Plant parasitic nematode survival and detection to inform biosecurity risk assessment

Plant parasitic nematodes (PPN) are known to survive periods of desiccation, an ability that increases the risk of them surviving unintentional transport between countries. To investigate nematode survival in soil subject to prolonged storage, soil collected from a native forest and an organic orchard was stored separately in cupboards at ambient temperature for 36 months. Subsamples were taken at 0, 3, 6, 12, 13, 24 and 36 months to determine the presence of plant parasitic and total nematodes using a standard misting technique. Pratylenchus was used as a model to determine if PPNs that had been under prolonged storage were able to infect plant hosts at 13, 24 and 36 months. Overall, the total number of nematodes recovered from stored soil declined over time, with differences in species diversity determined by molecular methods, related to soil origin. No PPN were recovered in soil stored beyond 13 months using the three-day misting technique. By comparison, Pratylenchus nematodes, using a baiting method, were found to successfully invade host plant roots (ryegrass and white clover) even after 36 months storage and were observed to produce offspring at 13 months. Baiting was not effective for Pratylenchus found in soil originally collected from the forest but was for orchard soil, a result attributed to the lack of suitable host plants for the Pratylenchus species found in forest soil. This study demonstrated, that in protected environments, nematodes could survive for at least 36 months and were observed to produce offspring at 13 months. Baiting with a host plant was more sensitive in detecting nematodes than using the misting extraction technique, although this approach only works where the host plant is known. Without a priori knowledge of the nematode-plant host association, plant baiting may also produce false negatives. In the context of plant biosecurity and providing an accurate risk assessment in soil contaminants, the development of a generic test for PPN that induces nematodes in a resting stage to emerge and respond to a cue would enhance the probability of detection. However, as assessments at the border are often time limited, a molecular based bioassay that can be used to indicate the presence of multiple species of live PPN species may be a more feasible option for risk assessments. Copyright Lee T. Aalders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. NeoBiota 36: 1–16 (2017) doi: 10.3897/neobiota.36.11418 http://neobiota.pensoft.net DATA PAPER Advancing research on alien species and biological invasions A peer-reviewed open-access journal


Introduction
Trade and tourism are important to the economic wellbeing of the world's global economy, but carry with it the real risk of introducing unwanted organisms that threaten the productive sectors of individual countries or regions because of production losses due to direct yield reduction or cost for pest control (Mack et al. 2000, Pimentel et al. 2000, Work et al. 2005, Hulme 2014). For natural habitats and native biota, invasive species can have both direct and indirect impacts through modification, displacement or eradication costs along with a general loss of biodiversity (Lee and Lee 2015). Impacts can therefore be economic, ecological and social with the impacts and costs prolonged for intractable invasive species. Plant parasitic nematodes (PPN) are estimated to cause billions of dollars (USD) of crop damage worldwide each year, many of which have known or potential phytosanitary importance (Singh et al. 2013).
Amongst PPNs there are three main types of parasitism, ectoparasitic, endoparasitic and semi-endoparasitic (Decraemer and Hunt 2006). For ectoparasites (e.g. Paratylenchus, Criconematidae), the nematodes remain within the soil feeding externally on plant roots. Two types of endoparasites feed within the roots; there are migratory ones such as Pratylenchus which do not form a permanent feeding site and can move in and out of the plant; and sedentary ones such as Globodera and Heterodera which form a permanent feeding site except for the infective second stage juveniles which are mobile within the soil. For semiendoparasites, such as Helicotylenchus, only the anterior portion of the nematode penetrates the root with the posterior portion remaining in the soil (Decraemer and Hunt 2006).
While their minute size and cryptic nature in plants and soil makes discovery more difficult when transported, the ability of many PPN species to survive periods of desiccation (Norton 1978), makes these nematodes a biosecurity issue as it increases the probability of establishment when inadvertently transported from one country to another via trade and tourism routes. PPNs have been detected in soil associated with shipments of imported seeds (Lal and Lal 2006), plant material (Tenente et al. 1996), contaminated footwear (McNeill et al. 2011), used machinery (Hughes et al. 2011, Aalders et al. 2012 and sea containers (Gadgil et al. 2000, McNeill et al. unpublished data).
In an effort to improve predictions on which PPN species will become invasive in a country or region before they arrive, a Pest Screening and Targeting (PeST) framework has been developed to provide a more structured and systematic approach for screening large numbers of species and identifying species likely to become invasive (Singh et al. 2015). PeST integrates heterogeneous information and data on species biogeography, biotic and abiotic factors to first determine a preliminary risk index. While species with better survival adaptations pose greater risks than those without the capability, a paucity of information on survival in transit represents an important knowledge gap when developing a pest risk profile for PPNs (Singh et al. 2015).
In this current study, soil was collected and stored in cupboards to mimic soil contaminants that may be stored in a protected environment (e.g. contaminated footwear, used equipment or camping gear) for a period of time before reuse. The study assessed not only nematode survival but the viability of nematodes recovered from soil that had been stored in a cupboard over a 36 month period. While the research commenced prior to the development of the PeST framework proposed by Singh et al (2015), this work provides a valuable contribution to our understanding of nematode survival over time. The hypothesis tested was that nematodes surviving long term desiccation would be able to subsequently invade plant roots ('baiting'), thus presenting a heightened biosecurity risk.

Collection and processing of soil
Soil was collected in late winter (23 August 2011) from two sites in the Canterbury region of New Zealand; a native forest reserve in Prices Valley, Banks Peninsula (S43.7669, E172.7140) and an organic orchard at Lincoln (S43.6508; E172.4559). At each site, a spade square soil sample (140 mm × 140 mm) was taken to a depth of 5 cm at three randomly chosen points within a 3 m radius of an arbitrarily designated central point. The soil sampled from these three locations at each site were treated separately throughout the experiment. Any vegetation was cut to ground level with scissors and loose litter was removed from the sample point prior to collection. The individual soil samples were mixed separately in a stainless steel tray and transferred to a plastic bag. The spade was cleaned with 70% ethanol between each site and location. Disposable laboratory gloves were worn at all times, and changed between sites. The work presented in this paper is part of a project published in , methods for collecting and processing the soil are the same as described in that paper.
In the laboratory, the soil was sieved (10 mm sieve) and a subsample taken for nematode counts and identification. The remaining soil from each site and sampling location (six individual soil sources) was divided amongst stainless steel steam trays (dimensions c. 400 × 200 × 50 mm (300 mm × 240 mm internal dimensions)), in which twenty × 4 mm drainage holes had been drilled into the base, then allocated to treatments (c. 700 g of soil per tray). The soil was spread roughly evenly onto the tray surface and gently pressed with a stainless steel pan to lightly compact the soil, resulting in a soil depth of approximately 40 mm . Soil from all sites contained plant root material.
The uncovered tray was then placed within a cupboard situated indoors at ambient temperature at Lincoln (S43.6279, E172.4704). The soil in the trays was subsampled at 3, 6, 12, 13, 24 and 36 months. Approximately 75 g soil was collected from each tray using a stainless steel spoon and placed in a 100 ml plastic screw cap container. The spoon was cleaned with 70% ethanol between sampling each tray.
In addition to the above, the two original locations were resampled at 3, 6, 12, 13, 24 and 36 months. This was to monitor the natural population in relation to counts taken from the stored soil to ensure that any decrease in population was due to storage. As per the original sampling strategy, at the three locations within each of the two original sites, soil was collected using 20 × 25 mm diameter × 100 mm deep cores, hand crumbled and mixed.

Extraction methodology
There are a range of accepted nematode extraction techniques (Hooper 1986, Hooper and Evans 1993, Hunt and De Ley 1996. Techniques are classified as either active methods such as the Whitehead and Hemming tray and misting, or passive such as centrifugation and flotation -sieving (McSorley and Walter 1991, Hooper and Evans 1993, Hunt and De Ley 1996. The misting method was chosen because it provided the capability for high throughput of the large number of samples. The limitation was that the method would not have extracted nematode cysts, but was the most efficient and cost effective method available.

Nematode survival and viability
To determine nematode survival over the duration of the study, at each storage time, a 25 g soil subsample was placed in a mistifier funnel for extraction and misted for 30 sec every 5 min over 72 hours at a water temperature of 20 °C. The water from the mister flushes the nematodes through the soil and into a test tube where they are collected. For the original day zero samples 100 g of fresh soil was placed on to extraction trays (Bell and Watson 2001) and extracted over a 72 hr period. At the three month sampling time the two methods of extraction were compared and no significant difference found (data unpublished). For the 36 month bioassay, samples were extracted for an additional 48 hours, in the expectation that prolonged storage could result in poor physiological condition so that more time may be required to extract surviving nematodes. Counts were taken for all nematodes (bacterivores, fungivores, omnivores, predators and plant parasites); PPNs were identified to genera where possible.
The endoparasitic nematode Pratylenchus was the only nematode extracted from soil after 12 months, so at 13, 24 and 36 months, the ability of Pratylenchus to invade plant roots was tested using both white clover (Trifolium repens L.) and ryegrass (Lolium spp.) as bait plants. To determine viability, plastic pots (50 mm × 50 mm × 120 mm), were part filled with 140 g of oven dried sand and topped with 46 g of soil from each ca. 75 g sample. The six original sites were also sampled to check plant host suitability of the sown seed for the nematode species present. This resulted in 24 pots of cupboard soil and 12 pots of fresh soil collected from the original six sites. Each pot was sown with three nil-endophyte ryegrass Lolium multiflorum (cv. Moata for 2012) and L. perenne (cv. Samson for 2013 and 2014) and 6-8 white clover Trifolium repens (cv. Sustain) seeds. The pots were randomised, placed into two forestry crates, maintained in a 20°C controlled environment room with a light: dark photoperiod of 16: 8 hours and watered as required. The forestry crates enabled the pots to be held separately from each other and above the bench to avoid cross contamination.
Twenty four days post-sowing, the ryegrass and white clover seedlings were removed from each pot, gently washed to remove adhering soil and counted before the shoots and roots were separated and weighed. For each pot, ryegrass and white clover roots were stained using aniline blue (Rohan et al. 2006), to determine if Pratylenchus nematodes had infected the seedling roots.

