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Changes in propagule pressure from single or multiple regions directly contribute to the success or failure of nonnative species establishment6,8,23,24. In this study, we collected and measured the quantity and diversity of seeds, over time obtained from the air-intake grilles of refrigerated containers, with two seasons for comparison. Targeting the trans-oceanic transport of a single commodity in this industrial trade system that serves as a transport vector for hitchhiking seeds provided reduced variation in which to quantify propagule pressure, including propagule size (Fig. 1) and propagule number (Supplementary Fig. 2) of plant species considered to be of high-risk to agriculture in the USA19. Our key finding is that influx is sufficient and reproduction of these species is high enough to represent a risk of population(s) establishment in and around the shipping port, even with the bottlenecks of escape from the shipping container, subsequent germination, and seedling survival (Figs. 2, 3).
Over 20,000 shipping containers are moved as import or export daily on the GCT 14, providing ample volumes for passive hitchhikers to establish at the GCT and surrounding areas. In fact, we found steady arrival of shipping containers over the approximately 32-week shipping season (Supplementary Fig. 2). Conversely, we found strong seasonal variation in propagule number (i.e., number of seeds per refrigerated shipping container; Fig. 1). We estimated for the FNW, S. spontaneum, that over 40,000 seeds entered GCT during the two shipping seasons (Table 1). This level of propagule pressure is clearly sufficient to represent introduction and establishment risk of a clonal, perennial, fecund species that likely does not require a large initial propagule size, even if the escape rate from the shipping containers is exceedingly small (Table 1; Fig. 2). In this study, the four focal monocotyledonous taxa all had similar seed sizes, and no other larger-sized propagative material of these species (e.g., rhizomatous material or cuttings) were encountered during our study at the GCT.
The theoretical literature postulates that increased numbers of propagules (i.e., propagule size6,8,9,10,11) and pressure (which includes propagule size and frequency as a rate) increases the likelihood of nascent population establishment and population size and diversity6,10; however, among our four focal taxa, a nascent population may establish from a single seed during arrival at a suitable terrestrial substrate, such as the GCT’s greenspaces19. Persistence of an extremely small population can, and is likely to, be facilitated by asexual propagation and spatial spread of these particular plant taxa. Theoretical population biology intrinsically includes propagule pressure within the invasion process6,10, and empirical studies measuring propagule pressure have demonstrated its importance as the most important and generalizable predictor of nonnative invasion success24. Propagule pressure in itself is also the factor most influenced by human activity8,9. Therefore, our study adds additional support to the importance of propagule pressure (see Figs. 2 and 3), and in this system, there is sufficient propagule pressure (i.e., influx from Fig. 3) for invasion success, even if escape rates from shipping containers, germination, and survival are low.
Though S. spontaneum is the only FNW we encountered, we were also able to identify Arundo donax L., a species that is listed as noxious by 46 of the USA’s 50 states25. We were not able to identify seeds to a taxonomic level sufficient to determine origin status for the other 28 taxa encountered (Table S4), but for our three additional focal species, we suggest that two species are native and one is likely introduced. We found Typha domingensis (Pers.) Steud. (native), Andropogon glomeratus (Walter) Britton, Sterns & Poggenb. and A. virginicus L. (both native), and Phragmites australis (Cav.) Steud. (nonnative), already established on-port at the GCT in a previous study that demonstrated that the Port of Savannah is a hub of nonnative species richness19.
For any of the species collected on the shipping containers, the propagules have the potential of being picked up en route to the GCT or, with the exception of S. spontaneum since it is not established there, at the GCT. Most of the taxa already have cosmopolitan distributions, and actual escape rates from the shipping containers are not yet known, meaning that the seeds could make multiple journeys on cargo ships across oceans before being released from a container. Also, the seasonality of seed dispersal coincides with dispersal time in the northern hemisphere, which may apply to seed sources in Panama, the Caribbean, or the USA. Andropogon glomeratus and A. virginicus occur throughout North and Central America (including Panama and the Caribbean)26. As native species to the southeastern USA, propagules escaping refrigerated shipping containers are not of significant concern, although they could be homogenizing genetic composition if genotypes from other portions of the parental established ranges are introduced here. Additionally, Andropogon propagules may result in introductions of nonnative species to South America if the seeds remain on the containers and are viable for return trips. Typha domingensis has nearly a global distribution, and though it is native to the southeastern USA, its presence at the Port of Savannah could also indicate the presence of admixed genotypes. Moreover, our morphological identification of the seeds could not distinguish to the species level, and T. angustifolia L., a nonnative species, could have been represented in our samples, though this species is well established and widely distributed already in the USA26. Phragmites australis, a noxious weed in 6 USA states25, is already found worldwide26. The genus Phragmites contains 4 species, of which only Phragmites australis is native to portions of North America; however, intra- and inter-specific hybridization among genotypes has resulted in the influx of nonnative lineages from Europe and Asia, which have spread to areas of the continent where it is not native27,28.
