCN) framework is based on a phenetic system of classification that does not reflect process (phylogenetic) characters of ecological systems. The PNA philosophy of conservation science is shown to be essentialist, rooted in traditional medieval Western metaphysics, and out of step with current developments in biogeography, systematics, and ecology. Panbiogeography is an appropriate global perspective for developing a conservation science because it meets the requirements of homology, monophyly, increased information content, and empirical testability whereas the PNA programme does not. Establishment of a Panbiogeographic Track Atlas is proposed as a suitable conservation framework for historical ecology and biogeography. The atlas could provide an empirical natural resource inventory to identify priority areas for conservation at an economically acceptable cost compared to the PNA Programme. Application of cladistic techniques to ecological and biogeographic patterns in relation to the Atlas can provide aphylogenetically sound hierarchical classification for conservation science. The potential benefits of the panbiogeographic approach for conservation education and tourism are discussed.     

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.     

Molecular and other biological evidence supports the recognition of at least three species of brown kiwi

The presence of morphologically crypticlineages within the threatened brown kiwi ofNew Zealand has confounded their taxonomy. Recent genetic studies (Herbert and Daugherty1994; Baker et al. 1995) revealed that atleast two phylogenetic species exist within thebrown kiwi, and suggested that further researchshould resolve the taxonomic problems. In thispaper we extend genetic analyses to includesequences from 58 brown kiwi representing fivephylogenetic lineages for four mitochondrialloci (control region, cytochrome b,ATPase 6 and ATPase 8). Major lineages ofbrown kiwi are shown to be reciprocallymonophyletic, and align with other biologicaldifferences in the ecology, behavior,morphology and parasites of kiwi. BecausemtDNA sequences of major lineages of kiwi arenot evolving in a clocklike manner, we used anew penalized likelihood method withrate-smoothing to date the divergence of NorthIsland brown kiwi and the geographicallyisolated Okarito population (rowi) at about 6.2Mya. These lineages diverged about 8.2 Myafrom the brown kiwi in Fiordland and Haast inthe southern part of the South Island, and arethus older than the species of spotted kiwi(5.8 Mya). Given their distinctness, long-termgeographical isolation, lack of hybridizationin introduced populations, and accumulation ofnew biological characters within theselineages, we hypothesize that reproductiveincompatibilities have probably arisen as well. We therefore recommend that these divergentlineages be formally recognized as fullspecies; Apteryx mantelli should bere-instated for the North Island brown kiwi,A. australis should be restricted to thetokoeka, and a new species A. rowiishould be erected to describe the rowi atOkarito. Tokoeka should be split into at leastthree conservation management units (Haast,Fiordland and Stewart Island [Rakiura]), butfurther research is required to determine theexact relationships and status of theselineages. Further investigation is alsorequired into the genetic structuring of theNorth Island brown kiwi to confirm conservationmanagement units on the North Island.     

Mitochondrial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves

The question of the origins of the dog has been much debated. The dog is descended from the wolf that at the end of the last glaciation (the archaeologically hypothesized period of dog domestication) was one of the most widespread among Holarctic mammals. Scenarios provided by genetic studies range from multiple dog-founding events to a single origin in East Asia. The earliest fossil dogs, dated approximately 17-12,000 radiocarbon ((14)C) years ago (YA), were found in Europe and in the Middle East. Ancient DNA (a-DNA) evidence could contribute to the identification of dog-founder wolf populations. To gain insight into the relationships between ancient European wolves and dogs we analyzed a 262-bp mitochondrial DNA control region fragment retrieved from five prehistoric Italian canids ranging in age from approximately 15,000 to approximately 3,000 (14)C YA. These canids were compared to a worldwide sample of 547 purebred dogs and 341 wolves. The ancient sequences were highly diverse and joined the three major clades of extant dog sequences. Phylogenetic investigations highlighted relationships between the ancient sequences and geographically widespread extant dog matrilines and between the ancient sequences and extant wolf matrilines of mainly East European origin. The results provide a-DNA support for the involvement of European wolves in the origins of the three major dog clades. Genetic data also suggest multiple independent domestication events. East European wolves may still reflect the genetic variation of ancient dog-founder populations.