Amplification success of multilocus genotypes from feathers found in the field compared with feathers obtained from shot birds

Effective DNA extraction methods from bird feathers have facilitated non‐invasive sampling, leading to the suggestion that feathers are a great source for genetic studies. However, few studies have assessed whether all feathers can be used or provide equal numbers of useful templates. In this study, feathers collected in various ways from Red Grouse Lagopus lagopus were examined to establish the quality of DNA extracted. Individual samples were classified into two categories according to whether they were collected from shot birds or found in the field. DNA was extracted from all samples and genotyped at 19 microsatellite loci. PCR products were analysed on a MegaBACE 1000. A total of 93% of the ‘shot’ category produced a genotype that was considered successful (i.e. 15 of 18 loci) and 23% of the ‘collected’ category produced successful genotypes under the same criteria. There was a significant difference between shot and collected samples in genotyping success and the observed number of missing loci. Recommendations and best practices are discussed along with the utility of bird feathers as a source of DNA for population and conservation biology.     

Neighbourhood analyses of tree seed predation by introduced rodents in a New Zealand temperate rainforest

House mice Mus musculus and other introduced rodents represent a novel source of predation on tree seeds in New Zealand forests. In the northern temperate forests where these rodents are native, spatial and temporal variation in tree seed production can result in dramatic fluctuations in the distribution and abundance of seed predators, with subsequent feedbacks on the distribution and abundance of seedlings. We use neighbourhood models to examine variation in rodent predation on seeds of 4 tree species of the temperate rainforests of New Zealand as a function of 1) spatial variation in local canopy composition and 2) spatial and temporal variation in mouse activity. We placed seeds throughout mapped stands of mixed forests in alluvial valley bottoms and on elevated marine terraces in the Waitutu Forest, South Island. The risk of predation on seeds of 2 dominant canopy trees – rimu Dacrydium cupressinum and mountain beech Nothofagus solandri var . cliffortioides – peaked in neighbourhoods dominated by those species and by silver beech N. menziesii , particularly in a year of plentiful seed rain from these species. The risk of predation on rimu and beech seed was also related to measures of local mouse activity. These relationships suggest that the highest local abundance of mice was concentrated in rimu and beech neighbourhoods because of the food provided by seed rain from those trees. Predation on seed of miro Prumnopitys ferruginea , which is eaten by rats but not mice, was low in rimu neighbourhoods and where mouse activity was high. These patterns may reflect spatial segregation in the activity of rats versus mice within stands. Our results suggest that the spatial distribution of canopy trees translates into predictable patterns of variation in mouse activity and seed predation. Heterogeneity in rodent activity and seed predation within stands may have important implications for tree population dynamics.     

Low genotyping error rates in non-invasively collected samples from roe deer of the Bavarian Forest National Park

Genetic wildlife monitoring is increasingly carried out on the basis of non-invasively collected samples, whereby the most commonly used DNA sources are skin appendages (hairs, feathers) and faeces. In order to guide decisions regarding future adequate ways to monitor the roe deer (Capreolus capreolus) population of the Bavarian Forest National Park in Germany, we tested these two different types of DNA source materials to compare their suitability for genetic monitoring. We determined the haplotypes (d-loop) of 19 roe deer and genotyped each individual (tissue, hairs, faeces) across 12 microsatellite loci. The amount of missing and erroneous microsatellite alleles obtained from hair and faeces samples, respectively, was estimated based on comparisons with the corresponding tissue sample control. We observed no missing alleles in hair samples, but in fecal samples PCR failed in 30 out of 228 instances (19 individuals x 12 loci), corresponding to a frequency of missing alleles of 13.2% across all loci and individuals. In genotypes generated from hairs erroneous alleles were detected in 2 out of 228 instances (0.9%), while genotypes retrieved from fecal samples displayed erroneous alleles in 6 out of 198 remaining instances (3%). We conclude that both hair and fecal samples are generally well suited for genetic roe deer monitoring, but that fecal sample based analyses require a larger sample size to account for higher PCR failure rates.     

