Ecology

Experimental design and community composition
We conducted a randomized-block experiment at the Russell E. Larsen Agricultural Research Center (Pennsylvania Furnace, PA, USA) with replicated mesocosm ponds. Mesocosms were 1100-L cattle tanks covered with 60% shade cloth. The spatial block was distance from a tree line in our mesocosm field. Three weeks before pesticide application, these mesocosms were filled with 800 L water, 300 g mixed hardwood leaves, and inoculations of zooplankton, periphyton, and phytoplankton homogenized from four local ponds. Just before pesticide application on the same day, each tank received two snail, three larval anuran, one larval dragonfly, one water bug, one water beetle, one larval salamander, and one backswimmer species (11 Helisoma (Planorbella) trivolvis, 10 Physa gyrina; 20 Hyla versicolor, 20 Lithobates palustris, 20 Lithobates clamitans; 2 Anax junius; 2 Belostoma flumineum; 5 Hydrochara sp.; 3 Ambystoma maculatum; 6 Nototeca undulata) (Fig. 1b). These community members naturally coexist and were applied at naturally occurring densities40. Initial conditions of some mesocosms varied in simulated pesticide treatments (see below).
We randomly assigned 18 treatments (12 pesticides, 4 simulated pesticides, 2 controls) with four replicate mesocosms of each treatment, which resulted in 72 total mesocosms (Fig. 1a). The 12 pesticide treatments were nested; we included two pesticide types (insecticide, herbicide), two classes within each pesticide type (organophosphate insecticide, carbamate insecticide, chloroacetanilide herbicide, triazine herbicide), and three different pesticides in each of four classes (Fig. 1a). To represent runoff of pesticides into freshwater systems following a rainfall event, we applied single doses of technical grade pesticides at environmentally relevant concentrations at the beginning of the experiment. To ensure our exposures represented environmental relevance, we used estimated environmental concentrations of pesticides, calculated by U.S. Environmental Protection Agency’s GENEEC v2 software, Supplementary Table 2). Our design also included water and solvent (0.0001% acetone) controls (Fig. 1a). Pesticides were obtained from ChemService (West Chester, PA, USA). Nominal concentrations of pesticides (μg/L) were: 64 chlorpyrifos, 101 malathion, 171 terbufos, 91 aldicarb, 219 carbaryl, 209 carbofuran, 123 acetochlor, 127 alachlor, 105 metolachlor, 102 atrazine, 202 simazine, and 106 propazine. We collected composite water samples 1 h after application to mesocosms and shipped samples on ice to Mississippi State Chemical Laboratory to verify these nominal concentrations. Measured concentrations of pesticides (μg/L) were: 60 chlorpyrifos, 105 malathion, 174 terbufos, 84 aldicarb, 203 carbaryl, 227 carbofuran, 139 acetochlor, 113 alachlor, 114 metolachlor, 117 atrazine, 180 simazine, and 129 propazine.
The four simulated pesticide treatments were top-down or bottom-up food web manipulations intended to mimic effects of actual herbicides and insecticides on community members. These manipulations occurred once and were concurrent with the timing of pesticide applications. Top-down and bottom-up simulated insecticide treatments were designed to reduce densities of zooplankton, simulating effects of insecticides on zooplankton survival. For top-down simulated insecticides, we doubled the densities of zooplankton predators by including six total A. maculatum larval salamanders and 12 N. undulata backswimmers per mesocosm. For bottom-up simulated insecticides (i.e., direct manipulation of a lower arthropod trophic level), we removed zooplankton with a net. Top-down and bottom-up simulated herbicides were designed to reduce algae, simulating effects of herbicides on survival and growth of algae. For top-down simulated herbicides, we doubled the densities of large herbivores to increase grazing pressure by including 22 H. trivolvis snails, 20 P. gyrina snails, 40 H. versicolor larval anurans, 40 L. palustris larval anurans, and 40 L. clamitans larval anurans per mesocosm. For bottom-up simulated herbicides, we covered mesocosms in three sheets of 60% shade cloth in an attempt to block light and reduce photosynthesis. The experiment ran for four weeks, from June to July.
Measurements of experimental responses
During the experiment, we sampled periphyton using clay tiles (100 cm2) oriented perpendicularly along the bottom of the mesocosm. Each mesocosm had two periphyton measurements: ‘inaccessible periphyton’ taken from caged clay tiles that excluded herbivores and ‘accessible periphyton’ taken from clay tiles that were uncaged, allowing herbivore access. We sampled phytoplankton from water samples taken 10 cm below the water surface. Periphyton was scrubbed from tiles and phytoplankton from water samples (10 mL) were filtered onto glass fiber filters (under low vacuum pressure, More

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Affiliations

Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia
Devi Veytia, Stuart Corney & Sophie Bestley

Australian Antarctic Division, Department of Agriculture, Water and the Environment, Kingston, Tasmania, Australia
Klaus M. Meiners & So Kawaguchi

Antarctic Climate and Ecosystems Cooperative Research Centre, University of Tasmania, Hobart, Tasmania, Australia
Klaus M. Meiners, So Kawaguchi & Sophie Bestley

