abstract: Various entities have been designated keystone resources, but few tests have been attempted and we are unaware of any experimental manipulations of purported keystone resources. Mistletoes (Loranthaceae) provide structural and nutritional resources within canopies, and their pervasive influence on diversity led to their designation as keystone resources. We quantified the effect of mistletoe on diversity with a woodland-scale experiment, comparing bird diversities before and after all mistletoe plants were removed from 17 treatment sites, with those of 11 control sites and 12 sites in which mistletoe was naturally absent. Three years after mistletoe removal, treatment woodlands lost, on average, 20.9 per cent of their total species richness, 26.5 per cent of woodland-dependent bird species and 34.8 per cent of their woodland-dependent residents, compared with moderate increases in control sites and no significant changes in mistletoe-free sites. Treatment sites lost greater proportions of birds recorded nesting in mistletoe, but changes in species recorded feeding on mistletoe did not differ from control sites. Having confirmed the status of mistletoe as a keystone resource, we suggest that nutrient enrichment via litter-fall is the main mechanism promoting species richness, driving small-scale heterogeneity in productivity and food availability for woodland animals. This explanation applies to other parasitic plants with high turnover of enriched leaves, and the community-scale influence of these plants is most apparent in low productivity systems.
author: David M. Watson; Matthew Herring\*Author for correspondence (<firstname.lastname@example.com>).
date: 2012-09-22
institute: Institute for Land, Water and Society, Charles Sturt University, PO Box 789, Albury, New South Wales 2640, Australia
references:
title: Mistletoe as a keystone resource: an experimental test

# Introduction

The idea of an ecological keystone was first transferred from an individual species to a generalized resource derived from multiple species by Leighton & Leighton \[1\] (see also Terborgh \[2\]) in their work on palm seeds, figs and nectar as food for birds and mammals in a Bornean forest. Since then, lichens, saguaro cacti, mineral licks, water, honeydew, mistletoes, salmon, acorns and various fruiting trees *inter alia* have been proposed to represent keystone resources, either within a particular region or generally \[3–5\]. With one notable exception \[6\], there have been no manipulations to quantify the direct and indirect influence of purported keystone resources. With most resources, removal would be logistically difficult, if not impossible, and various procedural difficulties confound the design or interpretation of supplementation experiments. Although our understanding of keystone species has been informed by numerous experiments (natural and controlled; removal and addition), the lack of manipulative tests represents a major impediment to advancing our understanding of how purported keystone resources influence diversity.

Engaged in a network of interdependencies with host plants, seed dispersers, pollinators and natural enemies, mistletoes are a group of highly interactive plants that have been proposed to represent keystone resources in forested ecosystems worldwide \[7\]. In addition to many regular and occasional consumers of their enriched foliage, abundant fruit and nectar resources offered when little else is available \[8–10\], mistletoe clumps are popular nest and roost sites \[11–13\] and numerous studies have reported positive relationships between mistletoe occurrence and species richness \[10,14\]. Mistletoe occurrence and density also affect several ecosystem processes: their abundant enriched litter has pronounced positive effects on nutrient dynamics and understorey composition \[15,16\] and changes to infected hosts increase colonization and functional diversity of mycorrhizal communities \[17,18\], increasing canopy complexity and changing fire behaviour and severity \[19\], thereby altering successional dynamics at the stand-scale \[20\].

Having called for removal experiments to test the keystone status of mistletoes, a series of predictions were outlined \[7\], detailing the short- and medium-term effects of mistletoe removal on community composition and structure. If mistletoe represents a keystone resource, areas where mistletoe has been removed would be expected to have:

1.  lower abundances of mistletoe-obligate frugivores and folivores, with local populations declining towards local extinction;

2.  lower abundances of regular mistletoe foragers (folivores, frugivores and nectarivores);

3.  lower abundances of species that nest in mistletoe clumps and hollows;

4.  lower richness of vertebrates generally; and

5.  increased sensitivity to drought and other extreme events, supporting fewer residents and displaying increased seasonal and inter-annual variability in species richness, compared with control plots with typical numbers of mistletoe plants (after Watson \[7\]).

These predictions were provisionally tested by comparing two adjacent eucalypt woodlands, from one of which all mistletoe plants had been removed from one woodland five years prior to study \[6\]. Marked differences in avian species richness and incidence were detected, consistent with the predicted effects of mistletoe on resource availability, offering preliminary support for the keystone resource hypothesis. This study had an effective sample size of one and no pre-treatment data were collected, so the recorded effects may include pre-existing habitat differences and may not be representative of the influence of mistletoe more generally.

Here, we report on a large-scale experiment evaluating the influence of mistletoe on community-level diversity and designed to test these five predictions. Pre-treatment bird diversities (estimated at the woodland scale) were compared explicitly with diversities three years post-treatment, contrasting changes in woodlands where all mistletoes were removed with those in control woodlands where mistletoe numbers were unmodified. As the first experimental test of a purported keystone resource, to our knowledge this 6-year study yielded a refined understanding of the direct and indirect effects of mistletoe on diversity and community composition. More broadly, this study contributes to a growing understanding of the role of parasitic plants as facilitators, affecting occurrence patterns of other plants and dependent animals through altered nutrient dynamics.

# Methods

## Study area and site selection

This study was conducted in the upper Billabong Creek catchment in southeastern New South Wales, Australia \[21\] (for a map, see electronic supplementary material, figure S1), located at the transition between xeric plains to the west and mesic highlands to the east \[22\]. Elevation ranges between 220 to 889 m a.s.l., and average annual rainfall increases from approximately 550 mm in the west to 900 mm in the east of the catchment. The study period (2003–2008) coincided with a prolonged drought affecting southeastern Australia, with annual rainfall totals for the nearby Albury weather station being approximately half of the long-term annual average of 770 mm.

Rather than individual trees or stands, entire woodland remnants were used as study units, isolated 80–100 years ago as surrounding habitats were cleared for agriculture. Sites qualified for selection if: canopy cover was more than 5 per cent; fragment area was 1.5–25 ha (larger woodlands were not considered because complete mistletoe removal was deemed prohibitively difficult); vegetation was classed as either Dry Foothill Forest or Grassy Box Woodland; the woodland had 'hard' edges *sensu* \[23\] and was at least 500 m from the nearest study site. The ground layer of all woodlands was dominated by grasses (both native perennials, and native and exotic annuals) with occasional shrubs (*Acacia* spp., *Exocarpus* spp.) in low-to-very-low densities. Mistletoe plants occurred at medium-to-low densities in these woodlands—primarily *Amyema miquelii* but with occasional *Muellerina eucalyptoides*, *Am. pendula* and *Am. miraculosa* (Loranthaceae). Except for the latter species (principally epiparasitic on *Am. miquelii* in this part of its range), these species are primarily eucalypt parasites, the plants forming dense pendulous clumps at the edge of the canopy \[24\].

## Bird surveys

To estimate bird species richness in the 40 woodlands, inventories were compiled from eight patch-scale surveys that were conducted each season in 2003/2004 prior to treatment and again in 2007/2008, three years after treatment. Surveys were conducted using the standardized search \[25,26\] with sample duration set at 20 min, using a quantitative, results-based stopping rule to determine the number of samples per survey. The stopping rule applied—stop sampling once observed richness of woodland-dependent species exceeds 80 per cent of predicted richness using the Chao 2 estimator \[27\]—yielded surveys of between three and six samples (i.e. 60–120 min of continuous sampling), with 260 of the 320 surveys tripping the stopping rule after three samples. Sampling involved walking throughout the woodland remnant and identifying all birds seen or heard within the woodland, including species flying beneath the canopy. Sampling was conducted only during favourable weather conditions, avoiding periods of heavy rain, strong wind or intense heat.

In addition to yielding richness estimates of uniform completeness, this approach generated incidence measures for each species in each woodland remnant, expressed as the proportion of samples in which it was detected \[25\]. Two incidence totals were calculated for each site in both years: summed incidence of all species that regularly feed on mistletoe nectar and/or fruit using the list compiled by Reid \[28\], and summed incidence of all species that nest in mistletoe clumps using \[12\]. In addition to total richness and richness of woodland-dependent species (after Watson \[6\] and references therein), a third richness measure was used for analysis—resident richness: those woodland-dependent species recorded in at least two seasons for the given year (excluding transients that were detected only in a single season). Analysis was restricted to changes in richness and incidence—interactions with patch and landscape-scale factors and comparisons of the individual responses of particular species and functional groups will be explored in subsequent contributions.

## Experimental design

The woodland remnants selected for study varied considerably in management history, area and degree of isolation from other remnant vegetation, potentially confounding the effects of mistletoe on diversity patterns. To guard against this, we ensured treatment and control groups were comparable, assigned treatment (mistletoe removal) using a blind stratified random approach and compared the effects of treatment in terms of proportional rather than absolute change. To assign treatment, woodland area was plotted against percentage tree cover, yielding a scatter plot of 40 points distinguishing larger and more densely wooded remnants from smaller, more open woodlands. Points closest to one another in graphical space were paired and treatment assigned by coin toss, yielding two groups of 20 woodland fragments with similar ranges of tree density and patch area (electronic supplementary material, table S1). A series of tests confirmed that these two groups exhibited comparable mean values for woodland area, percentage of tree cover in the surrounding 1 km radius buffer (an index of habitat openness), area of tree cover in the surrounding 1 km radius buffer (a measure of land-use intensity), mistletoe abundance and species richness of woodland-dependent birds prior to treatment. Some woodlands did not contain mistletoe, so, rather than a simple binary comparison, there were three groups: control woodlands with mistletoe (*n* = 11), treatment patches with mistletoe plants removed (*n* = 17) and woodlands from which mistletoe was naturally absent (*n* = 12); hereafter, they are referred as 'control', 'treatment' and 'mistletoe-free' woodlands, respectively.