The most interesting case is S. spontaneum, the FNW. This species is established only in Florida on the USA mainland, where it was introduced for historical and extant breeding programs with sugarcane29. This recent report29 showed that it was naturalized in only three counties, but we have documented it growing in six counties and in cultivation in one additional county (Supplementary Fig. 4). We did not find it growing at the GCT at the Port of Savannah. Yet, it is known that S. spontaneum, which is native to the Indian subcontinent, is well established in the Panama Canal region29,30 along the shipping route of interest. The number of propagules we intercepted and estimated, along with nontrivial germination rates and high survivorship of seedlings, indicate that this species represents a real threat of establishment outside of Florida. Combined with other modeled estimates that S. spontaneum can establish throughout the majority of the USA29, we suggest that this species represents a significant risk of negative invasive species impact, earning its FNW listing in the 1980s18,29.
All four of our focal taxa share common life history features that have been suggested to be characteristic of invasive plant species: asexual reproduction through rhizomes, persistence in a wide range of environmental conditions, prolific seed production (Table 1 and citations within), wind pollination, and wind dispersal31,32,33. These traits have the potential to enhance geographic spread into new ranges and rapidly lead to single-species domination of local plant communities. All of these taxa have a life history and ecology similar to the very successful southeastern USA invasive species cogongrass (Imperata cylindrica (L.) P. Beauv.) that has been demonstrated to benefit from intraspecific heterosis and multiple introductions34,35,36.
A previous study used molecular barcoding of seedlings germinated from seed collected from Season 1 in this study, and they identified some seedlings as: S. spontaneum, Typha sp(p)., Phragmites sp(p)., and Andropogon sp(p).37, as identified here. Seeds that were grouped as S. spontaneum in this study resulted in seedlings that returned haplotypes for the genus Phragmites (rbcL haplotype 1 and matK haplotypes 3 and 437) and Saccharum along with other genera37. There are two interesting and opposing forces at play here. First, in sorting seeds morphologically, there is the potential to group similar looking seeds of different species. The molecular barcode result that shows Phragmites haplotypes in seeds morphologically identified as S. spontaneum is evidence of misidentification and inaccurate sorting of seed. Second, some haplotypes showed equally correct molecular identification across multiple genera of grasses, indicating that these standard molecular barcode sequences for plants may not have the species-level resolution necessary for molecular identification of some of the highest threat invasive grass species.
There are two key approaches to mitigating the risk that propagules of nonnative taxa will become established: 1) prevent propagules from hitchhiking on transoceanic cargo ships, in this case, becoming attached to shipping containers at their point-of-origin or stops along the way (that result from “trans-shipping”), and 2) prevent viable propagules from entering and establishing in the USA, via inspection and interception by the “gatekeepers” of biosecurity at international points-of-entry. These agricultural inspectors are tasked with the interception of propagules of insects, fungi, and all other nonnative or “actionable” taxa, in addition to the seeds of plants. One potential solution to reduce invasion risk by vascular plant seed is to employ a scaled-up version of the research approach we implemented here of backpack vacuuming air-intake grilles of refrigerated shipping containers. Another possibility in lieu of labour-intensive vacuuming of intake grilles is to conduct research on efficacy of liquid pre-emergent herbicide application to the air-intake grilles. For either approach, our data support that these interventions may not be needed year-round for important species like S. spontaneum, which have a clear import seasonality on this particular commodity. For example, based on our data, seed removal measures may only be needed in October, November, and early-mid December.