Molecular identification of PPNs
DNA was extracted from single nematode specimens using the prepGEM TM tissue kit (ZyGEM Corporation Ltd, New Zealand) according to manufacturer's instructions.
DNA was amplified in 25 μl reactions using 1x buffer (Thermo Scientific Finnzymes), 0.2mM dNTPs, 0.3 μM of each primer, 0.2 mg /ml BSA and 0.5 units of Phusion Hot Start II Hi-Fi DNA polymerase (Thermo Scientific Finnzymes). Thermo cycling included an initial denaturing at 98 °C for 2 min, then 40 cycles of 98 °C for 10 sec, 57 °C for 30 seconds, and 72 °C for 60 °C, with a final extension step of 72 °C for 5 minutes. The product was purified using the GeneJET PCR Purification Kit (Thermo Scientific™). The fragments were sequenced by Massey Genome Service (Massey University, New Zealand) and cleaned using the computer programme Geneious TM 8.1.5 (Kearse et al. 2012). The sequences were compared to nematode sequences in the BLAST (Basic Local Alignment Search Tool) database (http://www.ncbi.nlm.nih.gov/blast.cgi).
Restriction fragment length polymorphism analysis (RFLPs) of the internal transcribed spacer (ITS) regions of ribosomal DNA was used to try and distinguish between the closely related Heterodera species to identify the Heterodera specimen isolated from the orchard soil in this study. Three reference sequences for each of H. trifolii, H. schachtii and H. betae were imported into Geneious to compare. In silico, the restriction enzyme MspI generated a RFLP profile that showed the

Soil Moisture
To determine soil moisture at the 6 and 12 month bioassay, a separate 20 g sub-sample of soil was taken from each sample and oven dried at 80 °C for 48 hours. The availability of the remaining soil was limited at 13, 24 and 36 months, so soil moisture was determined using the 25 g of soil following processing in the mistifier funnel. As with the earlier samples, the soil was oven dried at 80 °C for 48 hours. Temperature and humidity in the cupboards was measured using a Tinytag Ultra Temperature/Humidity logger (Gemini Data Loggers (UK) Ltd.).

Analysis
Data was analysed by split plot analysis of variance using GenStat (16th edition). Soil samples were the main plots and replicate trays the sub plots. Nematode data were log transformed to equalize the variance to better meet the normality assumptions of the analysis.

Results
Temperature and humidity in the cupboards averaged 12.5 °C (range 0.8-25.9 °C) and 76.9 % (38.4-100 %), respectively, over the course of the 36 month experiment. Soil moisture at the beginning of the experiment (day zero) was 34-38 % and 30-32 % for the forest and orchard soils respectively. At 13 months, the forest soil moisture ranged from 4.2-4.6 % compared to 3.3-3.5 % for the orchard soil (P < 0.001), with no significant change in moisture content from 13 to 36 months.

Total nematode numbers
The total number of nematodes extracted from the freshly collected forest and orchard soils was variable within each site (mean of 37.9 and 43.4/ g dry soil for forest and orchard, respectively), but with no significant difference between the two sites or the six different sampling times (Figure 1).
By comparison, for the stored samples, there was a difference between soil origin with the forest soil having significantly less nematodes than the orchard soil at 6, 12, 13 and 24 months (P <0.001) storage. After 36 months of storage, nematodes were only extracted from one sample and that was from orchard soil (1 of 12 trays) (Figure 1).

Plant parasitic nematodes
Fresh soil collected from the forest site contained the highest diversity of plant parasitic genera with a mean/g of dry soil of 1.9 Pratylenchus, 2.3 Paratylenchus, 0.3 Globodera, 0.3 Helicotylenchus / Rotylenchus and 0.1 for Criconematidae. By comparison, in the orchard soils, the plant parasitic genera consisted of 1.5 Pratylenchus, 1.8 Paratylenchus and 0.1 Heterodera spp. / g dry soil from the orchard site.
Over all sample times, Pratylenchus comprised 4.4% and 10.4% of the total nematode fauna in the forest and orchard soils, respectively. PPN populations were substantially larger in the fresh soil than were observed in stored soil (results not shown), especially so for the orchard samples.

Stored samples
Of the PPN taxa recovered at three months, Pratylenchus was the most common, found in seven of the twelve forest soil samples (58%) and in all of the orchard samples (12/12) ( Table 2). Small numbers of Paratylenchus, Globodera, Helicotylenchus /Rotylenchus and Criconematidae were also recovered from the forest soil at three months. Mean total number of nematodes per gram of dry soil (log e transformed) collected from either forest or orchard and stored in cupboards for up to 36 months or freshly collected from the original sites. Error bars are SEDs. Note: transformed data presented with back-transformed scale on right hand side for ease of conversion to actual numbers /g. Table 2. Age of soil from which plant parasitic nematode taxa were extracted using the three day misting technique, from 25 g of soil collected from either the forest or orchard location and stored in cupboards for 36 months.
Heterodera were present in very low numbers from the orchard site, with 1-10 nematodes extracted at the 3 month sampling (3/12 samples) despite none being extracted from the original sample (day zero). At six months, with the exception of a single Criconematidae from the forest soil and two Paratylenchus nematodes from the orchard soil, Pratylenchus was the only plant parasitic nematode extracted (Table 2). At both the 12 and 13 month sampling, the only PPN recovered were Pratylenchus spp.
Thirteen months after soil had been placed into cupboards, Pratylenchus were the only PPN recovered using the misting technique, and then only from the orchard soil. Of those recovered, both female and juvenile stages were present.
The number of Pratylenchus recovered over time decreased substantially using a three day misting interval for extraction, with no specimens detected from soil stored for 24 and 36 months ( Figure 2). An extra two days extraction time at 36 months did result in three Pratylenchus nematodes from one sample, all three of which were females.

Plant baiting
Sowing white clover and ryegrass seed resulted in Pratylenchus being recovered from more samples than with mistifier extraction at the 13, 24 and 36 month sampling intervals. At 13 months, Pratylenchus were found in four root samples (4 of 12 samples, 33%), but not in their respective misting samples. At 24 months, Pratylenchus were detected in five root samples (42%) and at 36 months in three samples (25%).
Reproductively mature Pratylenchus were evident in soil that had been stored for 13 months with eggs observed in white clover plant roots from two (c. 17%) of the stored orchard soil samples, demonstrating that not only could these nematodes survive in stored soil without a host plant but could also subsequently infect and reproduce in plant roots. No other PPN genera were detected using the plant baiting method.

Molecular identification of PPNs
The Pratylenchus DNA sequences from the forest soil matched P. bolivianus from the NCBI database and specimens were preserved to be confirmed morphologically. Pratylenchus sequences from the orchard soil indicated the presence of at least four species: P. crenatus, P. thornei, P. penetrans, and an unidentified Pratylenchus that had a poor match to Pratylenchus currently in the database (Table 3).
Specimens found in the orchard soil at the start of the experiment were most commonly P. penetrans with the unknown Pratylenchus sp. also being isolated, while P. crenatus and P. thornei were only isolated once the soil had begun to desiccate (Table 4). Specimens of P. crenatus were isolated at six and 13 months after storage but not from the 36 month samples. P. thornei, along with the unidentified Pratylenchus sp., were isolated at 12 and 36 months post storage.
When comparing the number of Pratylenchus present in the roots of white clover grown in fresh soil collected from the two original sampling sites, there was a significant difference between the two locations at 13 months (P = 0.004), 24 and 36 months (both P<0.001). For ryegrass, the number of Pratylenchus present in the roots grown in fresh soil was significantly different (P<0.001), at all three sample times.
Fewer Pratylenchus were found in the white clover grown in the fresh forest soil samples with a mean, median and range of 5.2, 4 and 1-17, compared to the fresh orchard soil (130.7, 144 and 39-224, respectively). Similar results were obtained for ryegrass growing in forest soil with a mean, median and range of 0.7, 0.5 and 0-3, respectively. This compared to a mean, median and range of 135.4, 115.5 and 24-291, respectively for orchard soils. For freshly collected forest soil, more Pratylenchus were recovered using the misting method than the baiting method.
For the forest soil, with the exception of Globodera zelandica, the PPNs were a poor match to the sequences found in the NCBI website (Table 5). Morphological and molecular identification found that the spiral nematodes observed in the samples were a mixed population consisting of both Helicotylenchus and Rotylenchus species. No Helicotylenchus specimens were sequenced.
For the Heterodera nematode extracted from the orchard soil, the DNA sequence did not give a clear distinction between H. trifolii, H. schachtii and H. betae. The sequence was compared to three reference sequences from NCBI of each species analysed in Geneious TM using the restriction site MspI. It produced a similar profile to H. trifolii and H. betae but not H. schachtii.