## Mistletoe removal

Mistletoe plants growing within treatment woodlands were systematically removed over a five-month period (winter and spring of 2004) by teams of volunteers using pole-mounted loppers and pruning saws. Unlike some groups of mistletoe (e.g. *Arceuthobium* spp., Viscaceae) that cause systemic infections throughout the host, these loranthaceous mistletoes can be removed by cutting proximal to the haustorium, the lack of cortical strands in these species precluding re-sprouting \[24\]. Hydraulic boom-lifts towed by four-wheel drive vehicles were used to access plants up to 18 m above the ground, the telescoping boom arm allowing access to most parts of the eucalypt canopies (electronic supplementary material, figure S2). Mistletoe plants and associated sections of host branches were left where they fell: beneath their former hosts. Sham removals were conducted in the control sites, driving within the woodland and removing branches from infected and non-infected trees, but leaving all mistletoe plants intact. Tree and branch selection was haphazard, determined partly by ease of access and partly to avoid any mistletoe clumps within the canopy. Comprehensive surveys conducted six months and three years after initial treatment revealed that not all mistletoe plants were removed, but less than four per cent of the original number of mistletoe plants remained or recolonized, which were subsequently removed in follow-up treatment in the summer of 2006/2007.

## Data manipulation and analysis

Responses to mistletoe removal were measured in terms of net change between pre- and post-treatment inventories. In some cases, this was simply the datum for the 2003/2004 subtracted from the equivalent datum for 2007/2008. To make these values more meaningful, many of these differences were expressed as proportions of the original (pre-treatment) value. The only mistletoe-obligate frugivore detected was the mistletoebird *Dicaeum hirundinaceum*, so prediction 1 was tested by comparing mistletoebird incidence before and after treatment. Predictions 2 and 3 were tested using summed incidence of mistletoe-feeding and mistletoe-nesting species (respectively), comparing control and treatment woodlands in terms of the difference in the net change of summed incidence. Tests were conducted on the actual values, while plots correspond to proportions of total incidence (pre-treatment) to place the differences in biological context. Prediction 4 was tested by comparing the change in species richness in control versus treatment woodlands, expressed as a proportion of the pre-treatment value (i.e. proportional change). For prediction 5, the study period coincided with a prolonged drought, with many species responding by leaving the study area completely or only occupying some habitats seasonally. The change in numbers of residents between the two years was expressed as a proportion of the initial value (i.e. the proportion decreasing as the number of transient species increased). Except for prediction 1, which entailed a paired *t-*test applied to the 17 treatment woodlands, all treatment effects were tested using Mann–Whitney–Wilcoxon tests (one-tailed) comparing mean values for the 17 treatment sites (with mistletoe plants removed) with the 11 control sites (with mistletoe left intact), with exact probabilities of less than 0.05 deemed significant (using spss v. 17.0). Tests were conducted using all species ('total species') and the subset of species considered to depend on woodland as their primary habitat ('woodland species'; excluding most raptors, aerial foragers, open country and exotic species, after Watson \[6\]). Non-parametric tests were used, as sample sizes were uneven and the data were generally heteroscedastic, and one-tailed tests were appropriate as all comparisons were testing explicitly directional predictions \[7\]. In addition to comparing treatment and control sites, values for mistletoe-free woodlands are presented to contextualize the changes associated with experimental treatment and represent background variation between sites and years not associated with experimental manipulation.

# Results

Mistletoe exhibited a highly irregular distribution across the 40 sites, being entirely absent from 12 sites and occurring in remaining sites at densities ranging from less than one per hectare to almost 200 per hectare. A total of 5493 mistletoe plants were removed: the great majority (5169; 94%) during the initial removal phase in mid-2004 and an additional 324 plants in follow-up removals in summer 2006/2007, most of which were immature plants.

Of the 75 woodland-dependent bird species recorded in the two years combined, 12 species were recorded only during pre-treatment surveys and two species were recorded only during post-treatment surveys—i.e. there was a net loss of 10 species, or 14 per cent of the pre-treatment diversity of woodland-dependent species across all 40 sites. Turnover in the other 41 species was more symmetrical, with seven species recorded only pre-treatment (2003/2004) and eight species recorded only post-treatment (2007/2008)—i.e. a net gain of one species (or 3% of the pre-treatment total). Additional detail on bird richness and mistletoe occurrence is summarized in the electronic supplementary material.

## Prediction 1: specialist frugivores

A significant difference in mistletoebird *D. hirundinaceum* incidence was detected in the 17 treatment sites (*t* = 1.825, *p* = 0.043, one-tailed), reflecting declines in the treatment woodlands following mistletoe removal—incidence decreased in two sites and the species became locally extinct in a further four. By contrast, mistletoebird incidence increased in five of the 11 control woodlands, decreased in three others and they were not recorded in the remaining three.

## Prediction 2: mistletoe foragers

No significant differences were detected between treatment and control sites in terms of net change in summed incidence of all 31 mistletoe foragers (*p* = 0.132, one-tailed), or for the 24 woodland-dependent mistletoe foragers (*p* = 0.373, one-tailed) for ranked data (figure 1*a*) \[28,12\]. There were decreases across all sites, with treatment sites losing an average of 8.7 per cent of summed incidence for total species and 10.6 per cent of woodland-dependent species (figure 1*a*), and control sites losing 2.9 per cent and 8 per cent, respectively. Note that these recorded losses were less than changes in summed incidence across all species in the treatment woodlands between years (decreases of 15.4% and 24.7%, respectively).

## Prediction 3: mistletoe nesters

Clear differences in summed incidence of the 67 mistletoe nesters were detected between treatment and control woodlands (*p* = 0.016, one-tailed), equating to a mean loss of approximately 9.4 per cent of total incidence (s.e. = 3.4) in treatment woodlands, becoming more marked if just the 48 woodland-dependent species were considered (*p* = 0.008, one-tailed; mean loss of 17.6% of total incidence, s.e. = 4.26). Although significantly greater than decreases in control woodlands (figure 1*b*), these changes were less than change in incidence across all species in the treatment sites (decreases of 15.4% for all species; 24.7% for woodland-dependents).

## Prediction 4: species richness

Total species richness decreased markedly in the treatment woodlands after experimental removal of mistletoe—a significant difference compared with control woodlands (*p* = 0.004), reflecting mean losses of 20.9 per cent of their original richness (s.e. = 4.04). This pattern was more marked for those species dependent on woodlands as their principal habitat (*p* = 0.002, one-tailed; compared with control sites), equating to mean losses of 26.5 per cent (s.e. = 4.58) of their original woodland-dependent species richness (figure 2*a*). This change compares with a mean decrease in total richness of 10.3% (s.e. = 6.04) in mistletoe-free woodlands and an increase of 4.7 per cent (s.e. = 8.38) in the control woodlands (for total species richness); with woodland-dependent species richness decreasing by an average of 6.5 per cent in mistletoe-free woodlands (s.e. = 9.63), while control woodlands gained an average of 10.2 per cent of their pre-treatment richness (s.e. = 12.68).

## Prediction 5: sensitivity to drought

For total species, clear-cut differences between control and treatment were detected (*p* = 0.008): treatment sites lost a mean proportion of 29.5 per cent (s.e. = 9.25) of their initial residents, while control sites exhibited an increase in the proportion of residents of 1.73 per cent (s.e. = 7.73). Differences in woodland-dependent species were greater (*p* = 0.002); control woodlands increased their proportion of residents (14.5%; s.e. = 12.34) while the proportion of residents in treatment woodlands decreased by 34.8 per cent (s.e. = 10.3), transient species becoming more dominant (figure 2*b*).

# Discussion

The influence of mistletoe on diversity was evaluated directly with a woodland-scale removal experiment, and the hypothesized status of these hemiparasites as keystone resources was strongly supported. Removing mistletoe plants from entire woodlands resulted in average losses of more than a quarter of the woodland-dependent bird species, with the number of resident species decreasing by more than a third. Over the same period, control woodlands with variable mistletoe densities exhibited moderate increases in woodland-dependent species richness, while no marked changes to richness or incidence were detected in naturally mistletoe-free sites. Rather than affecting just those species that feed on mistletoe or nest preferentially in mistletoe clumps, these changes were apparent across the community, being more pronounced for woodland-dependent species and residents. The magnitude of these changes within three years of mistletoe removal provides clear evidence that mistletoes directly modify resource availability in woodlands and forests, consistent with previous descriptive work relating mistletoe density to community-level diversity in a range of other ecosystems \[10,14\].