In the face of poorly resourced capacity for inspection and the potential of diminishing fiscal resources and human capital, consequences include acceleration of biodiversity loss, economic and environmental impacts, and on-going biotic homogenization. The interception efforts to prevent the entry of nonnative propagules of all nonnative taxa worldwide will ultimately conserve local endemism, biodiversity, economic output, and ecosystem services that are interrupted or extirpated by biological invasions1,3. This research aimed to identify key risks and highlights the need for improved strategies for efficacious prevention and interception of nonnative, particularly plant, propagules prior to establishment, though such prevention approaches can be designed and applied for many taxa. Enhancing the capacity, speed, and frequency of successful prevention programs will be required to minimize or eliminate the real risks posed by viable hitchhiking propagules associated with economic trade and sea/air transportation of commodities and people. More

When only local participation in fieldwork is possible, how effectively can remote collaborations be executed in the field sciences, when so much diverse expertise is required?
Foremost is a comprehensive investment in the creation of digital archives at different scales. Various government agencies and developer-led fieldwork as well as excavations in extreme locations have been using such methods and techniques for years14. However, practice is neither standardized nor mainstreamed across comparable research projects, and in the context of COVID-19 there are many reasons to push for such goals.
First, the creation of high-resolution photographic databases for photogrammetry is relatively easy to teach remotely, and is inexpensive, although post-processing time is substantial and requires investment in technicians. These techniques can record and visualize spatial relationships, stratigraphic sequences and, depending on the use of different light, may permit assessments that can even be superior to traditional by-eye illustrations14. Specialists can clearly mark sampling locations on the models for those on site, enabling group assessments. Second, the creation of three-dimensional (3D) geological and sedimentary archives also enables re-assessment of sequences in future. Third, the creation and sharing of both the archives and their interpretations will precipitate the much-needed standardization of sampling and analytical procedures. Within an open data framework, this working model will ensure that novice researchers and non-specialists learn from experts through collaborative, team-based inferences, rather than stitching together the results of individual specialists in a top-down approach.
Our existing international field season schedules also require change — a long-overdue adjustment in the face of increasing anthropogenically induced climate change. Fieldwork will be based locally and projects will require many short and closely spaced field seasons. Those who can access our field sites within a few hours can conduct short-distance trips, focused on discrete steps in the process of assessment, excavation, sampling and inference. For example, a short initial season would focus on building a high-resolution digital model of the field site that can be shared with remote collaborators to develop excavation strategies. A later season could focus solely on sampling, following remote collaborative assessment of digital archives. Such approaches also in part mitigate the problems faced by less-accessible field sites, where frequent online meetings and the exchange of information are impossible. Effective remote collaboration will require very clear scheduling among remote experts at each phase of the process to minimize the burden on local researchers.
At a landscape scale, the situation becomes more challenging. 3D models from unmanned aerial vehicles (UAVs), remote-sensing data15 and LiDAR16 are already widely used in prospection and analysis, and may facilitate effective collaboration between remote specialists and local participants. Remotely generated landscape-scale hypotheses and geomorphological maps can be tested at a later stage by specialists on the ground, whenever longer-distance travel becomes an appropriate option. The creation of 3D data using UAVs does not represent a considerable remote training challenge. A major new research focus should address the extent to which these methods and predictive models in geomorphology are able to replicate, complement and validate assessments of landscape-scale processes made in the field. More

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In this study, koalas from two geographically separated populations in SE Qld, at the Moreton Bay site (MB)9 and the Old Hidden Vale site (HV), underwent regular field monitoring and clinical examinations approximately every 6 months (or more frequently if required for health or welfare concerns). Blood samples, ocular conjunctival swabs and a urogenital tract swab were collected during each clinical examination. From these samples, C. pecorum load and genotype, koala MHC immunogenetics and KoRV proviral subtypes were determined. These results were evaluated in the context of clinical records compiled at the time of sample collection, which included chlamydial disease status.