Discussion
This study has confirmed the hypothesis that not only are Pratylenchus species able to survive soil desiccation, but after prolonged storage are able to successfully reproduce on host plants.
The ability of nematodes to survive desiccation has been known for some time (Norton 1978). Nematodes that can achieve anhydrobiosis have been divided into two groups, slow-dehydration and fast-dehydration strategists (Womersley 1987). The majority of nematodes require a slow, controlled rate of water loss to achieve anhydrobiosis (Womersley et al. 1998). The soil in this study was stored in cupboards reducing air flow over the samples slowing the rate of desiccation, allowing any nematodes present that had the ability to survive water deficits to achieve anhydrobiosis. The survival of PPNs was greater in soil stored in cupboards than soil stored in sea containers .
Nematodes have developed a number of means by which they can survive desiccation, which include survival stages such as eggs, cysts, and dauer larvae (Womersley et al. 1998;Wharton 2002). Nematodes from Globodera and Heterodera genera form cysts which can allow them to survive in the soil for many years, and some cyst species will not hatch unless stimulated by host root diffusates (Turner and Rowe 2006). This could potentially be the case for the G. zelandica and Heterodera juveniles that were extracted from the forest and orchard soil respectively that had been stored for three months. The other PPNs observed in this study, Paratylenchus, Helicotylenchus, Rotylenchus and Criconematidae also showed an ability to survive in stored soil albeit for a shorter period of time than Pratylenchus. Species from each of these genera have been found in previous studies to survive desiccation (Norton 1978). Other methods employed to slow the rate of water loss may include remaining in senescing plant tissue, swarming or forming aggregates and coiling (Womersley et al. 1998). Coiling has been observed in P. penetrans (Townshend 1984) and P. thornei (Glazer and Orion 1983). The soil in this current study included root fragments so it is possible they were a source of Pratylenchus nematodes able to withstand desiccation.
Pratylenchus species including P. penetrans and P. thornei have been recorded exhibiting anhydrobiosis (Glazer and Orion 1981;Townshend 1984;Townshend 1987;Anon 1997;Talavera et al. 1998;Ghaderi and Bideh 2011), but there is less information on the ability of P. crenatus to survive desiccation. Interestingly, Pratylenchus crenatus has been isolated from small quantities of soil associated with imported seed (Lal and Lal 2006). Survival of P. penetrans and P. thornei in air dried soil for up to 11 months has been pre-viously recorded and listed in a review by Norton (1978). Talavera et al. (1998) found P. thornei was able to penetrate roots after 75 days of desiccation. The current study isolated P. thornei from soil stored in a cupboard for 36 months (1097 days), but as an unidentified Pratylenchus species was also found after 36 months of storage, it is unclear which of the two species infected perennial ryegrass roots in the "baiting" experiment. Townshend (1984) found P. penetrans in slowly dried soil could survive up to 770 days (25.3 months) and that their infectivity and reproduction at 207 days was not affected by anhydrobiosis. The current study did not however find P. penetrans in the soil beyond six months (180 days). Conversely, P. crenatus was detected in soil that had been stored for 13 months but not at 24 or 36 months. Of the known Pratylenchus species identified from this study all three have a broad host range, particularly P. penetrans (Castillo and Vovlas 2007;Singh et al. 2013). However, Lolium perenne and L. multiflorum are considered less favourable hosts (Kimpinski et al. 1984;Townshend et al. 1984) than legumes such as red (Trifolium pratense L.) (Willis et al. 1982;Kimpinski et al. 1984) or white clover (T. repens L.) (L. Aalders, unpublished data). The plant host preference of the unidentified Pratylenchus species isolated from the orchard soil has yet to be determined.
The Pratylenchus isolated from the forest soils and tentatively identified as P. bolivianus, was only detected up to six months. Both white clover and ryegrass proved to be unfavourable hosts for this Pratylenchus sp. with root infection rates considerably lower than numbers extracted from soil using the misting technique.
Pratylenchus crenatus, P. penetrans and P. thornei are each regulated pests for at least one country globally (Singh et al. 2013), and this study has shown that P. crenatus and an unidentified species of Pratylenchus, along with P. penetrans and P. thornei, can also survive prolonged periods of desiccation. According to a review by Jones et al. (2013), Pratylenchus are ranked third only to Meloidogyne and Hetero deridae (includes Globodera and Heterodera) nematodes as having the greatest impact on crops worldwide, and coupled with their ability to survive desiccation their status as a biosecurity risk is increased with more than 80 Pratylenchus species described (Siddiqi 2000).
The study showed that for disturbed soil stored in protected environments Pratylenchus nematode populations can survive prolonged storage for up to 36 months (1095 days) and that in the presence of a suitable host plant, 'baiting' was a more sensitive technique in detecting Pratylenchus spp. than the misting extraction technique. However, this study demonstrated that the approach only works if a suitable host plant is available. Without a priori knowledge of the PPN-plant host association, plant baiting may also produce false negatives. For other PPN, the lack of a suitable host plant meant that the mistifier extraction method was more accurate. Where the host plant was not known, this provided the best option to assess presence /absence, although this method may not extract cyst nematodes. Extraction using flotation /sugar centrifugation would have extracted cysts as well as vermiform stages but the technique was not feasible with the high numbers of soil samples. Furthermore, examining only the roots of bait plants for parasitic nematodes will only show those endo-parasitic species present, it cannot be used to assess external root feeding species, which would require that the soil surrounding bait plants is also checked.
In the context of plant biosecurity and providing an accurate risk assessment for soil contaminants, the development of a generic test for PPN that induces nematodes in a resting stage to emerge and respond to a cue would enhance the probability of detection. Having a better understanding of PPN survival in soil inadvertently transported with commodities, freight, used machinery or humans (e.g. footwear) is important in the development of both scientifically valid pest risk analysis as well as cost-effective management strategies (Colunga-Garcia et al. 2013, Singh et al. 2015.

Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors contribution
LA: Developed the research concept, led and carried out the extraction and identification of nematodes, contributed to manuscript writing. MM: Developed the research concept, carried out the soil sampling and contributed to manuscript writing, NB: Contributed to development of baiting technique, identification of nematodes and writing of the manuscript. CC: Analyzed data.

Introduction
Recently, the focus of competition as the most important plant-plant interaction has been questioned as acknowledgement of the role of positive interactions (facilitation) in influencing neighbouring plants has been established Walker 1997, Brooker et al. 2008;Montgomery et al. 2010, Wright et al. 2014). Gaining advantages from neighbouring plants may increase growth opportunities over and above those gained through competitive superiority, particularly in highly stressful environments (stress gradient hypothesis: Bertness and Callaway 1994, Callaway and Walker 1997, Holmgren et al. 1997. Over the life of a plant, the relative strengths of competition and facilitation vary (Wright et al. 2014;Paterno et al. 2016) and interactions are influenced by other biotic interactions (e.g. herbivory, Hamilton III and Frank 2001). Consequently, understanding community processes involves investigating interactions through the life cycle. Early growth of plants is likely to be an important life stage where interactions are likely to quickly influence the success of seedlings, although this stage has not been investigated widely. Competition is often measured in the field as lower relative growth compared to the competing plant. However, competitive interactions that may be present are often confounded by species-specific differences in growth rates and resource use as well as a range of other positive and negative interactions amongst other species. Competition is a negative-negative interaction, whereby both species should do worse when growing with the other competitor than each does when growing alone. Competition is only possible when there is a limiting resource and few studies establish the limiting resource where competition is acting. In the field, measuring growth rates of individual plants growing alone is particularly difficult and thus identifying competition and the strength of competition is often not clear. Instead studies often focus on differentiating the relative differences between putative competitors. The outcome of these 'competitive interactions' in the field could therefore be caused by a range of other interactions which result in a difference in growth through better acquisition of non-limiting resources, rather than the result of competition with a neighbouring plant. Similarly facilitation is a positive interaction being experienced by at least one partner in the interaction compared to when growing alone and is usually associated with 'within guild' interactions (McIntire and Fajardo 2014). In the past, facilitation was traditionally associated with (+,0) or (+,+) interactions. What is difficult to tease apart in a field situation, is that while species might be facilitated, one species might still 'win' compared to the other species as a result of the strength of facilitation. Without adequate controls, this would be seen to be the result of competition. 'Winning' in a field situation, therefore, may be due to a number of reasons: competition for a limiting resource, better use of non-limiting resources, better facilitation or a combination of any of these. Changes in community structure are, therefore, a result of the relative advantages of facilitation versus competitive superiority that each species experiences when growing with each other (Montgomery et al. 2010). Laboratory experiments are, therefore, useful in teasing apart this complexity of interactions.
Lianas are considered to be strong competitors as they spend fewer resources on mechanical support allowing greater allocation to leaves, stem elongation and roots. They are also often considered to be structural parasites (Stevens 1987). In comparison to other growth forms, lianas have more leaves and a higher annual increment in new stem mass for a given aboveground mass (Wyka et al. 2013, Ichihashi andTateno 2015), and increased photosynthetic capacity (Pasquini et al. 2015). Lianas influence tree regeneration in tropical rainforests ) through both aboveground and below-ground competition (Schnitzer et al. 2005; Toledo-Aceves and Swaine 2008; Lobos-Catalan and Jiminez-Castillo 2014). Our understanding of lianas in forests is largely associated with work in the neotropics (summarised in Paul and Yavitt 2011) with very little work done elsewhere. In southern hemisphere temperate rainforests, Lobos-Catalan and Jiminez- Castillo (2014) suggested that resource competition was less influential as nutrients are greater than in the neotropics. Apart from Lobos-Catalan and Jiminez-Castillo (2014), little work has been done to determine whether lianas in other forest types follow similar trajectories and have similar impacts as those in the tropics. Furthermore, variable use of terminology such as competition and parasitism to describe the same attributes is confusing, highlighting the need to be very clear in attributing the direction of the interaction in studies.
Exotic, invasive lianas have the potential to be particularly harmful to rainforest habitats as the 'competitive' advantage of the liana life history may be coupled with the more competitive qualities of invasiveness. Theoretically, the advantage of being invasive is associated with escape from co-evolved pathogens and predators in the native habitat (Enemy Release Hypothesis, Keane and Crawley 2002) which frees up resources to be used in growth and reproduction. For many invasive species, growth is improved in host habitats where species have evolved improved competitive strategies (Blossey and Notzold 1995). Invasive species have faster growth strategies resulting in higher specific leaf area (SLA) and assimilation rates (Leishman et al. 2007) which causes higher relative growth rates (James and Drenovsky 2007). There are some examples of equivalent patterns associated with invasive lianas. Leicht-Young et al. (2011) found that under stronger competition the invasive liana, Celastrus orbiculatus, had greater relative growth rates, biomass and survival than the native Celastrus scandens. Osunkoya et al. (2010) found that four invasive lianas had higher respiration load, higher light compensation points and higher SLA indicating better carbon economy, but similar relative growth rates to four native species in tropical Australia. In contrast, while an invasive liana in the US, Cayratia japonica, had faster growth compared to a native, Parthenocissus quinquefolia, another invasive (Ampelopsis brevipedunculata) did not (Emerine et al. 2013). Native and invasive liana species in temperate forests in Australia have similar allometry of growth characteristics (French et al. 2016).
When lianas initially establish, advantages usually associated with their growth form may be less relevant, as establishment at the ground layer is likely to be associated with low light conditions where seedlings of all species have low biomass. During this stage of the life cycle, below-ground resources may be more important in determining the outcome of species that are seeking to occupy a site. Furthermore, positive interactions with neighbours may facilitate early growth in seedling lianas and could neutralise or outweigh the negative effects of resource competition. Increases in positive plant-plant interactions during this early stage, relative to negative competitive interactions, may improve growth of plants over and above growth when plants are grown alone (known as overyielding) and may buffer high levels of seedling mortality which would be otherwise experienced. Seedling lianas are unlikely to smother seedling trees as there is strong selection to invest in seeking a taller plant to reach the canopy and escape the darker ground level rather than continuing to grow large amounts of biomass at this level in the forest. Accordingly, in the early stages of establishment, the growth rates of seedlings of lianas and trees is likely to be more strongly affected by belowground resources (Toledo-Aceves and Swaine 2008) and positive plant interactions may become relatively more important. Being able to maintain high growth in lowlight environments in the early stages of growth would be advantageous, however the relative importance of low light, facilitation and competition for resources on growth at this early stage is unknown. Toledo-Aceves and Swaine (2008) found that although changes in light availability (reflecting gaps and interior conditions) did not influence below-ground competition there was an overall positive effect on early growth in 3 species. However, Osunkoya et al. (2005) identified an interaction between below-ground competition and light availability for two tree species suggesting that competition may well change depending on the position of seedlings in the rainforest.
Gaps and edges of rainforests are often areas where lianas are particularly prevalent (Putz 1984, Schnitzer et al. 2000, Schnitzer and Carson 2001, Schnitzer et al. 2012 and are areas where they compete intensely with trees, reducing tree growth and recruitment (van der Heijden and Phillips 2009, Carson 2010, Schnitzer et al. 2014). However, within forest interiors, resource limitation, particularly phosphorus, is important for both trees and lianas although lianas always outperform trees (Pasquini et al. 2015) and can suppress seedling growth and survival (Martinez-Izquierdo et al. 2016). Thus comparing competitive and facilitatory effects at the edges and interiors of rainforests may indicate the strength of competition, relative importance of facilitation and how effective lianas are in these early periods of growth.
We investigated plant-plant effects in low and moderate light conditions to simulate light conditions on rainforest edges and interiors. We compared two native and two invasive lianas grown with a common rainforest tree of temperate rainforests in Australia and predicted that both invasive lianas would be more competitive than native lianas with a better capacity to add biomass in competition with rainforest seedlings. We predicted that this interaction would be maintained in low and high light conditions. If facilitation occurred, then we predicted that lianas growing with another plant would show improved growth compared to when growing alone and that this effect would be greater for invasive than native lianas. If positive effects were evident in lianas, then native tree seedlings would also be positively (mutualism) or neutrally affected (commensalism) in line with the concept of facilitatory interactions.