The fear diet: Risk,refuge, and biological control by omnivorous weed seed predators

Weed seed biocontrol by omnivorous mice and insects can limit weed seedbanks, but this ecosystem service can be difficult to predict given the broad diet breadth of seed predators and their potential for intraguild predation. Seed foraging behavior is further modified by fluctuating cues of predation risk from higher trophic levels and the availability of refuge habitat. Uncertainty about whether co-occurring insects and mice additively contribute to weed biocontrol or interfere with each other via intraguild predation limits our ability to recommend habitat management strategies that reliably promote seed destruction. Using seed removal assays, fluorescent powder tracking, and stable isotope analyses, we assessed effects of a predation risk cue (moonlight) on mouse foraging patterns in a patchwork of vegetated and exposed plots in a cultivated field. Mouse foraging activity decreased on exposed ground during the full moon, compared to dark nights, yet foraging movements were unaffected by moon cycle within refuge patches. Weed seed consumption was more than three times higher in cover than exposed soil, and 78% of that difference was attributable to invertebrate granivores. Mice and invertebrate granivores both exhibited higher foraging activity in cover, indicating co-occurrence of intraguild predators and prey. However, stable isotope analyses of fecal samples revealed that mice captured in refuge habitats fed at slightly lower trophic levels than those in exposed habitats (suggesting minimal intraguild predation in refuge habitat), and mouse diet was unaffected by moonlight. Despite increased availability of invertebrate prey in cover patches, mice do not appear to preferentially exploit prey when avoiding their own predators or interfere with weed seed predation. Therefore, functional redundancy of mice and invertebrate seed predators in cover crops and other refuge habitats may strengthen and stabilize weed seed biocontrol.     

Intraspecific variation and phylogeographic patterns of Fagus crenata (Fagaceae) mitochondrial DNA

Mitochondrial (mt) DNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 17 populations distributed throughout the species' range. Total genomic DNA of samples from single trees representing each of 12 populations were digested with 18 restriction enzymes and hybridized with three probes containing coxI, coxIII, and atpA gene sequences. Thirty-four of the 54 enzyme/probe combinations showed polymorphisms and all the individuals were subsequently analyzed with six combinations of three probes and two enzymes. Restriction fragment length polymorphisms were evident around all three genes, allowing the identification of eight distinct haplotypes. Haplotype diversity within the populations was found to be very low (HS = 0.031), but population differentiation to be much higher (GST = 0.963). The mtDNA variation was strikingly different from allozyme variation (HS = 0.209; GST = 0.039). Gene flow for maternally inherited mtDNA should be restricted to seed dispersal while nuclear gene flow occurs by both seed and pollen dispersal. Therefore, the difference in the variation between mtDNA and allozymes may be largely a result of the much higher rate of gene flow associated with pollen dispersal than with seed dispersal. The mtDNA variation displayed strong geographic structure, which may reflect the species' distribution in the last glacial maximum and subsequent colonization, and probably also reflects intraspecific phylogeography of the species.     

Ancient DNA recovers the origins of Māori feather cloaks

Feather cloaks ("kakahu"), particularly those adorned with kiwi feathers, are treasured items or "taonga" to the Māori people of "Aotearoa"/New Zealand. They are considered iconic expression of Māori culture. Despite their status, much of our knowledge of the materials used to construct cloaks, the provenance of cloaks, and the origins of cloak making itself, has been lost. We used ancient DNA methods to recover mitochondrial DNA sequences from 849 feather samples taken from 109 cloaks. We show that almost all (>99%) of the cloaks were constructed using feathers from North Island brown kiwi. Molecular sexing of nuclear DNA recovered from 92 feather cloak samples also revealed that the sex ratio of birds deviated from a ratio of 1:1 observed in reference populations. Additionally, we constructed a database of 185 mitochondrial control region DNA sequences of kiwi feathers comprising samples collected from 26 North Island locations together with data available from the literature. Genetic subdivision (G(ST)), nucleotide subdivision (N(ST)) and Spatial Analysis of Molecular Variants (SAMOVA) analyses revealed high levels of genetic structuring in North Island brown kiwi. Together with sequence data from previously studied ancient and modern kiwi samples, we were able to determine the geographic provenance of 847 cloak feathers from 108 cloaks. A surprising proportion (15%) of cloaks were found to contain feathers from different geographic locations, providing evidence of kiwi trading among Māori tribes or organized hunting trips into other tribal areas. Our data also suggest that the east of the North Island of New Zealand was the most prolific of all kiwi cloak making areas, with over 50% of all cloaks analyzed originating from this region. Similar molecular approaches have the potential to discover a wealth of lost information from artifacts of endemic cultures worldwide.     