Chlamydial epidemiology at each study site
The overall prevalence of chlamydial infection and disease differed between the study sites
Longitudinal monitoring of 24 HV koalas (over 113 individual sampling points) identified 24 chlamydial infections for strain typing analysis and eight new chlamydial infections for disease progression analysis. This complemented longitudinal monitoring of 148 MB koalas (over 479 individual sampling points)9 that identified 76 chlamydial infections for strain typing analysis and 38 new chlamydial infections for disease progression analysis. Overall, there was a significantly higher prevalence of infection at HV (58%, 14/24) compared to MB (35%, 89/254)26 (Fisher’s exact test p = 0.028) (Table 1), as well as a significantly higher prevalence of disease at HV (58%, 14/24) compared to MB (27%, 75/279) 26 (Fisher’s exact test p = 0.002).
Table 1 A comparison of chlamydial epidemiology between the Moreton Bay site (MB) and the Old Hidden Vale site (HV).
Full size table

Chlamydial disease progression was common at both study sites
A total of eight HV koalas met our study inclusion criteria for disease progression analysis by having a new chlamydial infection detected at the ocular (n = 1) or urogenital tract site (n = 7) by quantitative polymerase chain reaction (qPCR) over a period of 18 months. These koalas had no evidence of chlamydial infection (infection loads below detection limit) or disease (clinical examination within normal limits) at that anatomical site at their previous clinical examination. If disease was detected at their first clinical examination, they were excluded from disease progression analyses only (unless it was their first sampling as an independent offspring, n = 1).
Interestingly, all of the new chlamydial infections at HV (100%, 8/8) progressed to disease, which was not significantly different to the number of new chlamydial infections at MB that progressed to disease (66%, 25/38)9 (Fisher’s exact test p = 0.084) (Supplementary Fig. S1). For six of these new chlamydial infections at HV (one ocular and five urogenital tract), the infection was detected at the same clinical examination as disease. For the other two new chlamydial infections at HV (both urogenital tract), the infection was present at a clinical examination 2.5 months and 4 months before disease was detected.
The urogenital tract infection load dynamics were similar at both study sites
The urogenital tract infection load (C. pecorum genome copies/µL) in both HV and MB9 koalas was significantly higher when infections were detected at the same clinical examination as disease (1,028,000 copies/µL, range 11,400–4,760,000 copies/µL), in comparison to infections that were present for one or more consecutive clinical examinations before disease was detected or infections that did not progress to disease (600 copies/µL, range 49–522,800 copies/µL) (Mann–Whitney U = 3, p = 0.030). Similarly, the urogenital tract infection load in both HV and MB9 koalas was significantly higher when koalas acquired a new chlamydial infection (within the last three months) (1,834,000 copies/µL, range 52,400–4,760,000 copies/µL), compared to koalas who had long-term infections (present for more than three months) (724 copies/µL, range 35–7,142 copies/µL) (Mann–Whitney U = 0, p = 0.010). Interestingly, the urogenital tract infection load was significantly higher at HV (1,028,000 copies/µL, range 11,400–4,760,000 copies/µL) compared to MB (3,824 copies/µL, range 138–1,340,000 copies/µL) when infections were detected at the same clinical examination as disease (Mann–Whitney U = 11, p = 0.003). In contrast, the urogenital tract infection load was not significantly different between the study sites (HV 600 copies/µL, range 49–522,800 copies/µL vs MB 794 copies/µL, range 16–13,900 copies/µL) when infections were present for one or more consecutive clinical examinations before disease was detected or infections did not progress to disease (Mann–Whitney U = 28, p = 0.703).
The prevalence of chlamydial strains, as determined by Multi-Locus Sequence Typing, differed between the study sites
Overall, 69 C. pecorum-positive samples, comprised of 45 samples from MB (4 ocular and 41 urogenital tract samples from 25 koalas) and 24 samples from HV (2 ocular and 22 urogenital tract samples from 14 koalas), were analysed using a C. pecorum-specific MLST scheme27. Three sequence types (STs) were detected in this study: ST 69, ST 202 and a novel ST (ST 281). ST 69 and ST 202 were detected at both study sites, however their prevalence at each study site was significantly different (Fig. 1). ST 69 was the most prevalent ST at HV, detected in 63% of total samples (15/24) and in 59% of urogenital tract site samples (13/22). ST 69 was significantly less prevalent at MB, detected in 11% of total samples (5/45) and in 12% of urogenital tract site samples (5/41) (overall and urogenital tract site Fisher’s exact test p  More