Two aspects of this study probably underestimate the measured influence of mistletoe, so reported effect sizes should be regarded as conservative. The study period coincided with the final years of a prolonged drought affecting southern Australia, so the pre-treatment inventories yielded lower species richness estimates than anticipated. Rather than supporting comparable bird diversities in the two years, the 11 control woodlands gained an average of 10.2 per cent woodland-dependent species because numbers of drought-sensitive species rebounded in 2007. So the mean-recorded loss of 26.5 per cent woodland-dependent species in the treatment remnants may actually reflect net losses closer to 36.7 per cent species richness once the confounding effects of regional climatic conditions are removed, comparable to the 34.8 per cent mean-recorded loss of woodland-dependent residents. Second, mistletoe densities within the study sites were low compared with other habitats in the region, the hemiparasites being completely absent from 12 of the 40 woodlands. The same mistletoe species are two to five times more abundant in adjacent riparian and floodplain woodlands and nearby box-ironbark forests, probably having more pronounced effects on diversity and community composition.

## Mistletoe as a direct nutritional resource

One of the main elements of the evidence base assembled to justify designation of mistletoe as a keystone resource \[7\] was dietary information for terrestrial vertebrates, with the popularity of mistletoe fruit, nectar and foliage deemed instrumental in explaining observed relationships with species richness. Although mistletoe specialists declined following mistletoe removal (in support of prediction 1), regular mistletoe foragers did not show significant reductions in occurrence or abundance following mistletoe removal (figure 2*a*). While it is likely that more of the bird species recorded in this study occasionally consume mistletoe fruit or nectar, Reid's \[28\] list corresponds closely with our own observations of nectarivory and frugivory, both within the study region and across southern Australia, and this negative result is considered robust.

We suggest that regular consumers of mistletoe fruit and nectar represent only a small component of the suite of species influenced by mistletoe occurrence in this system, and that direct nutritional effects comprise a relatively minor component of the community-level influence of mistletoe in general. Eucalypt forests are remarkable for their lack of fleshy fruited plants, and frugivorous species are only a minor component of the associated avifauna. Thus, changes in mistletoe diversity and abundance probably have more pronounced effects on frugivore occurrence in other systems, explaining temporal and spatial variation in frugivores numbers \[8,29\]. In terms of nectar, although 13 of the 75 recorded woodland-dependent species feed on mistletoe flowers and an additional seven visit mistletoe flowers opportunistically \[28\], none depends on mistletoes as their primary food source. Rather, eucalypts represent the dominant nectar-bearing plant in this region—the canopy-dominant sclerophyllous trees exhibiting mass flowering events that are highly variable through space and time, driving large-scale movements as nectarivores track peak flowering across large distances \[30\]. Hence, the direct influence of mistletoe fruit and nectar availability on community-scale diversity may not be as consistently great as predicted, and the influence of more abundant fruit and nectar-bearing plants in the canopy or understorey is probably far greater in most systems \[31,32\].

## Structural attributes

In addition to nutritional resources, mistletoes provide dense evergreen structures within forest canopies that are widely used by birds and mammals for nesting and roosting, and these structural attributes formed the second main justification for designating mistletoes as keystone resources. Accordingly, those species that use mistletoe for nest sites were expected to decrease in abundance following mistletoe removal (prediction 3). Although treatment sites displayed significant reductions in these species in terms of summed incidence, recorded changes were less than those exhibited across all woodland-dependent species—i.e. mistletoe-nesting species were no more likely to undergo declines following mistletoe removal than any other woodland species. This conclusion is supported by the relatively small number of nests found during mistletoe removal—fewer than one per cent of the plants removed had nests associated with them. So, although widely used as nesting and roosting sites, and preferentially selected by some species, we found no evidence that mistletoes were limiting as a nesting substrate, nor that woodlands with mistletoe can support greater abundances of mistletoe-nesting species.

Previous research has evaluated mistletoe nesting in more depth, and suggested that microclimatic factors may be important in explaining the widespread practice. Compared with nests elsewhere in the canopy, nests within mistletoe foliage experienced reduced fluctuation in both temperature and humidity, with the ambient climate ameliorated by the semi-succulent foliage of the hemiparasite \[13\]. The lack of signal detected here may reflect the temperate climate of the study region, and mistletoe is likely to have more pronounced effects in areas with more extreme climates. Indeed, research on wildlife use of dwarf mistletoe brooms in Arizona found evidence that mistletoes may be a limiting resource \[33\]. The influence of mistletoe on canopy structure may be better considered in terms of reproductive success rather than in terms of species richness, increasing the seasonal extent and geographical breadth within which successful fledging and recruitment can be achieved.

## Facilitation via litter-fall and nutrient concentration

Rather than direct nutritional supplementation or increased structural complexity in infected host canopies, we propose that the marked influence of mistletoe on diversity is mediated primarily via the abundant enriched litter shed by these hemiparasites, increasing productivity and promoting coexistence through bottom-up trophic dynamics of woodland and forest food webs. Previous work on the contribution of mistletoes to litter-fall and nutrient dynamics within the study area estimated that individual *Amyema miquelii* plants contributed an average of 544 g of leaf litter annually, with 0.81 ± 0.08 g of mistletoe litter produced per gram of mistletoe leaf biomass in the canopy \[15\]. The variation in mistletoe occurrence noted at the patch scale was even more pronounced within infected patches: mistletoe plants aggregated in discrete 'infection centres' and concentrated around the perimeter of remnants. Hence, rather than incremental changes in litter-fall across woodlands, mistletoe occurrence increases heterogeneity in litter-fall, effectively doubling or tripling total litter-fall in heavily infected stands. The effects of these qualitative changes in litter-fall are magnified by the enriched status of mistletoe litter compared with host litter, leading to marked increases in nutrient inputs \[16\] and pronounced spatial heterogeneity in nutrient returns.

In addition to affecting overall productivity and composition of understorey plant communities \[15\], these changes to the litter layer directly affect the litter-dwelling arthropods that form the principal food source for many woodland-dependent fauna. Previous work on the habitat preferences, dietary composition and foraging ecology of birds in eucalypt woodlands has consistently identified litter depth as a critical factor \[34 and references therein\], with deeper and more extensive litter beds considered to contain higher abundances of preferred prey \[35\]. Thus, we suggest that quantitative changes to litter-fall associated with mistletoe occurrence, plus qualitative changes in the chemical composition and associated rate of decomposition of resultant litter, result in fundamental changes to litter-dwelling arthropod communities, increasing and prolonging availability of prey for insectivores.

Rather than supplanting direct nutritional and structural factors, the facilitation explanation incorporates these pathways, with nutrient contributions by foraging and nesting animals adding to the inputs provided directly by mistletoes. Unlike litter-fall that represents reallocation of nutrients from the host to the litter-layer beneath the host, these animal-derived nutrients are drawn from a much larger pool, extending beyond the infected host and stand \[36\]. Similar mechanisms have been postulated to explain high concentrations of animal-derived elements near hollow trees, rock outcrops and copses of trees surrounded by grassland, summarized by Watson \[37\]. Rather than simply increased productivity, we suggest it is the heterogeneity of nutrient availability driven by the highly aggregated distribution of mistletoes that underpins their pervasive effects on diversity, boosting species richness by promoting species coexistence.

## Effects of parasitic plants on diversity

Rather than being unique to this system, nutrient enrichment is an attribute of parasitic plants generally, and numerous studies (of other mistletoes and various root parasites) have reported findings consistent with facilitation via enriched litter-fall. These findings involve a range of vegetation types, from alpine tundra \[38\] and arctic shrubland \[39\] to coniferous forests \[40\]. In addition to direct effects of hemiparasite litter on decomposition and nutrient availability, these studies demonstrate various indirect effects of parasitic plants, including the boosting of species richness by preferentially parasitizing competitively dominant species \[41,42\]. Parallel research on dwarf mistletoes (Arceuthobium: Viscaceae) has detected increased colonization of infected *Pinus edulis* by ectomycorrhizae \[17\] and increased functional diversity of fungal communities beneath infected *Pinus contorta* \[18\], suggesting that the fundamental shifts in soil microbial communities detected by Bardgett *et al*. \[43\] in grasslands experimentally infected with *Rhinanthus minor* may apply to parasitic plants more generally.

## Acknowledgments

All sampling was carried out under Scientific Research Permit S10921 issued by the Department of Environment and Climate Change (Parks and Wildlife Division) and the authority of Charles Sturt University Animal Care and Ethics Committee approval 05/054.

This research was supported by the Australian Research Council (DP034259), the Winnifred Violet Scott Trust and Charles Sturt University. We are indebted to the 18 landowners in the upper Billabong Creek catchment for granting access to their properties, supporting our research and sharing their insights about woodland dynamics. This work would not have been possible without our dedicated band of assistants: chief mistletoe removalist David Webb, Hugh McGregor, Scott Hartvigsen and teams from Australian Trust for Conservation Volunteers and Greencorps. We are grateful to two anonymous referees for their considered input and constructive comments.