Optimizing the use of shed feathers for genetic analysis

Shed feathers obtained by noninvasive genetic sampling (NGS) are a valuable source of DNA for genetic studies of birds. They can be collected across a large geographical range and facilitate research on species that would otherwise be extremely difficult to study. A limitation of this approach is uncertainty concerning the quality of the extracted DNA. Here we investigate the relationship between feather type, feather condition and DNA quality (amplification success) in order to provide a simple, cost-effective method for screening samples prior to genetic analysis. We obtained 637 shed feathers of the powerful owl (Ninox strenua) from across its range in southeastern Australia. The extracted DNA was amplified using polymerase chain reaction for a range of markers including mitochondrial DNA, ND3 and nuclear DNA, a simple sequence repeat (Nst02) and a portion of the CHD-1 gene (P2/P8). We found that feather condition significantly influenced the amplification success of all three loci, with feathers characterized as 'good' having greater success. Feather type was found to be of lower importance, with good quality feathers of all types consistently producing high success for all three loci. We also found that the successful amplification of multilocus genotypes was dependant on the condition of the starting material and was highly correlated with successful amplification of the sex-linked CHD-1 locus. Samples with low DNA quality have a higher probability of amplification failure and are more likely to produce incorrect genotypes; therefore, identifying samples with high DNA quality can save substantial time and cost associated with the genetic analysis of NGS. As a result, we propose a method for screening shed feathers in order to provide a subset of samples which will have a greater probability of containing high quality DNA suitable for the amplification of multilocus genotypes.     

Seasonal variation in the honeydew, invertebrate, fruit and nectar resource for bellbirds in a New Zealand mountain beech forest

To examine the seasonal availability of the major bellbird (Anthornis melanura) food sources in a mountain beech (Nothofagus solandrivar. cliffortioides) forest at Craigieburn, the invertebrate, honeydew, and mistletoe (Peraxilla tetrapetala and Alepis flavida) fruit and nectar resources were sampled over 12 months. The total available food varied 2.6-fold from a low in October (8798 kJ/ha) to a high in December (22,959 kJ/ha) with an annual mean of 15,782 kJ/ha. Invertebrates were available all year and represented 89% of the available food energy. Only 16% of the invertebrate resource was on beech foliage, and beech trunks with honeydew had 60% more invertebrate energy than trunks without honeydew. The energy value of honeydew at Craigieburn (0.9% of the total) was much lower than at lower altitude sites. The relative rankings of honeydew standing crops on 25 permanently marked trees were very constant. On an annual basis mistletoe nectar and fruit made up 6.3% and 4.9%, respectively, of total food energy, but P. tetrapetala nectar was 46% of available food in early January, and P. tetrapetala fruit was 25% of the total in March. Bellbirds spent less time foraging on invertebrates, and more time on the other foods, than energy values would predict. However, during the Peak of its short flowering season, P. tetrapetala nectar made up 46% of available energy but only 33% of bellbird foraging observations. At this site P. tetrapetala is pollen limited due to insufficient visits from pollinators. This may be because bellbirds require invertebrates for protein, or to feed to nestlings. Therefore the pollination mutualism is faltering, despite high investment in nectar by the plant.     

Molted feathers from clay licks in Peru provide DNA for three large macaws (Ara ararauna, A. chloropterus, and A. macao)

ABSTRACT Conservation genetic analyses of wildlife have increased greatly in the past 10 yr, yet genetic studies of parrots are rare because of difficulties associated with capturing them and obtaining samples. Recent studies have demonstrated that molted feathers can provide a useful source of DNA, but success rates have varied considerably among studies. Our objective was to determine if molted macaw feathers from Blue‐and‐yellow Macaws (Ara ararauna), Scarlet Macaws (A. macao), and Red‐and‐green Macaws (A. chloropterus) collected from rainforest geophagy sites called clay licks could provide a good source of DNA for population genetic studies. Specific objectives were to determine (1) how nuclear DNA microsatellite amplification success and genotyping error rates for plucked macaw feathers compared to those for molted feathers collected from clay licks in the Amazon rainforest, and (2) if feather size, feather condition, species, or extraction method affected microsatellite amplification success or genotyping error rates from molted feathers. Amplification success and error rates were calculated using duplicate analyses of four microsatellite loci. We found that plucked feathers were an excellent source of DNA, with significantly higher success rates (P < 0.0001) and lower error rates (P= 0.0002) than for molted feathers. However, relatively high success rates (75.6%) were obtained for molted feathers, with a genotyping error rate of 11.7%. For molted feathers, we had higher success rates and lower error rates for large feathers than small feathers and for feathers in good condition than feathers that were moldy and broken when collected. We also found that longer incubation times and lower elution volumes yielded the highest quality DNA when extracting with the Qiagen DNeasy tissue kit. Our study demonstrates that molted feathers can be a valuable source of genetic material even in the challenging conditions of tropical rainforests, and our results provide valuable information for maximizing DNA amplification success rates when working with shed feathers of parrots.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