British Antarctic Survey, Cambridge, UK
Eugene J. Murphy

Authors
Devi Veytia

Stuart Corney

Klaus M. Meiners

So Kawaguchi

Eugene J. Murphy

Sophie Bestley

Corresponding author
Correspondence to Devi Veytia. More

Deformation measurement accuracy
To evaluate the accuracy of ICT, synthetic deformation fields were added to the R-specimen datasets (Fig. 3a). For this purpose, constant strain was simultaneously introduced in both R and L directions ((varepsilon_{RR}),({ }varepsilon_{LL})). Absolute accuracy (hat{varepsilon }) was measured by adding synthetic deformation to the reference state #1 (#1 + synthetic), and then measuring with ICT strain of #1 + synthetic with respect to #1. Differential accuracy ({Delta }hat{varepsilon }) was measured by adding synthetic deformation to the deformed state #2 (#2 + synthetic), measuring with ICT strain of #2 + synthetic with respect to the reference state #1, and finally subtracting the ICT measured strain between #2 and #1.
Figure 3 shows ICT strain estimates for tracheids and wood rays. The accuracy is highest along the cell-cross section (RT for tracheids, LT for wood rays) and lowest in the cell longitudinal direction (L for tracheids, R for wood rays). Due to the tubular cell geometry (Fig. 2b,c) of both cell types, tracking is more challenging in the longitudinal cell axis, for which symmetry reduces the available landmarks (for instance, wood pits in Fig. 2) and deformation tracking accuracy. Absolute accuracy is limited by both cell segmentation and deformation parametrization. Differential accuracy is further influenced by experimental uncertainties between #1 and #2 acquisitions, such as vibration artifacts and sample relaxation strains. While absolute and differential accuracy are similar in cell-cross sections, differential accuracy is reduced with respect to absolute accuracy along the longitudinal cell axis. The sensitivity limit for strain measurements in tracheids is (varepsilon_{RR})  More

Collecting plant specimens
In the present study, 10 accessions assigned to 4 genera Atalantia, Luvunga, Paramignya, and Severinia were collected from different sites in Khanh Hoa and Lam Dong provinces of Vietnam (Fig. 1). Of these, six accessions of P. trimera (Oliv.) Burkill were collected at different sites in Khanh Hoa provinces including Ninh Van (PT1.NV, PT2.NV), Ninh Hoa (PT1.NH, PT2.NH), Dien Khanh (PT1.DK, PT2.DK); 1 accession of A. buxifolia (Poir.) Oliv. ex Benth collected in Van Ninh (PA.VN); 1 accession of S. monophylla (Lour.) Tanaka collected in Don Duong, Lam Dong province (PC.DD); two accessions of L. scandens (Roxb.), Wight, collected in Di Linh (PR.DL) and Cat Tien (PR.CT) in Lam Dong province (Fig. 1). The list of the collected accessions and information was summarized in Table 1.
Figure 1

Map of the sampling sites. Accessions of species P. trimera (Oliv.) Burkill, A. buxifolia (Poir.) Oliv. ex Benth, S. monophylla (Lour.) Tanaka, and L. scandens (Roxb.), Wight were collected at sites displayed as circles in the map. The map was created by using ArcGIS 10.3 using the color rendering and grouping tools built-in and Paintbrush version 2.5 (20190914) on mac OS Catalina.

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Table 1 List of the collected accessions and information.
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Taxonomic treatment
P. trimera (Oliv.) Burkill distributes in the high land areas in Khanh Hoa, Lam Dong provinces of Vietnam. P. trimera is scrambling shrub or erect, long, and curved spines, non-hairy stem. Leaves simple, typical narrow oblong, lamina 1.0–1.5 cm wide, 5–12 cm long; short petiole 0.5 cm long, leaf sub-vein 8–10 pairs; inflorescences axillary, fasciculate, peduncle 3–4 mm long, separate; calyx 3 lobes, 4 mm long; corolla 3; stamens 5, separate; ovaries 3, only 1 ovule, 2 locules in the ovary; globose fruit, 1.5–2.5 cm in diameter, 2 seeded. flowering time from May-Aug., fruiting Sep-Dec. Roots, leaves and stems were used as traditional medicine to treat liver diseases and cancers (Figs. 2, 4a).
Figure 2

The typical morphology and anatomy of Paramignya trimera (Oliv.) Burkill. Woody shrub 1–4 m or above (a); A flowering tree (b); Typical trimerous flowers (c); Green fruits (d); Ripen fruits (d); Opened ripen fruit with two seeds encapsulated by mucus endocarp (e).