# References


abstract: Writing a research manuscript is an intimidating process for many novice writers in the sciences. One of the stumbling blocks is the beginning of the process and creating the first draft. This paper presents guidelines on how to initiate the writing process and draft each section of a research manuscript. The paper discusses seven rules that allow the writer to prepare a well-structured and comprehensive manuscript for a publication submission. In addition, the author lists different strategies for successful revision. Each of those strategies represents a step in the revision process and should help the writer improve the quality of the manuscript. The paper could be considered a brief manual for publication.
author: Elena D. KallestinovaTo whom all correspondence should be addressed: Elena D. Kallestinova, Graduate Writing Center, Yale Graduate School of Arts and Sciences, Yale University, New Haven, CT; E-mail: <firstname.lastname@example.com>.
date: 2011-09
references:
title: How to Write Your First Research Paper

It is late at night. You have been struggling with your project for a year. You generated an enormous amount of interesting data. Your pipette feels like an extension of your hand, and running western blots has become part of your daily routine, similar to brushing your teeth. Your colleagues think you are ready to write a paper, and your lab mates tease you about your "slow" writing progress. Yet days pass, and you cannot force yourself to sit down to write. You have not written anything for a while (lab reports do not count), and you feel you have lost your stamina. How does the writing process work? How can you fit your writing into a daily schedule packed with experiments? What section should you start with? What distinguishes a good research paper from a bad one? How should you revise your paper? These and many other questions buzz in your head and keep you stressed. As a result, you procrastinate. In this paper, I will discuss the issues related to the writing process of a scientific paper. Specifically, I will focus on the best approaches to start a scientific paper, tips for writing each section, and the best revision strategies.

# 1. Schedule your writing time in Outlook

Whether you have written 100 papers or you are struggling with your first, starting the process is the most difficult part unless you have a rigid writing schedule. Writing is hard. It is a very difficult process of intense concentration and brain work. As stated in Hayes' framework for the study of writing: "It is a generative activity requiring motivation, and it is an intellectual activity requiring cognitive processes and memory" \[1\]. In his book *How to Write a Lot: A Practical Guide to Productive Academic Writing*, Paul Silvia says that for some, "it's easier to embalm the dead than to write an article about it" \[2\]. Just as with any type of hard work, you will not succeed unless you practice regularly. If you have not done physical exercises for a year, only regular workouts can get you into good shape again. The same kind of regular exercises, or I call them "writing sessions," are required to be a productive author. Choose from 1- to 2-hour blocks in your daily work schedule and consider them as non-cancellable appointments. When figuring out which blocks of time will be set for writing, you should select the time that works best for this type of work. For many people, mornings are more productive. One Yale University graduate student spent a semester writing from 8 a.m. to 9 a.m. when her lab was empty. At the end of the semester, she was amazed at how much she accomplished without even interrupting her regular lab hours. In addition, doing the hardest task first thing in the morning contributes to the sense of accomplishment during the rest of the day. This positive feeling spills over into our work and life and has a very positive effect on our overall attitude.

## Rule 1: Create regular time blocks for writing as appointments in your calendar and keep these appointments.

# 2. Start with an outline

Now that you have scheduled time, you need to decide how to start writing. The best strategy is to start with an outline. This will not be an outline that you are used to, with Roman numerals for each section and neat parallel listing of topic sentences and supporting points. This outline will be similar to a template for your paper. Initially, the outline will form a structure for your paper; it will help generate ideas and formulate hypotheses. Following the advice of George M. Whitesides, ". . . start with a blank piece of paper, and write down, in any order, all important ideas that occur to you concerning the paper" \[3\]. Use Table 1 as a starting point for your outline. Include your visuals (figures, tables, formulas, equations, and algorithms), and list your findings. These will constitute the first level of your outline, which will eventually expand as you elaborate.

The next stage is to add context and structure. Here you will group all your ideas into sections: Introduction, Methods, Results, and Discussion/Conclusion (Table 2). This step will help add coherence to your work and sift your ideas.

Now that you have expanded your outline, you are ready for the next step: discussing the ideas for your paper with your colleagues and mentor. Many universities have a writing center where graduate students can schedule individual consultations and receive assistance with their paper drafts. Getting feedback during early stages of your draft can save a lot of time. Talking through ideas allows people to conceptualize and organize thoughts to find their direction without wasting time on unnecessary writing. Outlining is the most effective way of communicating your ideas and exchanging thoughts. Moreover, it is also the best stage to decide to which publication you will submit the paper. Many people come up with three choices and discuss them with their mentors and colleagues. Having a list of journal priorities can help you quickly resubmit your paper if your paper is rejected.

## Rule 2: Create a detailed outline and discuss it with your mentor and peers.

# 3. Continue with drafts

After you get enough feedback and decide on the journal you will submit to, the process of real writing begins. Copy your outline into a separate file and expand on each of the points, adding data and elaborating on the details. When you create the first draft, do not succumb to the temptation of editing. Do not slow down to choose a better word or better phrase; do not halt to improve your sentence structure. Pour your ideas into the paper and leave revision and editing for later. As Paul Silvia explains, "Revising while you generate text is like drinking decaffeinated coffee in the early morning: noble idea, wrong time" \[2\].

Many students complain that they are not productive writers because they experience writer's block. Staring at an empty screen is frustrating, but your screen is not really empty: You have a template of your article, and all you need to do is fill in the blanks. Indeed, writer's block is a logical fallacy for a scientist ― it is just an excuse to procrastinate. When scientists start writing a research paper, they already have their files with data, lab notes with materials and experimental designs, some visuals, and tables with results. All they need to do is scrutinize these pieces and put them together into a comprehensive paper.

# 3.1. Starting with Materials and Methods

If you still struggle with starting a paper, then write the Materials and Methods section first. Since you have all your notes, it should not be problematic for you to describe the experimental design and procedures. Your most important goal in this section is to be as explicit as possible by providing enough detail and references. In the end, the purpose of this section is to allow other researchers to evaluate and repeat your work. So do not run into the same problems as the writers of the sentences in (1):

> 1a. Bacteria were pelleted by centrifugation.
>
> 1b. To isolate T cells, lymph nodes were collected.

As you can see, crucial pieces of information are missing: the speed of centrifuging your bacteria, the time, and the temperature in (1a); the source of lymph nodes for collection in (b). The sentences can be improved when information is added, as in (2a) and (2b), respectfully:

> 2a. Bacteria were pelleted by centrifugation at 3000g for 15 min at 25°C.
>
> 2b. To isolate T cells, mediastinal and mesenteric lymph nodes from Balb/c mice were collected at day 7 after immunization with ovabumin.

If your method has previously been published and is well-known, then you should provide only the literature reference, as in (3a). If your method is unpublished, then you need to make sure you provide all essential details, as in (3b).

> 3a. Stem cells were isolated, according to Johnson \[23\].
>
> 3b. Stem cells were isolated using biotinylated carbon nanotubes coated with anti-CD34 antibodies.

Furthermore, cohesion and fluency are crucial in this section. One of the malpractices resulting in disrupted fluency is switching from passive voice to active and vice versa within the same paragraph, as shown in (4). This switching misleads and distracts the reader.

> 4\. Behavioral computer-based experiments of Study 1 were programmed by using E-Prime. We took ratings of enjoyment, mood, and arousal as the patients listened to preferred pleasant music and unpreferred music by using Visual Analogue Scales (SI Methods). The preferred and unpreferred status of the music was operationalized along a continuum of pleasantness \[4\].

The problem with (4) is that the reader has to switch from the point of view of the experiment (passive voice) to the point of view of the experimenter (active voice). This switch causes confusion about the performer of the actions in the first and the third sentences. To improve the coherence and fluency of the paragraph above, you should be consistent in choosing the point of view: first person "we" or passive voice \[5\]. Let's consider two revised examples in (5).

> 5a. We programmed behavioral computer-based experiments of Study 1 by using E-Prime. We took ratings of enjoyment, mood, and arousal by using Visual Analogue Scales (SI Methods) as the patients listened to preferred pleasant music and unpreferred music. We operationalized the preferred and unpreferred status of the music along a continuum of pleasantness.
>
> 5b. Behavioral computer-based experiments of Study 1 were programmed by using E-Prime. Ratings of enjoyment, mood, and arousal were taken as the patients listened to preferred pleasant music and unpreferred music by using Visual Analogue Scales (SI Methods). The preferred and unpreferred status of the music was operationalized along a continuum of pleasantness.

If you choose the point of view of the experimenter, then you may end up with repetitive "we did this" sentences. For many readers, paragraphs with sentences all beginning with "we" may also sound disruptive. So if you choose active sentences, you need to keep the number of "we" subjects to a minimum and vary the beginnings of the sentences \[6\].

Interestingly, recent studies have reported that the Materials and Methods section is the only section in research papers in which passive voice predominantly overrides the use of the active voice \[5,7,8,9\]. For example, Martínez shows a significant drop in active voice use in the Methods sections based on the corpus of 1 million words of experimental full text research articles in the biological sciences \[7\]. According to the author, the active voice patterned with "we" is used only as a tool to reveal personal responsibility for the procedural decisions in designing and performing experimental work. This means that while all other sections of the research paper use active voice, passive voice is still the most predominant in Materials and Methods sections.

Writing Materials and Methods sections is a meticulous and time consuming task requiring extreme accuracy and clarity. This is why when you complete your draft, you should ask for as much feedback from your colleagues as possible. Numerous readers of this section will help you identify the missing links and improve the technical style of this section.

## Rule 3: Be meticulous and accurate in describing the Materials and Methods. Do not change the point of view within one paragraph.

# 3.2. Writing Results Section

For many authors, writing the Results section is more intimidating than writing the Materials and Methods section . If people are interested in your paper, they are interested in your results. That is why it is vital to use all your writing skills to objectively present your key findings in an orderly and logical sequence using illustrative materials and text.