Age-Specific Prevalence and a Possible Transmission Route for

The prevalence of infestation of the skulls of stoats with the parasitic nematode Skrjabingylus nasicola was previously described in a national survey by King and Moody (1982). Since then, more samples from Craigieburn Forest Park and from the Eglinton Valley, Fiordland, have been collected, and a method of determining the actual ages of adult stoats has been developed. The extended samples are here examined for a relationship between infestation and age, which could not previously be tested. Prevalence generally increases with age, significantly so at Craigieburn. Stoats which had lived through one or more beech (Nothofagus solandri:) mast years at Craigieburn were significantly more likely to be infested, when the effects of age were allowed for. The hypothesis is advanced that the paratenic host for S. nasicola in New Zealand is the feral house mouse, Mus musculus, which is more numerous after a heavy beech seed fall.     

Noninvasive genetic analysis in birds: testing reliability of feather samples

Noninvasive samples are useful for molecular genetic analysis of free‐ranging animals. I tested whether moulted feathers collected in the field are a reliable source of DNA for genotyping microsatellite loci. I prescreened extracts for DNA quantity and, using only samples with higher amounts of DNA, obtained reliable genotyping results. Polymerase chain reaction (PCR) amplification success was higher from extracts of plucked feathers than moulted feathers. DNA quantity in larger feathers was higher than that in smaller feathers. This study clearly demonstrates that moulted feathers could be used for genetic studies in birds.     

The Effects of a Natural Increase in Food-Supply on a Wild Population of House Mice

Changes in density and breeding of the house mouse (Mus musculus) in a New Zealand forest dominated by hard beech (Nothofagus truncata) were monitored for 2.5 years. Mice bred during winter and increased dramatically in density only during a beech mast year. Mice readily ate the endosperm and embryo of hard beech seed in die laboratory and chemical analysis showed it to be a very nutritious food source, similar in quality to Fagus beech seed in the northern hemisphere. Thus the mouse, introduced to New Zealand, responds to a Nothofagus mast year in a similar way to other rodent species in the northern hemisphere during a Fagus mast year.     

Noninvasive collection of fresh hairs from free‐ranging howler monkeys for DNA extraction

The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh‐plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low‐cost noninvasive technique for collecting fresh‐plucked hairs used to obtain DNA samples from free‐ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex–age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction‐based microsatellite marker analysis. The technique proved successful for collecting fresh‐plucked hairs of free‐ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species. Am. J. Primatol. 71:359–363, 2009. © 2009 Wiley‐Liss, Inc.     

Detecting invertebrate ecosystem service providers in orchards: traditional methods versus barcoding of environmental DNA in soil

1. The objective of this study was to assess barcoding of environmental DNA as a method for monitoring invertebrate ecosystem service providers in soil samples.
2. We selected 26 invertebrate ecosystem service providers that occur in New Zealand kiwifruit or apple orchards and produced mitochondrial cytochrome c oxidase gene subunit I (cytochrome oxidase I) and/or 28S ribosomal DNA sequences for each. Specific barcode primers were designed for each invertebrate ecosystem service provider and tested, along with generic barcoding cytochrome oxidase I primers, for their ability to detect DNA from invertebrate ecosystem service providers that had been added to sterilized and unsterilized soil samples.
3. Although the specific primers accurately detected the invertebrate ecosystem service providers in more than 96% of the samples, the generic cytochrome oxidase I primers detected only 37% of the invertebrate ecosystem service providers added to the sterilized samples and 2.5% in the unsterilized samples.
4. In a field test, we compared metabarcoding with traditional invertebrate trapping methods to detect the invertebrate ecosystem service providers in 10 kiwifruit and 10 apple orchards. All invertebrate ecosystem service providers were collected in traps in at least one orchard, but very few were identified by metabarcoding of soil environmental DNA.
5. Although the specific primers can be used as a tool for monitoring invertebrate ecosystem service providers in soil samples, methodological improvements are needed before metabarcoding of soil environmental DNA can be used to monitor these taxa.