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A. buxifolia (Poir.) Oliv. ex Benth distributed mainly in Van Ninh (Khanh Hoa) with several local names such as “Xao cua ga” or “Quyt gai” are medium climbing shrubs, up to 3 m tall; branches grayish brown, branchlets green; spikes axillary 0.5–1.2 cm or sometimes unarmed, apex yellowish; leaves simple, 2.5–3.5 cm wide, 3.5–4.5 mm long, petiole 4–8 mm, leaf blade ovate, obovate, elliptic, glabrous, coriaceous, midvein slightly ridged, apex rounded to obtuse at tip; inflorescences axillary, 1 to several flowers. Flowers 5 merous, petals white, 3–4 mm, stamens 10, calyx persistent. Fruit bluish black when ripe, globose, slightly oblate, or subellipsoid, 7–10 mm in diam., smooth, 1 or 2 seeded. Flowering from May-Aug., fruiting Sep-Dec. Roots, leaves and stems were used as traditional medicine to treat cough, lung diseases and kidney disorders (Fig. 4b).
S. monophylla (Lour.) Tanaka found in Don Duong (Lam Dong) was thorny shrub or small tree; spikes axillary 1–1.5 cm; leaves simple, ovate, apex round or retuse at tip, coriaceous, glabrous, round at base, short petiole; Inflorescences 4–6-flowered; calyx ca. 3.5–5 mm long; petals 4, petals white, oblong, obtuse, glabrous, stamens 8–10; filaments ca. 12 mm long, glabrous; anthers ca. 5 mm long, linear; ovary ca. 2.5 × 1.5 mm, long-ovoid, glabrous, 3-locular; style ca. 7 mm long, continuous with ovary, cylindric, glandular, glabrous; stigma capitate ca. 2.5 mm broad, glandular. Fruits yellow to orange, globose 1.5–2.0 cm in diameter, 1–2 seeded; flowering time from May-Aug., fruiting Sep-Dec. This species was used effectively for cough, expectorant, fever, anti-inflammatory, sciatica treatment and prevent aging of skin cells, roots and leaves used for skin disease, burning leaves to kill mosquitoes and insects (Fig. 2c).
L. scandens (Roxb.), Wight was discovered in Lam Dong of Vietnam with the local name “Xao leo”. L. scandens is woody climber or scrambling shrub; rough tufted from the ground with strong axillary sharp straight or slightly recurved spines. Leaves compound, digitately trifoliate or bifoliolate or simple; petioles 2–6 cm long, glabrous; lamina ca. 6.0–18.0 × 2.5–4.0 cm, variable, oblong-elliptic or oblanceolate, cuneate at base, shortly acuminate at apex, coriaceous, glabrous; secondary nerves 15 pairs; branches brown puberulent. No information from flowering time has been described. According to traditional experience, this plant is used to treat rheumatism, liver disease and ascites (Fig. 2d).
Phylogenetic relation analysis
The phylogenetic tree from ITS sequences included 3 groups (Fig. 5a). The first monophyletic group was only S. monophylla (PC.DD) as an out group. The second monophyletic group included 2 accessions of L. scandens (PR.DL and PR.CT). The third group was paraphyletic group with 9 accessions clustered in 2 sub-groups. The first sub-group included only P. trimera, whereas the second sub-group included 3 accessions P. trimera nested with P. confertifolia and A. buxifolia. In addition, in the second sub-group, the accessions of P. trimera collected in Dien Khanh, Vietnam (PT1.DK) and P. confertifolia from Mensong, China were in the same monophyletic clade whereas A. buxifolia (PA.VN) was clearly separated from others.
The unrooted tree from matK sequences included 3 groups in which the first monophyletic group were 2 species P. lobata and P. scandens (Australia), the second monophyletic group included only P. confertifolia (China) and the third group (paraphyletic group) included 3 sub-groups (Fig. 5b). The first sub-group included all accessions of P. trimera, the second sub-group included only S. monophylla and the third sub-group included L. scandens and A. buxifolia.
The unrooted tree from rbcL sequences included 2 main groups in which the first group included 3 species P. scandens, P. monophylla and P. lobata (Australia) and the second group (paraphilic group) included 5 species P. trimera, P. confertifolia (China), S. monophylla (Japan), A. buxifolia, and L. scandens (Fig. 5c). In this group, some accessions of P. trimera were nested in the paraphylic sub-groups because they did not share an immediate common ancestor.
The pattern of the phylogenetic tree constructed from the concatenated sequences was similar to that of ITS sequences (Fig. 5d). The tree included one monophyletic group with only L. scandens and one paraphyletic group with the accessions of P. trimera nested within P. confertifolia, A. buxifolia and S. monophylla.
Genetic distance analysis
The overall genetic distances for ITS, matK, rbcL and concatenated sequences were 0.11 ± 0.01, 0.29 ± 0.02, rbcL 0.48 ± 0.05 and 0.05 ± 0.0, respectively (Table 2). An overlap between the maximum intraspecific distances and the minimum interspecific distances were observed in the cases of ITS, rbcL and concatenated sequences (Table 2, Fig. 6a,c,d). In case of matK, a clear barcode gap was found between the maximum intraspecific distance (0.0028) and the minimum interspecific distance (0.0056). The histogram and ranked pairwise (K2P) distances demonstrated a significant difference in the cases of matK and rbcL (Fig. 6b,c).
Table 2 Intraspecific and interspecific distances across all data.
Full size table More

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