Your Results should be organized into different segments or subsections where each one presents the purpose of the experiment, your experimental approach, data including text and visuals (tables, figures, schematics, algorithms, and formulas), and data commentary. For most journals, your data commentary will include a meaningful summary of the data presented in the visuals and an explanation of the most significant findings. This data presentation should not repeat the data in the visuals, but rather highlight the most important points. In the "standard" research paper approach, your Results section should exclude data interpretation, leaving it for the Discussion section. However, interpretations gradually and secretly creep into research papers: "Reducing the data, generalizing from the data, and highlighting scientific cases are all highly interpretive processes. It should be clear by now that we do not let the data speak for themselves in research reports; in summarizing our results, we interpret them for the reader" \[10\]. As a result, many journals including the *Journal of Experimental Medicine* and the *Journal of Clinical Investigation* use joint Results/Discussion sections, where results are immediately followed by interpretations.

Another important aspect of this section is to create a comprehensive and supported argument or a well-researched case. This means that you should be selective in presenting data and choose only those experimental details that are essential for your reader to understand your findings. You might have conducted an experiment 20 times and collected numerous records, but this does not mean that you should present all those records in your paper. You need to distinguish your results from your data and be able to discard excessive experimental details that could distract and confuse the reader. However, creating a picture or an argument should not be confused with data manipulation or falsification, which is a willful distortion of data and results. If some of your findings contradict your ideas, you have to mention this and find a plausible explanation for the contradiction.

In addition, your text should not include irrelevant and peripheral information, including overview sentences, as in (6).

> 6\. To show our results, we first introduce all components of experimental system and then describe the outcome of infections.

Indeed, wordiness convolutes your sentences and conceals your ideas from readers. One common source of wordiness is unnecessary intensifiers. Adverbial intensifiers such as "clearly," "essential," "quite," "basically," "rather," "fairly," "really," and "virtually" not only add verbosity to your sentences, but also lower your results' credibility. They appeal to the reader's emotions but lower objectivity, as in the common examples in (7):

> 7a. Table 3 clearly shows that …
>
> 7b. It is obvious from figure 4 that …

Another source of wordiness is nominalizations, i.e., nouns derived from verbs and adjectives paired with weak verbs including "be," "have," "do," "make," "cause," "provide," and "get" and constructions such as "there is/are."

> 8a. We tested the hypothesis that there is a disruption of membrane asymmetry.
>
> 8b. In this paper we provide an argument that stem cells repopulate injured organs.

In the sentences above, the abstract nominalizations "disruption" and "argument" do not contribute to the clarity of the sentences, but rather clutter them with useless vocabulary that distracts from the meaning. To improve your sentences, avoid unnecessary nominalizations and change passive verbs and constructions into active and direct sentences.

> 9a. We tested the hypothesis that the membrane asymmetry is disrupted.
>
> 9b. In this paper we argue that stem cells repopulate injured organs.

Your Results section is the heart of your paper, representing a year or more of your daily research. So lead your reader through your story by writing direct, concise, and clear sentences.

## Rule 4: Be clear, concise, and objective in describing your Results.

# 3.3. now it is time for your Introduction

Now that you are almost half through drafting your research paper, it is time to update your outline. While describing your Methods and Results, many of you diverged from the original outline and re-focused your ideas. So before you move on to create your Introduction, re-read your Methods and Results sections and change your outline to match your research focus. The updated outline will help you review the general picture of your paper, the topic, the main idea, and the purpose, which are all important for writing your introduction.

The best way to structure your introduction is to follow the three-move approach shown in Table 3.

The moves and information from your outline can help to create your Introduction efficiently and without missing steps. These moves are traffic signs that lead the reader through the road of your ideas. Each move plays an important role in your paper and should be presented with deep thought and care. When you establish the territory, you place your research in context and highlight the importance of your research topic. By finding the niche, you outline the scope of your research problem and enter the scientific dialogue. The final move, "occupying the niche," is where you explain your research in a nutshell and highlight your paper's significance. The three moves allow your readers to evaluate their interest in your paper and play a significant role in the paper review process, determining your paper reviewers.

Some academic writers assume that the reader "should follow the paper" to find the answers about your methodology and your findings. As a result, many novice writers do not present their experimental approach and the major findings, wrongly believing that the reader will locate the necessary information later while reading the subsequent sections \[5\]. However, this "suspense" approach is not appropriate for scientific writing. To interest the reader, scientific authors should be direct and straightforward and present informative one-sentence summaries of the results and the approach.

Another problem is that writers understate the significance of the Introduction. Many new researchers mistakenly think that all their readers understand the importance of the research question and omit this part. However, this assumption is faulty because the purpose of the section is not to evaluate the importance of the research question in general. The goal is to present the importance of your research contribution and your findings. Therefore, you should be explicit and clear in describing the benefit of the paper.

The Introduction should not be long. Indeed, for most journals, this is a very brief section of about 250 to 600 words, but it might be the most difficult section due to its importance.

## Rule 5: Interest your reader in the Introduction section by signalling all its elements and stating the novelty of the work.

# 3.4. Discussion of the results

For many scientists, writing a Discussion section is as scary as starting a paper. Most of the fear comes from the variation in the section. Since every paper has its unique results and findings, the Discussion section differs in its length, shape, and structure. However, some general principles of writing this section still exist. Knowing these rules, or "moves," can change your attitude about this section and help you create a comprehensive interpretation of your results.

The purpose of the Discussion section is to place your findings in the research context and "to explain the meaning of the findings and why they are important, without appearing arrogant, condescending, or patronizing" \[11\]. The structure of the first two moves is almost a mirror reflection of the one in the Introduction. In the Introduction, you zoom in from general to specific and from the background to your research question; in the Discussion section, you zoom out from the summary of your findings to the research context, as shown in Table 4.

The biggest challenge for many writers is the opening paragraph of the Discussion section. Following the moves in Table 1, the best choice is to start with the study's major findings that provide the answer to the research question in your Introduction. The most common starting phrases are "Our findings demonstrate . . .," or "In this study, we have shown that . . .," or "Our results suggest . . ." In some cases, however, reminding the reader about the research question or even providing a brief context and then stating the answer would make more sense. This is important in those cases where the researcher presents a number of findings or where more than one research question was presented. Your summary of the study's major findings should be followed by your presentation of the importance of these findings. One of the most frequent mistakes of the novice writer is to assume the importance of his findings. Even if the importance is clear to you, it may not be obvious to your reader. Digesting the findings and their importance to your reader is as crucial as stating your research question.

Another useful strategy is to be proactive in the first move by predicting and commenting on the alternative explanations of the results. Addressing potential doubts will save you from painful comments about the wrong interpretation of your results and will present you as a thoughtful and considerate researcher. Moreover, the evaluation of the alternative explanations might help you create a logical step to the next move of the discussion section: the research context.

The goal of the research context move is to show how your findings fit into the general picture of the current research and how you contribute to the existing knowledge on the topic. This is also the place to discuss any discrepancies and unexpected findings that may otherwise distort the general picture of your paper. Moreover, outlining the scope of your research by showing the limitations, weaknesses, and assumptions is essential and adds modesty to your image as a scientist. However, make sure that you do not end your paper with the problems that override your findings. Try to suggest feasible explanations and solutions.

If your submission does not require a separate Conclusion section, then adding another paragraph about the "take-home message" is a must. This should be a general statement reiterating your answer to the research question and adding its scientific implications, practical application, or advice.

Just as in all other sections of your paper, the clear and precise language and concise comprehensive sentences are vital. However, in addition to that, your writing should convey confidence and authority. The easiest way to illustrate your tone is to use the active voice and the first person pronouns. Accompanied by clarity and succinctness, these tools are the best to convince your readers of your point and your ideas.

## Rule 6: Present the principles, relationships, and generalizations in a concise and convincing tone.

# 4. Choosing the best working revision strategies

Now that you have created the first draft, your attitude toward your writing should have improved. Moreover, you should feel more confident that you are able to accomplish your project and submit your paper within a reasonable timeframe. You also have worked out your writing schedule and followed it precisely. Do not stop ― you are only at the midpoint from your destination. Just as the best and most precious diamond is no more than an unattractive stone recognized only by trained professionals, your ideas and your results may go unnoticed if they are not polished and brushed. Despite your attempts to present your ideas in a logical and comprehensive way, first drafts are frequently a mess. Use the advice of Paul Silvia: "Your first drafts should sound like they were hastily translated from Icelandic by a non-native speaker" \[2\]. The degree of your success will depend on how you are able to revise and edit your paper.

The revision can be done at the macrostructure and the microstructure levels \[13\]. The macrostructure revision includes the revision of the organization, content, and flow. The microstructure level includes individual words, sentence structure, grammar, punctuation, and spelling.

The best way to approach the macrostructure revision is through the outline of the ideas in your paper. The last time you updated your outline was before writing the Introduction and the Discussion. Now that you have the beginning and the conclusion, you can take a bird's-eye view of the whole paper. The outline will allow you to see if the ideas of your paper are coherently structured, if your results are logically built, and if the discussion is linked to the research question in the Introduction. You will be able to see if something is missing in any of the sections or if you need to rearrange your information to make your point.