     

Stoat density, diet and survival compared between alpine grassland and beech forest habitats

In New Zealand, alpine grasslands occur above the treeline of beech forest. Historically stoat control paradigms in New Zealand?s montane natural areas have assumed alpine grassland is a marginal habitat that limits dispersal between beech forest stoat populations. We compared the summer-to-autumn (January?April) density, weight, diet and winter survival of stoats between these two habitatsduring years of low beech seedfall. Stoats were live-trapped, marked and released in alpine grassland and low-altitude beech forest in the Borland Valley, Fiordland National Park, during 2003 and 2004, and were caught and euthanased for necropsy in 2005. Stoat density was estimated using spatially explicit capture?recapture (SECR). The proportion of stoats marked in one year but recaptured in the next was used as a measure of ?observed survival?. Prey remains were identified from scats collected during 2003 and 2004 and stomachs from stoats killed in 2005. Stoat density was similar in both habitats over the two years, about one stoat per square kilometre. Observed survival from 2003?2004 was also similar, but survival from 2004?2005 was higher in alpine grassland than in beech forest. In 2003, male stoats were on average heavier in alpine grassland than in beech forest, although average weights were similar in the other years. Diet differed significantly between the two habitats, with stoats in alpine grasslands eating mainly ground weta (a large invertebrate) (72%) and hares (23%), while stoats in beech forest ate mainly birds (31%) and mice (19%). Collectively these results suggest that alpine grasslands are not a poor quality habitat for stoats. Traditionally it has been thought that stoats cannot survive on invertebrate prey alone. This research demonstrates that stoats relying largely on invertebrate prey can occur at similar densities and with equivalent survival to stoats relying on vertebrate prey.     

Radiation-induced DNA strand breaks and their repair in the developing rat brain.

Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated. The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min. Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions. The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite. The amount of DNA strand breaks induced per Gray (1--8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen. The DNA strand breaks in the rat brain induced by 4 Gray 60Co gamma irradiation were repaired 30 min after irradiation in all age groups studied.     

Predicting the incidence of mohua predation from the seedfall,mouse, and predator fluctuations in beech forests

Abstract

Predator control will be required to save many mohua (Mohoua ochrocephala) populations from extinction. However, control may be required only in years when stoat (Mustela erminea) densities are high. To manage local stoat populations effectively, a reliable predictor of high risk years is required. We examined whether different levels of beech seedfall and mouse capture rates were related to the levels of mohua predation recorded in the Hawdon Valley, Arthur's Pass National Park, and the Eglinton Valley, Fiordland National Park, between 1989 and 1994. During this period there was only one full beech mast year in each study area during autumn. The full mast seedfall in Hawdon Valley was predominantly of mountain beech (Nothofagus solandri var. cliffortioides) and red beech (N. fused), and in Eglinton Valley it was predominantly silver beech (TV. menziesii). During the following summer, mouse and stoat densities, and the predation rate of adult mohua, all increased considerably. There was very little predation on adult mohua in the summers following poor seedfalls when mouse and predator densities remained low. In 1993, a partial mast did not trigger a mouse or stoat irruption.

We conclude that counts of beech seedfall and indices of mouse density are potential predictors of an impending irruption of key predators. Winter mouse density appeared to be the most reliable indicator, because neither stoats nor mice respond to seedfall alone. A combination of these indicators could be used as a basis for management decisions on whether to undertake stoat control to protect mohua populations in the future. However, more information is required on the seedfall thresholds that may trigger sufficient increases in mouse and stoat numbers and, consequently, bird predation.     

Seed hairs of poplar trees as natural airborne pollen trap for allergenic pollen grains

The present article deals with the efficacy of seed hairs of poplar trees (Populus spp.) as a potent natural airborne pollen trap. Different species of Populus are commonly found planted along the streets in the cities of North China. The seed hairs and pericarp of poplar trees were collected from the trees and on the ground in Beijing Botanical Garden of Chinese Academy of Sciences and around Miyun Reservoir during May 2005 for pollen analysis. Different pollen spectra are recorded from different samples and are characterised by dominant occurrence of pollen grains of arboreal and anemophilous plants. In addition, pollen grains of non‐arboreal plants including grasses are also found trapped. Among the 46 trapped pollen grains, 26 are known to be allergenic. This study suggests that poplar seed hairs possibly make people feel uncomfortable due to the presence of allergenic pollen trapped in the hairs.