Study species
Seedlings of Guioa semiglauca (F.Muell) Radlk. (Sapindaceae) were used in growth trials in pots in a shade house. G. semiglauca is a common tree up to 18 m used as a host by lianas in a range of rainforest communities along Eastern Australia (Harden et al. 2006). It can grow in both edges and understorey areas in rainforests. Two native and two invasive lianas were compared in the trial: the native species were Cissus antarctica Vent. (Vitaceae) and Pandorea pandorana (Andrews) Steenis. subsp. pandorana (Bignoniaceae) and the invasive species were Araujia sericifera Brot. (Apocynaceae) and Anredera cordifolia (Ten.) Steenis (Basellaceae). All species were chosen as all can be abundant in disturbed rainforests. Cissus antarctica is a robust tendril climber reproducing from berries and spreading clonally through numerous stems (Fairley andMoore 2010, Harden et al. 2007). Pandorea pandorana subsp. pandorana is found throughout Australia in a range of vegetation communities including rainforests. Anredera cordifolia is a succulent climber from South America which was first introduced to Australia in the early 1900's (Vivian-Smith et al. 2007). It is currently listed nationally as a Weed of National Significance. It primarily spreads vegetatively through smaller aerial tubers and extensive subterranean tuber networks (Swarbrick 1999). Araujia sericifera is a common stem twiner native to Peru and declared a noxious weed in many areas in eastern Australia. It produces numerous wind-dispersed seeds from large pear-shaped fruit (Harden et al. 2007, Pellow et al. 2009).
All native plants were bought commercially as tube stock. The exotic species were obtained from the field as seedlings (Ar. sericifera) or tubers (An. cordifolia) and grown in a glasshouse for approximately three months prior to the experiment. Plants were potted (12cm diam pots) in coarse river sand to facilitate final harvest of belowground biomass, and given 5 g of slow-release native fertiliser (Osmocote® native) at the beginning of the experiment. Lianas were supplied with wire and rope trellises in the same cardinal direction, hence pots were not rotated during the experiment. We accounted for this by randomly allocating pots to competition treatments within each of the light treatments (see below).

Experimental design
Plants were grown under experimental conditions over spring and summer from August 2011 to February 2012 (24 weeks). Seven replicate pots of each experimental condition were set up in a shade house. To measure maximum growth under no competition, control pots contained a single plant of each species (liana or host). Intraspecific competition was measured in pots that contained two individuals of a species and interspecific competition was measured in pots that contained one individual of the host species, G. semiglauca, and one individual of a species of liana (Fig. 1).
For the experiment, the two light treatments were created by constructing two adjacent shadehouses using standard shadecloth. Plants were grown in either medium light (ML, 33% daytime PAR) or low light (LL, 7% daytime PAR) to simulate the available PAR in forest edges and interiors respectively. Measurements at various points in each shade house showed them to be an average of 33±2% and 7±1 % full PAR. Readings were made using two Spectrosense dataloggers attached to quantum sensors (Skye Instruments Ltd, Llandrindod Wells, Powys, UK).
Initial measurements of stem, leaf and belowground biomass were obtained from four randomly chosen plants of each species prior to placing in light treatments. Each plant was divided into stem, leaf and belowground portions, washed and oven dried at 60°C for five days before being weighed. Destructive measurements of specific leaf area (SLA = leaf area/ dry weight) were conducted after four weeks from five leaves of each species, using spare plants. Each leaf was labelled and its area measured with a Li-Cor leaf area meter (Model Li-3000A, Lincoln, Nebraska, USA), before being dried and weighed.
During the six months, all pots were watered daily via an automatic mist irrigation system and soil was maintained at field capacity. The lianas were allowed to climb freely onto trellises but were prevented from growing onto adjacent hosts by moving stems away from adjacent plants every few days. Aerial tuber production on Anredera cordifolia plants was monitored and recorded.
After 24 weeks, final measurements of leaf number were made before all plants were harvested and then biomass assessed (see below). Aerial tubers from A. cordifolia were removed before measurements to prevent them from falling off their stems. These were dried and weighed separately. No plants died during the experiment.

Analysis
For the host plant, we investigated changes in biomass by analysing accumulated biomass, above-and below-ground biomass, stem and leaf biomass. We also investigated effects on SLA and leaf number. For plants grown with a conspecific we chose a single plant randomly from each pot as the focal individual to be used for analysis. For comparisons between species we calculated relative growth rate per month using the following equation: (ln DW f -ln DW i )/no. months, where DW i is the average dry weight of 4 plants sacrificed at the beginning of the experiment, DW f is the weight of an individual seedling at the end of the experiment, and no. months is the amount of time, in months, over which plants were in the experiment (5.6 mo).
We undertook two different analyses to test questions about how lianas influence seedling trees. Using two factor ANOVAs, we investigated whether any of our measures of growth for G. semiglauca varied with competition or light level (JMP Pro 11). Secondly, we used a linear mixed effects model fitted using restricted maximum likelihood to investigate how changes in biomass of G. semiglauca varied with origin of the liana species and light levels. Species of liana were treated as random effects and nested within origin (exotic, native). Only interspecific competition treatments were included in this analysis.
For each liana species, we tested whether inter-or intra-specific competition influenced growth rates using a two factor ANOVA with competition and light level as fixed factors, comparing each liana species grown alone with those grown with another conspecific or with G. semiglauca. Tubers of An. cordifolia were analysed in two ways. Initially we undertook a nominal logistic model to investigate the probability of producing tubers associated with different competition and light levels and tested the effects using a likelihood test. Secondly, for those plants that produced tubers, we investigated whether dry biomass of tubers varied with competition or light using a two factor ANOVA. Finally, we compared differences in growth amongst the four liana species and G. semiglauca using an ANOVA on relative growth rates. We included light level as a factor.
As data fitted the assumptions of normality and homogeneity we did not transform any variables. Tukeys HSD multiple comparisons were used to determine where differences lay in significant factors in the ANOVAs. We used nominal logistic models on pairs of levels of competition when the overall nominal logistic model was significant for tuber production associated with competition, and corrected probability values to α = 0.017 (a Bonferroni correction) to account for Type 1 errors.

Effect of plant-plant interactions and light on native tree seedlings
Guioa semiglauca seedlings were not significantly affected by intraspecific competition (Table 1, Fig. 2) although plants grown with a conspecific grew to only 68% of the  biomass of plants grown alone. Liana species differed in their ability to affect biomass of G. semiglauca. Anredera cordifolia reduced overall biomass of G. semiglauca to 42% of that when it was grown alone, while Araujia sericifera and Pandorea pandorana did not appear to impact biomass in G. semiglauca seedlings at all (Table 1, Fig. 2a). Where biomass of G. semiglauca was reduced by interspecific competition (with An. cordifolia) both above-and below-ground biomass accumulation appeared to be impacted although the biomass was often not different to growing alone or in intraspecific competition (Table 1, Fig. 2b, c). There was no evidence of facilitation with any of the lianas. For G. semiglauca, a reduction in light influenced growth, reducing total biomass through reductions in both above-ground and below-ground biomass (Table 1, Fig. 3). Above-ground changes were most apparent with an increase in stem growth for plants grown in ML conditions. In deep shade (LL), overall growth was reduced to 70% of growth in the ML treatment. Below-ground biomass was more affected by LL and was reduced to 62% of growth in the ML conditions, while above ground biomass was reduced to only 71%.
The number of leaves produced was not affected by competition or light levels while SLA showed a typical increase in LL conditions (Table 1, Fig. 2e,f ). In LL, SLA was 167 + 59 (s.d.) while in the ML treatment it was 116 + 93. The origin of the competing liana had no effect on any growth parameter of G. semiglauca (Table 1).