The next step is to revise each of the sections starting from the beginning. Ideally, you should limit yourself to working on small sections of about five pages at a time \[14\]. After these short sections, your eyes get used to your writing and your efficiency in spotting problems decreases. When reading for content and organization, you should control your urge to edit your paper for sentence structure and grammar and focus only on the flow of your ideas and logic of your presentation. Experienced researchers tend to make almost three times the number of changes to meaning than novice writers \[15,16\]. Revising is a difficult but useful skill, which academic writers obtain with years of practice.

In contrast to the macrostructure revision, which is a linear process and is done usually through a detailed outline and by sections, microstructure revision is a non-linear process. While the goal of the macrostructure revision is to analyze your ideas and their logic, the goal of the microstructure editing is to scrutinize the form of your ideas: your paragraphs, sentences, and words. You do not need and are not recommended to follow the order of the paper to perform this type of revision. You can start from the end or from different sections. You can even revise by reading sentences backward, sentence by sentence and word by word.

One of the microstructure revision strategies frequently used during writing center consultations is to read the paper aloud \[17\]. You may read aloud to yourself, to a tape recorder, or to a colleague or friend. When reading and listening to your paper, you are more likely to notice the places where the fluency is disrupted and where you stumble because of a very long and unclear sentence or a wrong connector.

Another revision strategy is to learn your common errors and to do a targeted search for them \[13\]. All writers have a set of problems that are specific to them, i.e., their writing idiosyncrasies. Remembering these problems is as important for an academic writer as remembering your friends' birthdays. Create a list of these idiosyncrasies and run a search for these problems using your word processor. If your problem is demonstrative pronouns without summary words, then search for "this/these/those" in your text and check if you used the word appropriately. If you have a problem with intensifiers, then search for "really" or "very" and delete them from the text. The same targeted search can be done to eliminate wordiness. Searching for "there is/are" or "and" can help you avoid the bulky sentences.

The final strategy is working with a hard copy and a pencil. Print a double space copy with font size 14 and re-read your paper in several steps. Try reading your paper line by line with the rest of the text covered with a piece of paper. When you are forced to see only a small portion of your writing, you are less likely to get distracted and are more likely to notice problems. You will end up spotting more unnecessary words, wrongly worded phrases, or unparallel constructions.

After you apply all these strategies, you are ready to share your writing with your friends, colleagues, and a writing advisor in the writing center. Get as much feedback as you can, especially from non-specialists in your field. Patiently listen to what others say to you ― you are not expected to defend your writing or explain what you wanted to say. You may decide what you want to change and how after you receive the feedback and sort it in your head. Even though some researchers make the revision an endless process and can hardly stop after a 14th draft; having from five to seven drafts of your paper is a norm in the sciences. If you can't stop revising, then set a deadline for yourself and stick to it. Deadlines always help.

## Rule 7: Revise your paper at the macrostructure and the microstructure level using different strategies and techniques. Receive feedback and revise again.

# 5. It is time to submit

It is late at night again. You are still in your lab finishing revisions and getting ready to submit your paper. You feel happy ― you have finally finished a year's worth of work. You will submit your paper tomorrow, and regardless of the outcome, you know that you can do it. If one journal does not take your paper, you will take advantage of the feedback and resubmit again. You will have a publication, and this is the most important achievement.

What is even more important is that you have your scheduled writing time that you are going to keep for your future publications, for reading and taking notes, for writing grants, and for reviewing papers. You are not going to lose stamina this time, and you will become a productive scientist. But for now, let's celebrate the end of the paper.

###### Outline — Level 1

|                                            |
|--------------------------------------------|
| 1\. What is the topic of my paper?         |
|                                            |
| 2\. Why is this topic important?           |
|                                            |
| 3\. How could I formulate my hypothesis?   |
|                                            |
| 4\. What are my results (include visuals)? |
|                                            |
| 5\. What is my major finding?              |

###### Outline — Level 2

|                                                          |
|----------------------------------------------------------|
| **Introduction**                                         |
|                                                          |
| 1\. Why is your research important?                      |
|                                                          |
| 2\. What is known about the topic?                       |
|                                                          |
| 3\. What are your hypotheses?                            |
|                                                          |
| 4\. What are your objectives?                            |
|                                                          |
| **Materials and Methods**                                |
|                                                          |
| 1\. What materials did you use?                          |
|                                                          |
| 2\. Who were the subjects of your study?                 |
|                                                          |
| 3\. What was the design of your research?                |
|                                                          |
| 4\. What procedure did you follow?                       |
|                                                          |
| **Results**                                              |
|                                                          |
| 1\. What are your most significant results?              |
|                                                          |
| 2\. What are your supporting results?                    |
|                                                          |
| **Discussion and Conclusions**                           |
|                                                          |
| 1\. What are the studies major findings?                 |
| 2\. What is the significance/implication of the results? |

###### Moves in Research Paper Introductions

|  |
|----|
| **Move 1. Establish a research territory** |
|  |
| a\. Show that the general research area is important, central, interesting, and problematic in some way; |
|  |
| **Move 2. Find a niche** |
|  |
| a\. Indicate a gap in the previous research, or extend previous knowledge in some way. |
|  |
| **Move 3. Occupy the niche** |
|  |
| a\. Outline purposes or state the nature of the present research; |
|  |
| b\. List research questions or hypotheses; |
|  |
| c\. Announce principle findings; |
|  |
| d\. State the value of the present research; |
|  |
| e\. Indicate the structure of the research paper. |

Adapted from Swales and Feak \[11\].

###### Moves in Research Paper Discussions.

|  |
|----|
| **Move 1. The study's major findings** |
|  |
| a\. State the study's major findings. |
|  |
| b\. Explain the meaning and importance of your finding. |
|  |
| c\. Consider alternative explanations of the findings. |
|  |
| **Move 2. Research Context** |
|  |
| a\. Compare and contrast your findings with those of other published results. |
|  |
| b\. Explain any discrepancies and unexpected findings. |
|  |
| c\. State the limitations, weaknesses, and assumptions of your study. |
|  |
| **Move 3. Closing the paper** |
|  |
| a\. Summarize the answers to the research questions. |
| b\. Indicate the importance of the work by stating applications, recommendations, and implications. |

Adapted from Swales and Feak and Hess \[11,12\].


abstract: A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.
author: Joan Politz; Roel van Driel; Markus Sauer; Ana Pombo
date: 2003
institute: 1University of Massachusetts Medical School, Department of Biochemistry and Molecular Pharmacology, 377 Plantation Street, Worcester, MA 01605, USA; 2Swammerdam Institute for Life Sciences, University of Amsterdam, PO Box 94062,1090 GB Amsterdam, The Netherlands; 3Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany; 4MRC - Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK; Corrspondence: Ana Pombo. E-mail: firstname.lastname@example.com
title: From linear genome sequence to three-dimensional organization of the cell nucleus

In the modern era of biochemical science, the isolation, identification and characterization of biologically relevant molecules have become routine. The challenge remains, however, to discover how these molecules are assembled within cells to form the dynamic network of molecular machines that supports living organisms. Because molecular machines are only several tens of nanometers in size (for example, a ribosome is about 30 nm wide), new optical methods with both increased sensitivity and higher resolution are required to define the spatial relationships and dynamic interplay between interacting biomolecules. Furthermore, techniques that provide dynamic, three-dimensional images of individual molecules *in vivo* will be necessary if we are to study the detailed organization of 'nanomachines' within the cell. The 'Advances in nanostructural genomics II' meeting at The Jackson Laboratory (Bar Harbor, ME, USA) brought together workers from the diverse areas of research that address these challenges. The topics covered ranged from genome sequence analysis and gene expression and organization of the cell nucleus to high-resolution imaging methods, nanolithography and mathematical modeling.

One of us (R.v.D.) opened the meeting by summarizing the current state of research on gene expression, chromatin structure and nuclear organization, and highlighting the areas that would benefit from a multidisciplinary approach. These include studies of chromatin structure *in vivo*, the function of subnuclear structures, the relationship between gene clustering and functional nuclear domains, and how the mobility of nuclear components relates to their activities. A newly assembled magneto-microscope was also described; it was developed by a consortium of groups (called 'Cellular Submarine') who came together after the 'Genomics meets nanoscience' meeting last year (Politz and Pombo, *Genome Biol* 2002,**3:**reports4007.1-4007.3). It can guide micro-magnetic beads that have factors or enzymes bound through the nucleus, like an 'intracellular submarine', to measure and/or interfere with nuclear function at the nanoscale level.

