DOI: 10.5061/dryad.f7m0cfxsg
title: Data from: Allelopathy and its co-evolutionary implications between native and non-native neighbors of invasive Cynara cardunculus L.

description: Invasive plants apply new selection pressures on neighbor plant species by
 different means including allelopathy. Recent evidence shows allelopathy
 functions as remarkably influential mediator for invaders to be successful
 in their invaded range. However, few studies have determined whether
 native and non-native species co-occurring with invaders have evolved
 tolerance to allelopathy. In this study, we conducted germination and
 growth experiments to evaluate whether co-occurring native Juncus pallidus
 and non-native Lolium rigidum species may evolve tolerance to the
 allelochemicals induced by Cyanara cardunculus in Australian agricultural
 fields. The test species were germinated and grown in pots filled with
 collected invaded and uninvaded rhizosphere soil of C. cardunculus with
 and without activated carbon (AC). Additionally, a separate experiment was
 done to differentiate the direct effects of AC on the test species. The
 soil properties showed invaded rhizosphere soils had higher total phenolic
 and lower pH compared with un-invaded soils. We found significant
 reduction of germination percentage and seedling growth in terms of
 above-and below ground biomass, maximum plant height and root length of
 native in the invaded rhizosphere soil of C. cardunculus, but little
 effect on non-native grass species. Even soil manipulated with AC showed
 no significant differences in the measured parameters of non-native except
 aboveground biomass. Taken together, the results indicate allelochemicals
 induced by C. cardunculus exert more suppressive effects on native than
 non-native linking the co-evolved tolerance of those.
description: MATERIALS AND METHODS Our experiments were
 set to test the allelopathic effects of <em>C. cardunculus
 </em>on germination and growth of grass species that co-occurs in
 agriculture field in Victoria, Australia. We used field-collected
 rhizosphere soils to imitate natural conditions through exposing of target
 species that minimize uncertainties in plant extract concentrations or
 exclusion of other possible edaphic effects (Gómez-Aparicio &
 Canham, 2008). As the allelopathic effects are species-specific (Callaway
 & Ridenour, 2004; Gomez-Aparicio & Canham, 2008), we also
 devised the experiments to determine the evolved tolerance of associated
 native versus non-native grass species to the allelopathic potential of
 <em>C. cardunculus </em>in its invaded community. In addition,
 chemical properties (total phenolic content and pH) of <i>C.
 cardunculus</i> rhizosphere were assessed to test the differences
 among the populations. Overall, this study aimed to evaluate allelopathy
 and co-evolutionary effects of rhizosphere soil induced by <i>C.
 cardunculus</i> on co-occurring native and non-native grass species
 in its invaded range. Measurement of field soil
 chemical properties To test whether invasive plant
 species <i>C. cardunculus</i> changes chemical soil
 properties, we collected rhizosphere soil at the end of November, 2018
 during its highest biomass production from three invaded and three nearby
 non-invaded populations in agricultural land at 26.7 m above sea level in
 Werribee South, Victoria, , Australia (37°54'44.5"S
 144°42'00.8"E). We designed our sampling protocol to minimize
 the effects through selection of homogenous soil profile, and plant
 density and diversity in uninvaded and invaded sites of each population,
 which were also close to each other. The location of each population
 including soil from both invaded and nearby non-invaded was separated from
 the others by a distance of at least 500 m. At each location, we collected
 five samples from five individuals of <i>C. cardunculus</i> as
 invaded soil samples and five from nearby non-invaded population where
 other species such as <i>Juncus pallidus</i>,
 <i>Maireana decalvans</i>, <i>Themeda triandra</i>
 etc. except <i>C. cardunculus</i> were present. After that,
 the soil samples were transported into the laboratory, sorted and
 homogenized for composite sample. Then, we took five sub-samples from each
 population for measurement of soil properties and measured total phenolic
 content (TPC) and pH in soil as it is assumed those properties might play
 a significant role in testing allelopathy, though rhizosphere has also
 numerous aspects (Scavo, Abbate, et al., 2019). Soil pH was determined
 with a pH meter (Pocket digital pH meter, 99559, Dick Smith Electronics,
 Australia) in a 1: 2.5 w/v (soil: distilled water) ratio (Paz‐Ferreiro,
 Trasar‐Cepeda, Leirós, Seoane, & Gil‐Sotres, 2007). Soil phenolics
 was determined by sampling 100 mg of air dried soil following the
 Folin-Ciocalteu method (Blainski, Lopes, & De Mello,
 2013). Germination experiment
 However, the multiple populations provide the possibility to
 check the variation among populations through increasing the reliability
 of the results, and better representing the condition of each treatment,
 but we avoided using multiple populations in this occasion due to
 complexity in research design and the possibility of confusion. The
 collected homogenized rhizosphere soil from only one population was used
 in the experiment as no attempt was made to check variation among
 populations. Half of the soil (invaded and non-invaded) was thoroughly
 mixed with activated carbon (AC) and the mixture was placed into the pots
 as a treatment. The remaining soil was considered as a treatment without
 AC. The AC has special characteristics with high absorptivity of organic
 complexes such as allelochemicals, and weak affinity for inorganic
 molecules, such as those present in nutrient solution. Many allelopathic
 studies related to <i>C. stoebe </i>including<i> C.
 maculosa</i>, <i>C. rhenana, C. muretii, C.
 vallesiaca</i> and <i>C. stoebe</i> subsp. have
 demonstrated through reducing the suppressive effects of root exudates
 (Callaway, Ridenour, Laboski, Weir, & Vivanco, 2005; Lyytinen
 & Lindstrom, 2019; Ridenour & Callaway, 2001). The 100 g
 soils were placed in punnets (9.75 cm by 6.75 cm) with five replicates.