Effect of plant-plant interactions and light on biomass of lianas
Both invasive lianas showed similar patterns of biomass change in response to plantplant interactions, although changes in biomass were only significant for An. cordifolia (Fig. 4, Table 2). For An. cordifolia, growth appeared to be facilitated by growing with G. semiglauca (Fig. 4). Growth increased nearly 4-fold from 6.0 + 3.0 g when grown alone to 22.6 + 7.9 g when grown with G. semiglauca. For An. cordifolia, growth alone was not different from growth with a conspecific. Increased growth with G. semiglauca was similar for both above-ground and below-ground biomass and was influenced by a doubling in the number of leaves produced (see Appendix 1).
In contrast, total biomass in Ar. sericifera was less influenced by plant-plant interactions. Again, facilitation was evident when this species was grown with G. semiglauca compared with a lower increase in biomass when grown with a conspecific. This increase could not be assigned to an increase in above-or below-ground biomass but appeared largely influenced by changes in overall leaf biomass (Table 2, Fig. 4).
The two native lianas were quite different in their responses to plant-plant interactions (Table 2, Fig. 4). Like both invasive species, C. antarctica, plants appeared to be facilitated by growing with G. semiglauca adding twice the biomass compared to when grown alone or with a conspecific (Fig. 4). Both below-and above-ground biomass were affected similarly. For C. antarctica, the interaction of competition type with light (Table 2) identified that plants grown alone put on biomass to similar levels to when grown with G. semiglauca at LL but did not compete as well under ML and had similar growth to plants grown with  While light level had some moderate impacts on interactions for C. antarctica, light level alone did not influence biomass accumulation in lianas, with the exception of P. pandorana (Table 2). This species responded with an increase in leaf mass under ML conditions (av. ML, 7.8 + 3.7 g; LL, 5.5 + 2.8 g). Plants in LL had increased SLA Figure 4. Average a total biomass b above-ground biomass c below-ground biomass of two invasive lianas, Araujia sericifera, Anredera cordifolia, and two native lianas, Cissus antarctica and Pandorea pandorana grown under different competition treatments: alone (white bars), with a conspecific (grey bars) and with G. semiglauca (black bars). Letters above each group of bars are results from Tukeys multiple comparison tests where different letters represent significant differences within each set of bars. compared to those in ML, and SLA was not affected by growth in competition in either invasive species although it was in both native species (Table 2).
Anredera cordifolia developed tubers over the course of the experiment. The probability of tubers developing was influenced by plant-plant interactions (χ 2 = 14.78, p <0.001). When grown with G. semiglauca, 93% of plants produced tubers whereas 57% of plants produced tubers when grown alone, however these did not differ in the likelihood of producing tubers (χ 2 = 6.24, p = 0.013, α=0.017). When grown in competition with another An. cordifolia, 29% of plants produced tubers and the probability of producing tubers did not differ from plants grown alone (χ 2 = 2.37, p = 0.124, α=0.017), however there was a higher probability of producing tubers when growing with G. semiglauca than when growing with a conspecific (χ 2 = 14.77, p <0.001, α=0.017). For plants producing tubers, there was no difference in tuber biomass per plant amongst treatments (F 2,19 = 1.68, p = 0.212) or light environments (F 1,19 = 0.013, p = 0.910). On average plants accumulated 1.45 + 1.58 g dry biomass of tuber which amounted to 10% additional biomass when in competition with G. semiglauca, 59% when in competition with a conspecific and 38% additional biomass when grown alone.

Comparison of growth rates of lianas and G. semiglauca
Relative growth rates differed amongst species (F 4,120 = 7.17, p <0.0001). Under LL conditions G. semiglauca had the lowest relative growth rates when grown without competition with all lianas having relative growth rates about 10 times higher, however, this difference was not evident when plants were grown with a conspecific, with G. semiglauca having a higher relative growth rate (Fig. 5). There were no differences amongst species in the ML treatment (F 1,120 = 0.661, p = 0.418).

Discussion
Our prediction that invasive lianas would be more competitive than native lianas with a better capacity to add biomass in competition with rainforest seedlings was not supported and light had little effect on the responses. We found strong evidence of facilitation although not all lianas benefited. However, the facilitation of growth in lianas was coupled with a loss of growth in the rainforest seedling.

Are invasive or native lianas good competitors in rainforests?
The success of these invasive lianas in establishing in habitats is not based on an improved capacity to compete in early establishment, although for An. cordifolia, early competition may contribute to invasion success. The two invasive lianas did not show consistent patterns in their effects on native seedling growth, suggesting that invasive lianas are not always more competitive than native lianas in reducing growth of a native rainforest seedling. However, no liana showed any positive effect on native tree seedlings, suggesting no facilitation. Exotic An. cordifolia had the capacity to reduce both above-ground biomass and leaf mass in the rainforest tree, however, the other exotic species, Ar. sericifera, had no impact on growth of the rainforest tree in these early stages. Native P. pandorana and C. antarctica did not influence growth. At this stage in the life cycle, most lianas were as competitive as a conspecific seedling for G. semiglauca. Osunkoya et al. (2010) identified a number of traits in invasive lianas which may provide them with competitive advantages but also noted that there were few differences in a range of traits associated with plasticity between native and invasive lianas. In temperate regions, our study suggests that competitiveness against a common rainforest tree seedling is not related to being invasive. Similar allometry has been found between these invasive and native lianas, identifying few differences in life history strategies between these two groups (French et al. 2016).
Light had no effect on growth of lianas nor on the impact of competition. As expected, plants that grew at low light increased their SLA through an increase in the area of leaf relative to the biomass of the leaf, however, all lianas grew equally well in both light treatments and there was no increase in the proportional effect of neighbouring plants on biomass. This suggests that these lianas grow equally quickly in both the interior and edges of rainforests and, in a similar way to lianas in tropical forests, have the capacity to impact on tree seedlings in gaps (Schnitzer and Carson 2010) and in understorey (Martinez-Izquierdo et al. 2016). While Toledo-Aceves and Swaine (2008) also found no interaction between competition and light, they did find that growth was enhanced with higher light in tropical rainforests. All lianas maintained high relative growth rates in different lighted and competition treatments and appeared quite resilient to these factors and able to successfully maintain growth despite these limiting resources. Similar relative growth rates between native and invasive lianas, including some of the same species as our study, were found in tropical areas of Australia (Osunkoya et al. 2010).
In contrast, the rainforest tree, G. semiglauca, showed improved growth under the higher light treatment, associated with both below-ground and above-ground increases, suggesting that it should show improved growth in gaps and along edges of rainforests. This species is clearly more light-limited in the interior of the rainforest although it is not restricted to edges in rainforests. If lianas are increasing in abundance in temperate rainforests, as they are in the neotropical rainforests (Schnitzer and Bongers 2011), then lianas are likely to impact on recruitment rates of this rainforest tree. While this is the first experiment to test the effect of such interactions in these temperate forests, there is the potential for a range of other tree species to be negatively affected.
Our results suggest that the invasive, An. cordifolia is a particularly strong competitor in rainforest environments and a serious invasive weed at early stages of growth. Three results particularly highlight this; overyielding in the presence of G. semiglauca, coupled with its strong negative effect on G. semiglauca and the increased growth of tubers while growing with the native tree seedling. Within 6 months, this plant had the capacity to spread in both edge-simulated light levels and interior-forest light levels through the release and dispersal of tubers.
As rainforest communities in temperate Australia are naturally patchy in distribution, edges are important sources of recruitment. Temperate rainforests are likely to be particularly affected by the predicted increase in drought and extreme temperatures in the future and they are already faced with significant threats from habitat clearing. If native and exotic lianas also increase in abundance, then the recruitment capacity at edges and within forests may well be hampered. There is much research to be done on how lianas may interact with rainforest trees within this future environment.

Variation in plant-plant interactions
When lianas were grown with a rainforest tree, rather than experiencing a decrease in biomass (relative to growing alone), three species had enhanced accumulation of biomass; both exotic species and the native C. antarctica. Overyielding in An. cordifolia and C. antarctica occurred in both above-ground and below-ground biomass and in tuber growth in An. cordifolia. For Ar. sericifera, overyielding could not be attributed to above-or below-ground biomass, as the magnitude of difference compared to plants grown alone was not as large. This suggests that positive plant interactions were far more influential on growth of these three liana species than for the other native liana species and the tree seedling.
While not often done (e.g. Montgomery et al. 2010, Dohn et al. 2013, we have measured both sides of the interaction in this experiment, and the positive effect was only seen for one of the participating species (the liana) with a strong negative effect for the native tree seedling. The interaction is, therefore, more associated with the directions of advantage associated with a parasitic interaction (+,-), a term not usually applied to plant-plant interactions where both species are physiologically independent. This is the first time such a plant-plant interaction has been reported to our knowledge; we have termed this, parasitic facilitation. Without measuring each individual species response to being grown alone, an understanding of the direction of the interaction would not be able to be distinguished from competition or facilitation, highlighting the complexity of interactions amongst species, and the difficulty of identifying true interactions without suitable controls.
The mechanism for parasitic facilitation is currently unknown but a number of possibilities can be identified. It is plausible that the parasitic-style interaction that is shown by the three species of liana, is mediated by some change in the soil environment rather than above ground. While facilitation was seen in Brazilian Restinga communities where shrubs facilitated the abundances of vines through providing trellises for initial growth (Garbin et al. 2012), we consider that in early stages of growth there was no facilitatory effect of structural support by the native seedling as we did not observe smothering or shading to any great extent. Our results may be associated with coupling through shared mycorrhizae (Simard and Durall 2004;Giovannetti et al. 2004, Walder et al. 2012 lending some weight to the idea that the plant-plant interaction is being mediated by a third taxa (mycorrhizal fungae). If this link is present, then the liana could be viewed as being parasitic on the symbiotic mycorrhizae, and the term parasitic facilitation is useful.
One other possibility is that G. semiglauca changes other soil microflora to enhance release of nutrients which benefit the lianas as well (Hooper andVitousek 1998, Zak et al. 2003). In an example of this, Hamilton III and Frank (2001) showed that, when two species were grown together, defoliation increased root exudation of carbon in one species which increased N pools in the soil improving soil resources for neighbouring plants. Likewise, increases in nutrients by nitrogen-fixing species can enhance and cause overyielding in co-occurring crop species (Li et al. 2007). If lianas are competitively superior then they may gain greater access to these freed resources at the expense of the native seedling; perhaps more indicative of a parasitic interaction.
An alternative interpretation is that the liana may be clearly superior in gaining resources from the fungi, which could be viewed as highly asymmetric resource competition where the liana is better at using resources provided by the fungi, than the native seedling. There are a range of studies which have identified changes in mycorrhizal communities associated with invasive plants that influence neighbouring native species (e.g., Stinson et al. 2006;Zhang et al. 2010, Shannon et al. 2014. Marler et al.(1999) showed that the presence of mycorrhizae increased the negative effect of the invasive Centaurea maculosa on native bunchgrass, Festuca idahoensis. Competitive interactions usually result in negative effects whereby plants when grown without another plant do better than when grown with a plant. Given the overyielding identified in three of our species (a positive response), a competitive interaction is less accurate as a description, although resource limitation is at the base of our interpretation of the mechanism. It is clearly important to measure responses of both species to distinguish between negativenegative and positive-negative interactions. Using appropriate terminology will be an important factor in understanding plant-plant interactions. Distinguishing between facilitation, competition and other more complicated interactions such as parasitism is difficult experimentally, and confirms that describing accurately many plant-plant interactions is necessary to understand the underlying mechanism of invasion.