# Higher-order chromatin organization

The availability of the nearly complete human and mouse genome sequences has increased the need for robust tools for sequence annotation. Carol Bult (The Jackson Laboratory, Bar Harbor, USA) and Ross Hardison (Pennsylvania State University, University Park, USA) described the development of annotation methods, such as those available at the websites of the Penn State Bioinformatics Group <http://bio.cse.psu.edu/> and of the UCSC Genome Bioinformatics Group <http://www.genome.ucsc.edu>; they pointed out that currently no single search program can consistently identify all coding sequences within a region, and that the detection of functional non-coding regions is even more difficult. As Tim O'Brien (also at The Jackson Laboratory) explained, more detailed information can currently be obtained from specific genomic regions when computational and genetic methods are combined. As a model for functional genomic analyses he used the complex, 40 Mb large piebald region encoding coat color on mouse chromosome 14. Five coding genes per megabase were found in this region, together with a large number of non-coding sequences, some of which are essential. Although further studies are needed to determine which of these are associated with biological function, initial studies have shown that experimentally defined auto-regulatory elements and putative transcription-factor-binding sites are contained within these conserved blocks of sequence. Giacomo Cavalli (National Centre for Scientific Research (CNRS), Institute of Human Genetics, Montpellier, France) and Doug Engel (University of Michigan, Ann Arbor, USA) showed that long-range interactions *in trans* between different sequences, such as locus control regions and Polycomb repressor elements, affected gene activity in *Drosophila* and mouse. Cavalli demonstrated that these sequences physically interact within the three-dimensional space of the *Drosophila* nucleus, and Engel echoed the idea that our definition of a gene must change, if we wish it to include all sequences that control a particular gene's transcription.

Long-range interactions were further discussed by Job Dekker (Harvard University, Cambridge, USA) who presented a novel high-throughput method, chromosome conformation capture or '3Cs', used to study the conformations of chromosomal sequences *in vivo*. In brief, sequences that interact *in vivo* are fixed within whole nuclei, chromatin is digested, and crosslinked fragments are ligated at low concentration to promote intra-molecular ligation. Ligated DNA fragments were quantified by PCR using oligonucleotides spanning a genomic area of interest. Dekker used this method to determine the frequencies of physical interactions between pairs of genomic loci on chromosome III of *Saccharomyces cerevisiae* and found the chromosome to be quite flexible with a circular configuration of only 300 nm in diameter; the most active domains occurred in more extended conformations. Siegfried Janz (National Cancer Institute, National Institutes of Health (NIH), Bethesda, USA) and Michael Difilippantonio (Center for Cancer Research, NIH, Bethesda, USA) discussed interchromosomal interactions that result in oncogenic chromosome translocations and gene amplifications. They used the murine t(12;15) translocation as a model for human Burkitt's lymphoma, in which interchromosomal DNA rearrangements lead to aberrant expression of the oncogene c-*myc* and to increased propensity for chromosome segregation errors and tumorigenesis. Difilippantonio reported that the joining of non-homologous broken chromosome ends may trigger the aberrant DNA rearrangement, as non-homologous end joining is involved in repairing programmed double-strand breaks in DNA that occur naturally during lymphocyte development, for example, when V, D and J gene segments in the immunoglobulin loci are rearranged to create the variable antigen receptor region (VDJ recombination), or when the gene segments that define different antibody classes are recombined (isotype switching). Janz also studied the fine structure of the t(12;15) translocation but used mice that have intact non-homologous end-joining pathways by using c-*myc* knock-ins, which he argued can mimic translocations between the c-*myc* and the immunoglobulin H (IgH) gene more accurately than previous mouse models. He found that DNA rearrangements were most frequently caused by aberrant isotype switching, and rarely by VDJ recombination.

Understanding the complexities of chromatin and its interactions with chromatin-modifying factors were highlighted in a thought-provoking talk by Chris Woodcock (University of Massachusetts, Amherst, USA). If we are to fully understand the nucleus we will need to describe its biochemical and biophysical parameters. We have begun to understand the substrates (chromatin being perhaps the main one), the catalysts (enzymes that act on the substrates), and the kinetics of interaction, but it remains a daunting challenge to understand the complexity of the kinetic parameters of a long polymer with local modifications that is acted upon only locally by specific catalysts. Woodcock highlighted that a wide range of techniques will be necessary (with examples from his own work on the yeast SWI/SNF nucleosome remodeling complex) if we are to define chromatin structure at four levels - at the primary, nucleosome level; the secondary level of the 30 nm DNA fibre; the tertiary folding beyond the 30 nm fibre; and the quaternary structure of the whole chromosome within the nucleus. Astrid Visser (Scripps Research Institute, La Jolla, USA) discussed the need for combined efforts to sequence the histone modification 'code', which posits that different combinations of histone modifications signal different chromatin states, to give insights into higher-order chromatin structure. This could include capture of specific chromatin conformations, followed by linearization of the chromatin and reading of the histone code. Chromatin configurations could also be captured either after DNA stretching and visualization by light or electron microscopy, or possibly with technology that might allow sequencing the histone code in the future.

# Improvements in nanotechnology

Although research on nanostructures is one of the most rapidly growing fields in modern science, the lack of higher-resolution substrate patterning methods has prevented direct examination of these structures at the level of resolution needed to study macromolecular activity within the cell. Joachim Spatz (University of Heidelberg, Germany) demonstrated that non-conventional methods of patterning surfaces onto glass coverslips allow the preparation of 5 nm gold dots that are separated by distances of 30-100 nm - a scale at which molecular interactions within and between cells can be monitored.

Rapid advances in microfluidics and microinstrumentation are also taking place. Gabriel Lopez (University of New Mexico, Albuquerque, USA) described the development of high-throughput microfluidic devices that allow cells that are cultured in 96-well plates to be washed completely between medium changes, by using flow, rather than diffusion, as the mixing impetus. Scott Collins (University of Maine, Orono, USA) discussed refined instrumentation for single-cell experiments, such as microperfusion chambers that can be used to mimic arteries in arteriosclerosis studies and optical traps that can be used to manipulate individual cells. He also discussed a nano-sequencing technique in which DNA is electrophoresed through a nanopore and the sequence determined by measuring the current modulation caused by different bases as they move through the pore. Currently, however, only purines and pyrimidines can be differentiated. Watt Webb (Cornell University, Ithaca, USA) demonstrated that the activity of single polymerase molecules that are immobilized inside a zero-mode waveguide (subwavelength holes in a metal film) can be followed by timing the fluorescence bursts that are emitted from labeled nucleotide analogs that enter the active site of the enzyme and are incorporated into the growing DNA strand. This technique is also being developed into a new DNA sequencing method.

Magnetic tweezers are a promising tool in cell biology; they allow objects inside cells to be moved while the forces exerted by biological systems in the pico- to nano-Newton range are measured. In contrast to optical tweezers, they do not use laser light that is potentially harmful, particularly when objects are below the micrometer size range. Gunther Reiss (University of Bielefeld, Germany) described a magnetic trap with which the two-dimensional and three-dimensional position of magnetic nanoparticles can be controlled with high sensitivity and precision.

Horst Weller (University of Hamburg, Germany) and Avi Ulmann (Brooklyn Polytechnic Institute, New York, USA) discussed how nanoparticles can be used for biolabeling and new surface chemistry techniques, for example, biotin-conjugated particles can be used to visualize streptavidin that is used in immunolabeling procedures or that is attached to a glass coverslip. Advances in the chemical synthesis of highly luminescent semiconductor nanocrystals, such as gold nanocrystals coated with silica, often referred to as quantum dots, allow the development of alternative fluorescent or luminescent labels that can be used instead of standard organic dyes, such as fluorescein isothiocyanate (FITC).

# Improvements in microscopy

Conventional fluorescence microscopy can be used to determine the positions of objects only when they are separated by distances greater than a few hundred nanometers - a restriction imposed by the diffraction limit of light. In contrast, fluorescence resonance energy transfer (FRET) techniques can be used to measure distances within one to ten nanometers. To visualize objects whose distance lies between these limits, alternative techniques are being developed. One of us (M.S.) discussed the use of dyes, such as Cy5 and JF9, that have different fluorescence life times and of time-resolved fluorescence spectroscopy to distinguish and measure distances between two dye molecules that are separated by less than 30 nm. With fluorescence correlation spectroscopy (FCS) several properties of fluorescent labels can be measured and the mobility of single labeled molecules in live cells can be monitored. Samuel T. Hess (National Institute of Child Health and Human Development, NIH, Bethesda, USA) and Thomas Weidemann (German Cancer Research Center, Heidelberg, Germany) used this technique to study the properties of proteins tagged with green fluorescence protein (GFP) and their intracellular mobility in living cells. Hess discussed different properties of fluorescent proteins and their application as biological markers. For example, enhanced GFP (eGFP) flickers between dark and bright states at low pH, such that it may be used as a pH sensor. Weidemann used FCS to determine the concentration of nucleosomes labeled with H2B-eYFP in the HeLa cell nucleus and in mitotic chromosomes.

Other new microscopy techniques, such as laser holography and X-ray microscopy, are being used to study the three-dimensional organization of the nucleus. Jürgen Kreuzer (Dalhousie University, Halifax, Canada) showed that laser holography allows the acquisition of detailed images of living organisms without prior labeling. The image not only looks 'real' but appears to float in space and to move when the viewer moves, as with a real object. So far, Kreuzer has imaged heart cells and diatoms, and has tracked swimming algae using this technique. Günther Schmahl (University of Göttingen, Germany) and Gerd Schneider (Lawrence Berkeley National Laboratory, Berkeley, USA) discussed the application of X-ray microscopy to studying the nuclear distribution of the *Drosophila* dosage compensation protein MSL-1, which elevates the transcriptional activity of the X chromosome in males. Structural preservation is excellent under cryogenic conditions and the natural contrast between macromolecules and water in the cell allows the visualization of fine structural details. The distribution of immuno-gold stained MSL-1 protein was resolved to a level of about 60 nm; for example, the protein was observed to localize close to, but not within, the nucleolus. Michael Grunze (University of Heidelberg, Germany) discussed developments in the use of newly available synchrotron radiation sources of high brilliance to yield X rays and other types of radiation for electron- and soft X-ray-based microscopy of biological systems. With such a source of radiation, separate imaging of different chemical elements may become possible and we may see the development of novel types of microscopic imaging with spatial resolution at the atomic level.