 The punnets were moistened and kept at room temperature for 24 hours to
 activate the soil microbes. The sterilized seeds (collected from fields
 invaded by <i>C. cardunculus</i> L.) of native <i>Juncus
 pallidus</i> and non-native <i>Lolium rigidum</i> grass
 species were then seeded at a rate of 20/pot. The tested seeds were
 selected due to the following reasons: i) they are co-occurring plant
 species of <i>C. cardunculus</i> L.; ii) allelopathic
 interactions between invasive alien and coexisting plant species (native
 and non-native) seem to be one of the underlying mechanisms for the
 invasion success of some invaders; and iii) testing the evolved tolerance
 of the co-existing species. We followed a complete randomized block design
 in this study. The pots were kept in shade for 3 days, and then
 transferred to the natural lit greenhouse at 23 ± 3° C day and 12 ± 2° C
 night temperatures. The pots were watered with automated irrigation system
 twice in a day. Pots were randomly shuffled every week to minimize the
 spatial effects. Then, after four weeks, the germinant was counted for
 each treatment. Growth and establishment
 experiment The soils were prepared as per the
 abovementioned germination experiment and the 100g soils were placed in
 punnets (9.75 cm by 6.75 cm) for growth experiment. However, the size of
 the punnets may seem tighten for growth of whole period of the experiment,
 but the plants can adopt with it, and it may not interfere the findings as
 it was applicable for all replicates of both tested species. About 300
 (three hundred) sterilized seeds of each grass species (native and
 non-native) were germinated in a tray (30 cm by 28 cm) filled with
 sterilized sand. One seedling of both species was then transplanted into
 the prepared punnet by twenty-one replicates and pots were grouped by
 target species following a randomization across treatments and kept as per
 the abovementioned condition. Dead seedlings were replaced after a period
 of two weeks, to allow the experiment to continue. The pots were watered
 using an automated irrigation system twice in a day and fertilized
 fortnightly with liquid fertilizer (Hyponex, N-P-K, 6-10-5, Hyponex Inc.,
 Japan) at a concentration of 2ml/1 of water. Pots were randomly shuffled
 every week to minimize the spatial effects, and after 3.5 months, the
 plants were harvested for biometric parameters including maximum plant
 height and root length, aboveground biomass (AGB), belowground biomass
 (BGB), total biomass (AGB plus BGB), and AGB-BGB ratio
 We conducted a separate experiment along with the main
 experiments to assess the direct effect of AC on seedlings growth of
 native and non-native used grass species with sterilized sand and AC
 treatments. We used five replicates per treatment (with and without AC)
 for both of grass species. The direct effect of AC on plant growth has
 been assessed due to its complication on allelopathy (Lau et al., 2008).
 This experiment was carried out adopting the same procedure as the
 aforementioned experiment and measured biometric parameters (Appendix
 S1). Data analyses An independent
 sample t-test was performed to check the differences of soil phenolics and
 pH level among un-invaded and invaded sites for each population of
 <i>C. cardunculus </i>L. Two-way ANOVAs were also conducted to
 test the differences of those variables as functions of populations and
 invasion status (un-invaded and invaded). We again conducted two-way
 ANOVAs to test the effect of soil allelopathy as functions of activated
 carbon (with and without AC) and invasion status (un-invaded and invaded)
 on AGB, BGB, AGB-BGB ratio, total biomass, maximum plant height and root
 length of native and non-native grass species. Target species (native and
 non-native) were not considered as third fixed factor in the univariate
 general linear model due to avoid complexity. For example, the test does
 not provide which specific species is significantly different from each
 other. As a result, separate analysis (species-specific) was done to get
 direct understanding of the allelopathic effects on each of them.
 Moreover, an independent sample t-test was performed to compare the
 significant differences between AC treatments (with vs. without carbon)
 and species (native vs. non-native) in both of germination and growth
 experiments including separate growth experiment. To maintain homogeneity
 of variances and normality of the used data for these analyses, we used
 data transformations techniques:: a square root (soil phenolics, AGB, BGB,
 total biomass for native; AGB fornon-native) and an arcsine-square root
 function (germination percentage for native) We also applied Levene’s test
 for homogeneity of variance and Komolgorov-Smirnov (K-S) test for
 normality of the used data. All data were analyzed using SPSS version 20.0
 (IBM Corporation, NY, USA).