Comparing functional traits and abundance Introduction
A major cause of global change is the pervasive introduction of alien species (Wardle et al. 2011;Simberloff et al. 2013) and the increase in global biotic homogenization is not predicted to diminish any time soon (Seebens et al. 2017). Once established in new locations, alien species with strong impacts can change ecosystems in many ways, including altering the biology and ecology of native competitors. The results from interactions between invasive and native competitors can range between coexistence, survival by differential use of resources that arise occasionally or periodically, and competitive displacement of the native species that can remain at low densities or become extinct (see Boivin et al. 2008).
The nature and extent of ecological impacts of alien species depend on the functional ecology of these species in the context of the structure, diversity, and evolutionary experience of the recipient community . Evaluation of functional traits of invasives and co-existing natives is one means for understanding the effects of invasions, and especially the extent that an invasive is impacting native communities (Mouillot et al. 2013). Although impacts must be evaluated in the context of communities, functional traits such as rapid exploitation of resources and elevated fecundity have been associated with high-impact invasives . Invasive species that have the greatest impact have also been suggested as being those introducing novel traits and becoming dominant members of communities (Rodriguez-Cabal et al. 2015;Callaway and Ridenour 2004).
Arthropods constitute the most diverse group of organisms (Pimm et al. 1995) and our understanding of the biologies and ecologies of the majority of native species in native ecosystems is frequently limited for many arthropod species prior to invasions. Yet, invasive arthropods have had and are presently having enormous impacts in forests (Liebhold et al. 1995;Gandhi and Herms 2010). Woodwasps are generally not considered forest pests and relatively little is known about their functional traits, although the aggressive invasive Sirex noctilio is a major exception. Sirex noctilio is not a pest where it is native in Eurasia and North Africa, but has caused economic damage in plantations of exotic pines (Pinus spp.) after introductions in the southern hemisphere (Slippers et al. 2015). Established populations of S. noctilio were first reported in northeastern North America in 2004 (Hoebeke et al. 2005) and this invasive now occurs in eight eastern US states (CERIS 2017) and two eastern Canadian provinces (de Groot et al. 2006, CFIA 2009 and it is expected to continue to spread. In the southern and northern hemispheres, S. noctilio typically oviposits in suppressed pines (Neumann andMinko 1981, Dodds et al. 2010;Ayres et al. 2014). However, populations can increase and, especially in overstocked stands and during droughts, S. noctilio can kill pines that are considered healthy (Madden 1988). Sirex noctilio kills pines by injecting an obligate symbiotic white rot fungus, Amylostereum areolatum (Ryan and Hurley 2012), and a phytotoxic venom (Bordeaux et al. 2014) in association with laying eggs into trees. Wood-boring S. noctilio larvae can only develop if the white rot fungus is present, as it serves as an external rumen for larvae (Thompson et al. 2014).
The establishment of S. noctilio in eastern North America is the first time that this invasive has invaded an area hosting native Pinus and Sirex communities, including the congeneric native pine-specialist S. nigricornis which has never been considered a pest in North America. Sirex nigricornis is associated with one of two fungal symbionts: either the same fungal symbiont as S. noctilio (A. areolatum) or Amylostereum chailletii Olatinwo et al. 2013). Sirex nigricornis can co-occur in pines with S. noctilio (Ryan et al. 2012a;Hajek et al. 2013). Most adults of S. nigricornis emerge to mate and oviposit approximately 1-2 months after S. noctilio (Ryan et al. 2012a;Foelker et al. 2016;Suppl. material 1: B). Both species can be highly variable in size which depends on sex, parasitism and larval developmental conditions (Ryan and Hurley 2012;Kroll et al. 2013;Haavik et al. 2016a), but in North American areas of sympatry, S. noctilio adults are usually slightly larger and carry more eggs than S. nigricornis (Haavik et al. 2016a;AEH and JCH unpublished data). While S. noctilio is more aggressive and can attack healthier trees, both species will oviposit in pines of poor vigor or that are recently dead (see Suppl. material 1: A). The resources needed by these species are ephemeral and often patchily distributed; in agreement, studies have shown that populations of S. noctilio are limited by the availability of acceptable pines (Haavik et al. 2016b). To investigate these two Sirex species in pines after establishment of S. noctilio, we compared the functional traits of S. noctilio and S. nigricornis. Here, we compare the sizes of venom glands of these two species in relation to female sizes, and their pine species use and voltinism. Using trees with evidence of S. noctilio infestation, we present the relative densities of the two Sirex species across time, beginning three years after the first detection of S. noctilio in the northeastern US. Our goal is to compare functional traits between the native and invasive to begin to understand the effects of the S. noctilio invasion on native siricid communities.

Rearing procedures
Red pines (Pinus resinosa) and scots pines (Pinus sylvestris) with resin beads characteristic of S. noctilio attack (Dodds et al. 2010) were located from October to December in New York and Pennsylvania. These trees were often suppressed and patchily distributed. In spring, infested trees were felled, areas of trunks with resin beads were cut into bolts, and ends of bolts were coated in sealant (Waxlor, Willamette Valley Company, Eugene, Oregon, USA). For the majority of studies, 70 cm long bolts were placed in fiber barrels (91 cm high × 61 cm diam) covered securely with window screening, in an unheated barn. Once adult wasp emergence began (in early to mid-July), barrels were checked daily for two months and then subsequently 3-4 times per week. Sirex that emerged were placed in 29 ml clear plastic cups at 4°C. All Sirex emerging were identified using characters described in Schiff et al. (2012).
To cover the emergence periods for both Sirex species, checking for emergence finished in early November, and, at that time, barrels were completely checked for any additional specimens.
For studies comparing the abundance of Sirex species by tree species, Sirex were reared from 50 cm long bolts placed in horizontal cardboard rearing tubes. Bolts were spaced from tube sides using nails and were housed at ambient environmental conditions in a screened outdoor insectary. Glass emergence jars were fitted to the tube ends, oriented facing screened windows for a natural photoperiod, and emerging Sirex were collected daily.

Adult size and venom gland measurement
To quantify the sizes of emerging adults, we followed Madden (1974) and Nahrung (2016) in using the apical width of the prothorax. Measurements were made using digital calipers (Traceable Products, Webster, Texas, USA). Infection with the parasitic nematode Deladenus siricidicola negatively impacts adult size (Kroll et al. 2011;Haavik et al. 2016a), so we diagnosed whether each of the adults being measured was parasitized by these nematodes. Males and females were dissected alive under a dissecting microscope at 10× magnification. The abdomen was removed and cut lengthwise to open it, without damaging the venom glands in females, and presence of nematodes within the abdominal cavity and reproductive tissues was recorded. Eggs from females were counted at 63× magnification and venom glands were removed and immediately weighed on a microscope cover slip.
To compare sizes of S. noctilio adults with (n = 92) and without (n = 412) nematodes, we used randomly selected individuals emerging from barrels in 2012-2014. There were far fewer S. nigricornis emerging during this period and nematode infections were uncommon in S. nigricornis, so analyses included only S. nigricornis of both sexes without nematodes (n = 36).
To assess weights of venom glands, a total of 51 S. noctilio were randomly selected in 2014-2016 for dissection and 21 of these contained nematodes. Since few female S. nigricornis emerged from rearings from the northeast during 2014-2016, 30 S. nigricornis females from Arkansas and Louisiana that had been caught in panel traps were used for venom gland weights. We were concerned that these S. nigricornis from the southeast both had been flying before being trapped and were from a different geographic region. To test the accuracy of using southeastern S. nigricornis as replacements for northeastern S. nigricornis, we compared the relationship between numbers of eggs and body size for S. nigricornis females collected from traps in the southeastern vs. emerging from wood from the northeastern USA. Southeastern S. nigricornis carried the same number of eggs in relation to prothorax width as S. nigricornis emerging from wood in the northeastern US (t = -1.10; P = 0.2713), so we used venom gland weights from southeastern S. nigricornis for comparisons. Sirex were reared from trees as described above.

Voltinism
On 4 June 2013, 12 Sirex-infested P. resinosa were collected from a plantation on River Road, Warren Co., New York (43°31'59.7"N 73°49'30.8"W). Trees were cut into 219 70 cm long bolts and woodwasps were reared as described above, with barrels checked every 1-2 days to collect individuals very soon after emergence; females were never found to be ovipositing when collected this soon after emergence. In November 2013, barrels were thoroughly checked for dead Sirex so that any first year emergers were not mistaken for second-year emergers. Barrels were stored in an unheated barn over the winter and were checked for emergence throughout the 2014 flight season. This procedure was repeated for a third year, through the 2014-2015 winter and 2015 flight season. As a continuation to this study, in Tioga Co., Pennsylvania, in spring 2014 we harvested 23 infested P. resinosa and, in spring 2015, 29 P. resinosa. Wood from trees cut in 2014 and 2015 was maintained and emergence was checked for two years.

Densities and co-occurrence of S. noctilio and S. nigricornis
Pines with resin beads indicative of S. noctilio attack were harvested in central and northern New York State and north-central Pennsylvania and Sirex were reared from them. Even within the same region, it was rare that the exact same site was sampled more than one year. Pinus resinosa were harvested from plantations in Tioga Co., Pennsylvania yearly, from 2011 and 2013-2015, with 20-30 trees harvested each year. Sirex were also reared from mixtures of P. resinosa and P. sylvestris from natural forests in northern New York State and from mature plantations in central New York State. In 2007 only P. sylvestris was sampled as described in Long et al. (2009). Trees included in analyses were those from which either one or the other or both Sirex species emerged.

Data analysis
A general linear mixed model with year as a random effect was used to compare sizes of Sirex. To compare body size versus venom gland mass, the significance of difference between slopes was calculated (Cohen et al. 2003). Comparisons of body size across species, sex and nematode parasitism were conducted using mixed models with year as a random effect and least squares means were used for post hoc comparisons (SAS 2014). Because very few S. nigricornis were reared that were parasitized by nematodes, comparing body sizes for individuals that were parasitized versus not parasitized was only possible for S. noctilio. Tree species use analysis was conducted using a mixed model with (ln +1)-transformed densities by volume of wood for S. nigricornis versus S. noctilio (SAS 2014). Sirex species, tree height, tree diameter and tree species were explanatory variables, with tree nested within site and site as random effects. The interaction of Sirex species with tree species was initially included but was removed as it was not significant. Wilcoxon signed ranks tests were used to compare numbers of S. noctilio versus S. nigricornis adults emerging from trees in 2007-2014.