# Nuclear biology

Kevin Sullivan (Scripps Research Institute) summarized the current understanding of the DNA sequence and protein content of kinetochores, which form at the centromeres of mitotic chromosomes. Although centromeres are rich in satellite DNA, the sequence itself does not specify the function of centromeres, which is instead inherited epigenetically. CENP-A, a histone-like protein that binds alphoid satellite DNA, is made during G2 phase of the cell cycle and incorporated into the inner kinetochore plate until mitosis. Using stretched DNA fibers, it was found that centromeres contain a mixture of CENP-A and histone 4. The Aurora B kinase, which phosphorylates CENP-A and H3, assembles at pericentric heterochromatin in a CENP-A-dependent process; Aurora moves to other chromosomal regions only after phosphorylation is complete at this site. This mechanism ensures an epigenetic mark of centromeres. Jim McNally (National Cancer Institute) addressed the relationship between transcriptional activation and chromatin decondensation. Several genetically engineered systems that consist of clusters of tandemly repeated genes display such relationship, but this behavior could be specific to artificially amplified gene clusters. McNally therefore looked for naturally occurring clusters of genes that are transcriptionally coactivated, for example, clusters that are controlled by extracellular signals. Such a cluster was found on mouse chromosome 22 in a microarray gene-expression analysis, and McNally showed that this naturally occurring cluster shows a decondensation after transcriptional activation that is very similar to that of the artificial gene clusters.

Active RNA polymerase I transcription sites are spatially organized into superstructures within the nucleolus. Making use of quantitative high-resolution light and electron microscopy, another of us (A.P.) reported that polymerases II and III also seem to form clusters of approximately eight enzymes in the nucleoplasm; such 'factories' are about 50 nm in diameter and each is surrounded by a cloud of nascent RNA. Antibodies against phosphorylated (active) polymerase II provided evidence that the factories contain different forms of polymerase.

The main cytological features of a eukaryotic nucleus are the nucleolus, chromosome territories, and the ill-defined interchromatin space. Thoru Pederson (University of Massachusetts Medical School, Worcester, USA) reviewed our current understanding of the interchromatin space, focusing mainly on the diffusion properties of RNA and other particles. He argued that interchromatin domains are active regions that are defined by and simultaneously interact with genes from different chromosomes. One of us (J.P.) showed that both the RNA and proteins that make up the signal recognition particle (SRP), which is required for targeting membrane and secretory proteins to membranes, are present within nucleoli, suggesting that the assembly of SRP might occur in this compartment. SRP RNA was found concentrated in subnucleolar domains that do not overlap with sites of ribosome assembly, suggesting that the nucleolus contains previously unrecognized domains devoted to novel functions.

Christoph Cremer (University of Heidelberg) first described that small, gene-dense chromosomal territories are located near the center of the nucleus, whereas small, gene-poor chromosomal territories are located near the periphery. He also found that larger chromosomes tend to be located closer to the periphery of the nucleus. He explained that current modeling techniques cannot account for all of these observations. In an overview of new and forthcoming approaches in the nuclear structure field, he then discussed new microscopic technologies that are being developed in his group that will allow structural analysis to a precision level of 10 nm. Cremer stressed that beyond the development of new microscopy methods, quantitative modeling of biological structures and processes must be refined and developed, and better methods for labeling biomolecules must be discovered if we are to visualize specific proteins and RNA and DNA sequences *in vivo* with minimal disturbance of biological structures. So far, we have only had a glimpse at cellular structures. But with the rapid development of methods that allow us to study nanostructures, we may soon get a clearer picture.


abstract: In their recent study, Sohn and colleagues identify multiple plasma proteins in the synovial fluid of patients with osteoarthritis (OA) and demonstrate the capacity of several of the proteins to activate macrophages via the innate immune receptor Toll-like receptor-4 (TLR-4). The authors speculate that the plasma proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells within the synovium. These results demonstrate a novel mechanism by which synovial inflammation can be initiated in patients with OA and how this process may contribute to the pathogenesis of OA joint pathology.
author: Steven R Goldring; Carla R Scanzello
date: 2012
institute: 1Hospital for Special Surgery, Weill Cornell Medical Center, New York, NY, USA; 2Section of Rheumatology, Departments of Immunology/Microbiology and Biochemistry, Rush University Medical Center, Chicago, IL 60612, USA
references:
title: Plasma proteins take their toll on the joint in osteoarthritis

See related research by Sohn *et al.*, <http://arthritis-research.com/content/14/1/R7M>

The view of osteoarthritis (OA) as strictly a non-inflammatory joint disease has evolved as imaging and histopathological studies have demonstrated that synovial inflammation is present in many patients, and importantly, is associated with increased pain, poorer joint function, and accelerated deterioration of joint structures. In the current article by Sohn and colleagues \[1\], the authors advance the understanding of how inflammation may be initiated by demonstrating that certain plasma proteins present in OA synovial fluids (SFs) can activate macrophages via the innate immune receptor Toll-like receptor (TLR)-4.

The authors began with a proteomic analysis, utilizing two-dimensional PAGE followed by liquid chromatography-mass spectrometry, to survey proteins in SF from five OA patients. The analysis identified 108 proteins, many of which previously were shown to be increased in OA SF compared to normal, including proteins related to inflammation and immunity and products of extracellular matrix degradation and remodeling \[2\]. Of these, 64 proteins were novel and included histonerelated and macrophage-associated proteins, proinflammatory receptors and proteins related to the proinflammatory transcription factor nuclear factor kappa B. As the approach was not optimal for detection of small molecules such as cytokines, they went on to perform multiplex bead-based immunoassays to measure cytokines and chemokines involved in inflammatory processes and compared the levels in normal sera, OA and rheumatoid arthritis sera and OA and rheumatoid arthritis SF. In general, the levels in OA sera samples were only slightly higher than the normal levels and those in the rheumatoid arthritis sera were markedly higher. Some novel mediators were identified by this comparison, including T cell attracting chemokine (CTAK) and IL-9, which were significantly higher in the OA compared to the normal sera, supporting a role for T cells in OA inflammation. Among the OA samples, multiple proinflammatory cytokines and chemokines were much higher in the SF compared to the sera, consistent with overproduction of these cytokines in the joints.

Interestingly, they found that over one- third of proteins identified in the proteomic screen of OA SF were plasma proteins. This is not unexpected, since SF is partially a plasma filtrate, but many of the plasma proteins were increased in OA compared to normal SF. Together with evidence that another plasma protein, fibrinogen, can incite inflammation by stimulating the innate receptor TLR-4, they tested whether other plasma proteins identified in the screen could do the same and demonstrated that three (α1m, α2m, and Gc-globulin) induced tumor necrosis factor-α, IL- 1β, IL-6, and vascular endothelial growth factor release from cultured macrophages. Very recently, other members of this group demonstrated that the complement membrane-attack complex plays a role in the development of inflammation and joint degeneration in an animal model of OA \[3\]. As plasma can be a source of many proteins, including complement components, the authors speculated that these proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells such as macrophages and synovial fibroblasts.

The authors demonstrated that the effect of these plasma proteins is mediated through TLR-4, as macrophages from mice lacking TLR-4 were unresponsive to the protein treatments. These results are significant for several reasons. First, the authors present data linking SF proteins present in OA joints, including plasma proteins, to activation of inflammatory processes. Second, TLR activation has previously been demonstrated to impact cartilage breakdown \[4,5\]. As macrophages are important mediators of synovial inflammation in OA, the present results extend the potential impact of this pathway to synovitis in OA. Finally, reports of TLR activation by non-microbial products often have been called into question, as low-level endotoxin contamination can often lead to false positive results. This issue was addressed using two different approaches. First, they demonstrated that treatment of the cultures with polymyxin B, which binds lipopolysaccharide by charge interactions and prevents it from associating with TLR-4, reverses the lipopolysaccharide response, but does not significantly affect the response to α1m, α2m, and Gc-globulin. Secondly, they demonstrated that proteinase K treatment fully or partially reversed the macrophage response to the three plasma proteins, while not affecting lipopolysaccharide.

In conclusion, the article by Sohn and colleagues contributes to our understanding of how TLRs may be activated locally in the setting of OA, by pointing to potential stimuli beyond matrix-derived and stress-related molecules derived from joint tissues. The observation that plasma proteins present in the OA joint can stimulate macrophages provides important rationale for further study of this pathway in OA models, but also may implicate this pathway in a variety of other local joint conditions that may produce alterations in synovial diffusion properties.

# Abbreviations

IL: interleukin; OA: osteoarthritis; SF: synovial fluid; TLR: Toll-like receptor.

# Competing interests

The authors declare that they have no competing interests.

## Acknowledgements

CRS is supported by 1K08 AR057859, Mentored Clinician Scientist Career Development Award from the National Institute of Arthritis, Musculoskeletal and Skin Diseases.