Trait-based studies: Comparing voltinism
Nearly half of adult S. nigricornis emerged from wood during the first season in our rearings, over half emerged during year 2 (Table 1), and no S. nigricornis emerged during year 3. In contrast, emergence of S. noctilio adults primarily occurred in year 1 ( Table 1). Numbers of S. noctilio emerging from wood in 2013-2014 were low but among these, 26.7% emerged during the second year. For wood harvested in 2014 or 2015 and reared for two years, from 1.5% to 10.9% of the S. noctilio emerged during year 2.

Densities and co-occurrence of S. noctilio and S. nigricornis
Between 2010 and 2015, S. nigricornis densities were lower than densities of S. noctilio in central New York State and north central Pennsylvania, where most pines that were sampled had been purposefully planted (Table 2). However, when sites in central and northern New York State were sampled within 2-3 years of the first reports of S. noctilio in those areas (see Table 2 footnotes), numbers of S. nigricornis emerging did not differ significantly from S. noctilio, although they were usually numerically lower. When S. nigricornis emerged, they more commonly co-occurred in the same trees as S. noctilio and rarely were the only Sirex species emerging from a tree. In 2015, no S. nigricornis emerged from infested material from north central Pennsylvania and during this study, densities of S. nigricornis from this same area were always low.

Comparing functional traits
The overall body sizes of S. noctilio as well as the sizes of their venom glands were significantly larger than the bodies and venom glands of the native S. nigricornis. The venom glands of S. noctilio are also larger than those of seven other European siricids (Spradbery 1977) and experimentation has shown that venom from S. noctilio had greater phytotoxic activity than venom of these European siricids (Spradbery 1973). While a key component of the venom in the S. noctilio gland has been characterized as noctilisin (Bordeaux et al. 2014), the identities of compounds in the venom glands of S. nigricornis, or glands of any other siricids, are not known (Wang et al. 2016).
Regardless, the fact that the S. noctilio venom glands are larger (relative to adult body size) than venom glands known from other siricids is consistent with the fact that this is the most aggressive siricid, reported as able to kill relatively healthy pines where it is adventive (see Suppl. material 1: A), while other woodwasps are considered relatively benign secondary pests in forests (Schiff et al. 2012). Haavik et al. (2016a) found that S. nigricornis carries more eggs per body size than S. noctilio and our studies have documented the same trend (AEH and JCH, unpublished data). However, the present study demonstrated that on average S. noctilio females with or without nematode infections were larger than S. nigricornis females and the same relationship holds for males. So, although S. nigricornis has the potential to carry more eggs than S. noctilio, when we calculated the numbers of eggs per female based on sizes of females that emerged from wood in our studies, using the regression equations in Haavik et al. (2016a), the number of eggs for average-sized S. noctilio female was 166 but 138 eggs would be produced by S. nigricornis. Thus, the larger body size of S. noctilio emerging from trees compensated so that on average females of the invasive carried more eggs than S. nigricornis. It was unexpected that while nematodeinfected S. noctilio females were smaller than healthy females, nematode-infected males were larger than healthy males, an association also found by Haavik et al. (2016a).
The densities of the two Sirex species emerging from P. resinosa and P. sylvestris did not differ by tree species. Studies have found a trend of P. sylvestris being colonized more frequently by S. noctilio compared with P. resinosa (Dodds et al. 2010;Foelker 2016) and S. nigricornis was not included in those studies. Largely based on the southern hemisphere where many North American pines have been introduced, susceptibility of pine species to S. noctilio varies by tree species (Ryan and Hurley 2012;Nahrung et al. 2015). Our result was somewhat unexpected because P. sylvestris is native to Europe, where it is assumed to have coevolved with S. noctilio (Ayres et al. 2014). However, we do not know exactly where the genotypes of S. noctilio introduced to New York and Pennsylvania originated and, given the broad native geographic range of S. noctilio, it is possible that these genotypes of this woodwasp did not co-evolve with P. sylvestris (Boissin et al. 2012;Bittner et al. 2017). On the other hand, P. resinosa and S. nigricornis co-evolved in North America and in this case there was no preference for the native pine over the introduced pine.
Sirex noctilio and S. nigricornis also differed significantly based on the percentages of the populations emerging from wood after 1 vs 2 years. In our studies, S. noctilio mainly emerged in year 1; from 1.5-27.7% of S. noctilio emerged the second year (Table 2) while in Ontario, Ryan et al. (2012b) found 2.2% emergence during year 2. Second-year emergence of S. noctilio was 15-25% in New Zealand and Australia, including Tasmania (Morgan 1968;Taylor, 1978), 1.6% in Eurasia (Spradbery and Kirk 1978), and 24% in Galicia, Spain (Lombardero et al. 2016). In all cases, percent emergence for S. noctilio in the second year of rearing was less than that recorded for S. nigricornis in our study, for which close to 50% emergence occurred in both years 1 and 2. Differences in voltinism between S. noctilio and S. nigricornis could have large impacts on their population dynamics. Based on our results, future studies of S. nigricornis, at least in northeastern North America, should allow two years for emergence from infested wood.

Comparing abundance of Sirex species
In northeastern North America S. noctilio is now the most abundant woodwasp attacking pines (Long et al. 2009;Ryan et al. 2012a;Foelker et al. 2016). Unfortunately, there are no records of the population densities or tree use by S. nigricornis before S. noctilio arrived but now, use of suppressed pines by S. nigricornis is much less than use of this resource by S. noctilio (e.g., Table 2).
Sirex noctilio has a temporal advantage over S. nigricornis as many emerge 1-2 months before S. nigricornis, although there is overlap in emergence between these species in northeastern North America (Ryan et al. 2012a;Haavik et al. 2013;Hartshorn et al. 2016;Suppl. material 1: B). Sirex noctilio is found colonizing trees at higher densities than S. nigricornis. Therefore, each year S. noctilio will occupy what is considered as being their preferred resources (i.e., suppressed trees that are not yet dead; see Suppl. material 1: A) before S. nigricornis adults have emerged. Among all pines from which Sirex were reared, only S. noctilio emerged from 56% (across the 9 years of this study). However, after the invasive had been present for a few years in an area, when the two species both emerged from trees, numbers of S. nigricornis were lower than S. noctilio and we very rarely found only S. nigricornis emerging from a tree. Based on the low numbers of S. nigricornis emerging from infested trees, we hypothesize that population densities of S. nigricornis emerging could be low because when adults of the native species emerge, the transient resource of suppressed trees had already mostly been exploited. However, S. nigricornis could have an advantage in situations where pines unacceptable to S. noctilio for some reason could be available for use by S. nigricornis or when pines could become weakened (e.g., by lightning strikes) after the main flight of S. noctilio adults and before or during the S. nigricornis flight time. In addition, the fact that the transient resource of suppressed trees are often patchily distributed (e.g., Ayres et al. 2014) could lead to coexistence of these species via differential spatial distribution of use of suppressed pines. Monceau et al. (2015) have shown that some degree of niche differentiation between native and invasive hornets can minimize competition. We did not sample recently dead trees in this study but experiments have shown that S. nigricornis will oviposit into wood from trees that have recently been cut, although oviposition was minimal as soon as 30 days after cutting the wood (Hartshorn 2012). We do not know to what extent S. noctilio will choose to oviposit in wood from recently cut trees but when siricids were reared from diverse types of wood sampled over eight years from Europe, Turkey and North Africa, S. noctilio only emerged from samples from standing trees and timber and never from fallen trees or wood on the ground (Spradbery and Kirk 1978). While niches of these two Sirex species probably differ to some extent relative to the health or condition of pines that are preferred or acceptable for oviposition and development, there is also niche overlap (see Suppl. material 1: A). Based on relative densities and traits, we hypothesize that after the invasion of S. noctilio, S. nigricornis could develop in suppressed trees less often than prior to the invasion and the native might now more frequently use recently fallen, dead trees, as S. noctilio would have already exploited the majority of standing suppressed pines. Alternatively, suppressed pines further weakened by S. noctilio attack could at times provide an increased resource for S. nigricornis populations. Knowledge about normal densities of S. nigricornis before invasion by S. noctilio is necessary to further understand the impact of the native on the invasive woodwasp. In addition, further studies investigating the health of pines associated with oviposition and development of these two Sirex species are needed to clarify the overlap in niches of these two species with regard to tree health.

Conclusions
In this study, we investigated aspects of the biology and ecology of these now-sympatric native and invasive siricids toward predicting the impact S. noctilio might be having on this congeneric native species also utilizing suppressed pines (Suppl. material 1: A). The invasive S. noctilio uses the same tree species as the native S. nigricornis but venom glands of S. noctilio are larger. Although S. nigricornis carries more eggs than S. noctilio per body size, S. nigricornis adults are significantly smaller than S. noctilio, resulting in greater fitness for S. noctilio. In other systems, fecundity has been shown to be an important functional trait for predicting success of invasives (e.g., Boivin et al. 2008;Capellini et al. 2015). In addition, the phenology of the invasive and its faster speed of development also allow S. noctilio to exploit common resources before S. nigricornis.
We cannot definitively answer to what extent the presence of S. noctilio results in more or less habitat in which S. nigricornis can develop. Little is known of the biology and ecology of S. nigricornis but we know that it will oviposit and develop in pines already attacked by S. noctilio. Attacks by S. noctilio could create more suppressed trees acceptable to S. nigricornis by pre-injecting the symbiont plus venom and thereby disabling tree defenses and thus creating more habitat for S. nigricornis. However, abundance data suggest that few S. nigricornis emerge from trees that they co-inhabit with S. noctilio. As an alternative, since S. noctilio mostly flies and oviposits before S. nigricornis, perhaps this invasive attacks the best of the transient resource of weakened trees that could potentially be used by either species and S. noctilio thus uses most of this resource before S. nigricornis adult females would have emerged to oviposit. The extent that co-occurring S. nigricornis and S. noctilio will compete for recently dead trees remains to be determined. Further data documenting the ecology and naturally occurring densities of S. nigricornis in the southeastern US, where pine forests are extensive, will assist with predicting to what extent S. nigricornis niches and abundance will be altered when S. noctilio spreads into this region.
Seven years of NeoBiotathe times, were they a changin'? Ingolf Kühn 1,2,3 ,Petr Pyšek 4,5 ,Ingo Kowarik 6,7 Background During the NEOBIOTA conference 2010 in Copenhagen (see http://www.neobiota. eu/conferences for an overview of all conferences), the attendants decided to transform the serial of the European Group on Biological Invasions Neobiota, edited by Ingo Kowarik and Uwe Starfinger, into an international, open access journal. In the following year, NeoBiota was relaunched under the same name, but with an upper case 'B', by Pensoft Publishers. In the editorial of the first issue, a large group of co-editors claimed for openness in covering a broad range of issues in invasion science, including the intersections with applied and social sciences, and referring to different groups of taxa and geographical regions (Kühn et al. 2011). What happened since then? We think that it is now time to shortly reflect how the new NeoBiota journal has developed in the first years of its infancy -based on some data on the published papers, the addressed topics and the geographical background of our contributing authors.
First of all, we are pleased with the increasing visibility of NeoBiota -thankworthy to many papers by our esteemed authors that you, our readers, found interesting. Since the relaunch in 2011, we passed through two stages with respect to visibility in major bibliometric databases, namely ISI Web of Science (since 2017 Clarivate Analytics, http://www.webofknowledge.com) and Scopus (https://www.scopus.com/home.uri). We were scrutinised for the first five years by both companies. All papers published since 2015 by NeoBiota are now listed by Web of Science as well as Scopus. But also papers published before 2015 are well visible: up to October 2017, they were cited on average, more than 6 times in Web of Science, the more conservative of the two bibliometric databases recognised in this study.

Some early highlights
Indeed, a range of papers seems to have clearly raised timely scientific interest and hence contributed to get successfully listed in both bibliometric databases. For brevity, we just present some prominent examples (cited at least 15 times in Web of Science), starting with the two most cited NeoBiota papers. The first was on the support of major hypotheses in invasion biology by Jeschke et al. (2012). This was some sort of seminal work, leading also to further analyses on this topic (Jeschke 2014). The second was a conceptual framework on prioritising alien species for management (Kumschick et al. 2012), based on the approach introduced by Nentwig et al. (2010), that also had several follow-up papers (Kumschick et al. 2015, Kumschick and Richardson 2013. Bridging the two aforementioned topics is the study of Colautti et al. (2014). They used hypotheses in invasion biology and improved tests of these by introducing a simple mathematical framework to quantify the invasiveness of species. Also the work of Atwood and Meyerson (2011) was based on favourite hypotheses in invasion biology. They argue that the lack of consensus across studies that test EICA (evolution of increased competitive ability; Blossey and Nötzold 1995) may be in part due to the lack of consistent definitions and varying experimental designs. They provide a design framework that will increase data harmony across future studies and will facilitate examinations of any potential selection pressure driving evolution in the invaded range. Humair et al. (2014) featured an essay on why experts disagree on common concepts and risk assessments. Gassó et al. (2012) modelled the potential distribution range of invasive plant species in Spain.
The work of Kowarik and von der Lippe (2011) on secondary wind dispersal of an invasive species in urban road corridors is one of a suite of related papers on dispersal of alien plant species along urban roads (von der Lippe and Kowarik 2007aKowarik , b, 2008 and was followed by another experimental study (von der Lippe et al. 2013). Saul et al. (2013) provided a seminal study that lead to a more detailed one (Saul and Jeschke 2015) on the role of ecoevolutionary experience in invasion success. The study of MacNeil et al. (2013) shows how analysing the functional response of alien gammarid species enhances understanding of the success or failure of invasions in the face of various resident predators. Lastly, checklists of alien species are used by many others, such as the inventory of invasive alien species in China (Xu et al. 2012).

Submissions before and after 2015
Here we analyse whether certain characteristics have changed for papers submitted to NeoBiota before getting listed on Web of Science and Scopus in 2015 and after getting listed. In particular, we will explore whether rejection rates, paper lengths, countries of authors and topics have changed. We considered all papers submitted to NeoBiota from 2011 until September 2017. Although looking hard, we did not find any publication trying this sort of analysis for other papers of new journals with sufficient time before and after being listed in the relevant bibliometric databases.
The number of submissions was rather stable (Figure 1), with slightly more papers submitted per year after being listed (not accounting for incomplete 2017). Until 2014, 162 papers were submitted to NeoBiota (i.e. before being listed) and 112 since 2015 until September 2017 (after being listed). In the prelisting phase, we accepted 89 papers, after that 52 (see Figure 2), resulting in a rejection rate of 45.1% and 53.6%, respectively. The difference is not significant, though (χ 2 = 1.59, df = 1, p = 0.21). The length of the published papers did not change significantly (mean±standard deviation: 17.1 ±6.9 vs 19.0 ±8.1, t = -1.34, p = 0.18) between the two periods.
Although a lower number of papers were published so far in the second period, the number of individual authors of published papers increased from 168 to 191; the median number of authors increased only insignificantly (W = 2073, p = 0.3)  from 3 to 4 per paper. Yet, there were some remarkable changes in the countries of the institutions the submitting authors were affiliated with ( Figure 3, Table 1). In the years 2011-2014, especially submissions coauthored by researchers from Spain, but also Canada, China, Ecuador, Germany, The Netherlands, Switzerland, UK, USA were over-represented compared with the second period. In 2015-2017, especially Austria, New Zealand, South Africa were over-represented compared with the first period. These changes were significant (χ 2 = 133.7, df = 38, p < 0.0001). Still, in terms of the total number of submissions we observe the geographical bias stated by , i.e. Europe, North America and Australia are over-represented, many parts of Africa, Asia and South America are under-represented.

Topics covered
The topics covered by NeoBiota range across a variety of issues (Figure 4). Most prominent, among the papers rejected as well as accepted in both periods, is the term "plant", indicating a taxonomic bias . Modelling studies as well as distributional analyses are more represented in those papers that were rejected. This mirrors the availability of methods and data. The Global Biodiversity Information Facility GBIF (http://gbif.org) is a great source of information. Unfortunately, it does have many biases in occurrence records as well as taxa (Beck et al. 2014, Meyer et al. 2015. Similarly, MaxEnt (Elith et al. 2011) is an extremely powerful tool for distributional analyses, if properly used (Merow et al. 2013, Kramer-Schadt et al. 2013. Combining not revised GBIF data with standard settings of MaxEnt, though, leading to poor ecological results, can be a reason for an immediate rejection. But also successful distributional analyses were published on taxa that were not mainstream, until recently. Saltmarsh et al. (2016) published an analysis on the distribution and abundance of exotic earthworms in Alaskan forests. A combination of ecophysiological models with a correlative model to project coypu (Myocastor coypus) distribution under climate change was presented by Jarnevich et al. (2017). Tabak et al. (2015) modelled the distribution of Norway rats (Rattus norvegicus) on offshore islands in the Falkland Islands.
Also quite prominent in both periods were papers on risk assessment. This topic even made it into the so far (December 2017) only "highly cited" paper, i.e. among the top cited papers of their publication cohort: namely Kumschick et al. (2017), Table 1. Geographical background of authors, illustrated by the number of countries of the institutions the submitting authors are affiliated with (multiple affiliations can result in multiple countries per author) submitted to NeoBiota before and after being listed in Web of Science and Scopus in 2015.  prioritisations for Amur honeysuckle (Lonicera maackii) and conclude that the flexible AHP tool could be useful for prioritizing management of exotic plant invasions. Laypersons' perceptions of invasive alien plant species and their attitudes towards their management were analysed by Lindemann-Matthies (2016) in Switzerland. Few participants could correctly identify pictures of alien species. Knowing a species, though, resulted in a higher positive attitude towards their management, but the perceived beauty of a species inhibited support of their management. Planted forests are a major source of invasive alien trees in Europe. Therefore Brundu and Richardson (2016) introduced the 'Code of Conduct on Planted Forest and Invasive Alien Trees' relevant to stakeholders and decision makers in the 47 Member States of the Council of Europe. Panetta and Gooden (2017) review different management options for biodiversity, recognizing impact and action thresholds for invasive plants in natural ecosystems. They conclude that economic and ecological considerations are aligned when invaders are sustainably maintained at relatively low abundances.

Some highlights since 2015
NeoBiota always claimed to be open minded and aimed at facilitating scientific discussion (Kühn et al. 2011). We therefore always welcome papers raising scientific discourse. One of the most controversial papers probably was that of Hoffmann and Courchamp (2016). The authors argued that human-mediated invasions are part of the spectrum of species movements, not a unique phenomenon, because species self-dispersing into novel environments are subject to the same barriers of survival, reproduction, dispersal and further range expansion as those assisted by people. They proposed an all-encompassing framework of species range expansion, including alien species. This paper was challenged by Wilson et al. (2016), who state that invasion science now is not only a biological phenomenon, but that the human dimension of invasions is a fundamental component in the social-ecological systems in which invasions need to be understood and managed.
Other NeoBiota highlights published since 2015 that were well perceived, cover several different aspects: Using data from the DAISIE database (www.europe-aliens.org) (DAISIE 2009), Pergl et al. (2017) address whether established alien plants, mammals, freshwater fish and terrestrial invertebrates with known ecological impacts are associated with particular introduction pathways (release, escape, contaminant, stowaway, corridor and unaided; Hulme et al. (2008). Woodford et al. (2016) review problems arising from the management of biological invasions and argue that they can be either tame (with simple or obvious solutions) or wicked, where difficulty in appropriately defining the problem can make complete solutions impossible to find. On a similar topic Kuebbing and Simberloff (2015) surveyed land stewards of a major conservation NGO. Their results indicate that these managers are selective rather than profligate, targeting species that are having a demonstrable impact or are likely to do so. Another aspect of impacts of alien species are human health problems, reviewed by Schindler et al. (2015) for Europe.

Outlook
So far, NeoBiota seems to be well perceived by the invasions science community. We have found some differences regarding submissions before and after the listing of NeoBiota by Web of Science and Scopus. It would be interesting to see, how submission rate, rejection rate, involved countries and featured topics would change in the future, not only following recent advances in the scientific literature (Ricciardi et al. 2017), but also in response to increasing Scopus CiteScores or receiving an Web of Knowledge impact factor.
Despite the broad range of issues addressed by the previous contributions of 463 individual authors from 38 countries to NeoBiota, we are still short of papers covering social, legal or economic aspects. We thus strongly encourage further submissions also from these topical areas. Still we are confident that NeoBiota will gain an increasing role in all aspects related to the multi-disciplinary topics of invasion science and its interconnections with other disciplines.