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No improved resistance against generalist herbivores found for invasive

genotypes: a case study of Senecio vulgaris (Asteraceae)and a generalist

snail (Achatina fulica)

Viet Thang Nguyen 1,2, *, Noel Ndihokubwayo 1,3, *, Dandan Cheng4, **

1 School of Environmental Studies, China University of Geosciences (Wuhan), Wuhan, Hubei,

430074, China 2 Faculty of Biology and Agricultural Techniques, Thai Nguyen University of Education, No. 20,

Luong Ngoc Quyen Street, Thai Nguyen City, Vietnam 3 Ecole Normale Supérieure, Département des Sciences Naturelles, Boulevard du 28 Novembre,

B.P 6983 Bujumbura, Burundi 4State Key Laboratory of Biogeology and Environmental Geology, China University of

Geosciences, Wuhan, Hubei, 430074, China

* contributed equally to the article

** Correspondence: E-mail: [email protected]

Abstract

Invasive plants escape from some natural enemies as predictions of Enemy Release

Hypothesis (ERH). However, in fact they still have to face the pressure of generalist

herbivores in introduced ranges resulting in the maintenance or enhancing of resistance

ability to generalist herbivores. In this study, we carried out a general feeding bioassay in

a laboratory with leaves of Senecio vulgaris to test the difference in resistance between

ranges. White jade land snails (WJLD, Achatina fulica) were fed with the leaves of

Pakchoi (Brassica chinensis), Lettuce (Lactuca sativa), native and invasive plants of S.

vulgaris. The feeding experiment with S. vulgaris leaves was carried out in two waves.

We found that both native and invasive S. vulgaris plant were resistant again to WJLD

compared to Pakchoi and Lettuce. However, there were no significant differences

between native and invasive plants of S. vulgaris in relation to the resistance against

WJLD. The results prove the maintenance of chemical defense against generalist

herbivores in invasive plants in introduced range. The success of S. vulgaris to invader

China could not be explained by releasing from natural enemies but possessing of

defense ability against herbivores before it introduced to China.

Key words: Palatability, natural enemy release, EICA, herbivore resistance

PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1696v1 | CC-BY 4.0 Open Access | rec: 2 Feb 2016, publ: 2 Feb 2016

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1. Introduction

The introduction of plants in new range the alien plants subject to the change of their

natural enemies which may have an important role in the success of their invasion. To

explain the success of biological invasion, it is necessary to take into account of the

interaction between exotic plants and herbivores in introduced ranges. If in introduced

ranges, invasive plants escape from herbivores as predicted by the enemy release

hypothesis (ERH) (Keane & Crawley, 2002), this may result in the increase of growth

and/or reproduction by allocation of resource from defense to growth as the predictions

of the evolution of increased competitive ability (EICA) hypothesis (Blossey & Notzold,

1995). However, the invasive plants do not completely escape from herbivores in the

introduced ranges (Müller-Scharer, Schaffner & Steinger, 2004; Parker, Burkepile & Hay,

2006). The invasive plants in fact are attacked by generalists in introduced ranges

resulting in improvement of qualitative defense, which leading better defense generalists

(Müller-Scharer, Schaffner & Steinger, 2004; Joshi & Vrieling, 2005).

Recently, studies found a phenomenon that invasive populations resist against generalist

herbivores better than the native ones. For examples, Oduor et al. (2011) found that the

invasive populations of Brassica nigra s resisted against herbivore better than native

ones. Introduced populations of Defense against both specialists and generalists of

introduced populations of Centaurea maculosa (Asteraceae) were better than their native

counterparts (Ridenour et al., 2008). In addition, the invasive Verbascum thapsus

populations grew better than the native ones but the defense between the two range were

remarkably similar (Alba et al., 2011). Therefore, a pattern of both

maintenance/increasing resistance and growth in invasive populations should be

considered (Oduor et al., 2011).

To our knowledge no documentation has reported how native plants of S. vulgaris differ

from the invasive ones in relation to the resistance ability to generalist herbivores. Hence,

the main aim of this study is to examine whether the native plants could resist against

generalist herbivores and whether there is an evolution of defense against generalist

herbivores happened to invasive plants of S. vulgaris in China. Following questions were

addressed:(1) Are S. vulgaris less palatable to a generalist herbivore, white jade land snail


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(WJLS, Achatina fulica) than Brassica chinensis and Lactuca sativa? If yes, then (2) Are

the resistance of S. vulgaris plants against WJLS depending on the range or the

populations?

2. Material and Method

2.1 Study species

Common groundsel (Senecio vulgaris)

Common groundsel (S. vulgaris L., Asteraceae), is native to southern Europe (Kadereit,

1984). In the 18th century, it spread to North Africa, North America, South America,

Hawaii, Asia, Australiaand New Zealand (Hulten, 1968) (Robinson et al., 2003). It was

first recorded in China in 19th century (Xu et al., 2012) and nowadays mainly distributes

in the south - western and north – eastern part of China (Cheng & Xu, 2015). Senecio

vulgaris contains pyrrolizidine alkaloids (PAs) (Cano et al., 2009; Yang et al., 2011),

which served as deterrent or toxic to herbivores and pathogens (Boppré, 1986; Schneider,

1987; Macel, 2003; Molyneux et al., 2011). In addition, S. vulgaris causes great damage

to agricultural plants (barley, rape, strawberry, etc.), fruit trees and lawns in low latitudes.

Unfortunately, some biotypes of S. vulgaris can resist herbicides such as simazine,

atrazine (Ryan, 1970; Radosevich & Appleby, 1973; Radosevich & Devilliers, 1976),

atrazine, bromacil, pyrazon, buthidazole (Fuerst et al., 1986), linuron (Beuret, 1989).

Therefore, S. vulgaris is considered as a troublesome weed, especially in horticulture

where frequent cultivation occurs (Holt & Lebaron, 1990).

White Jade Land Snail (WJLS, Achatina fulica)

White jade land snails are albino morphs of the normal giant African land snails (A.

fulica, Achatinidae). They differ from the giant African land snails by having a paler shell

and a white body. The WJLS that is a species of molluscan pest, is believed to be native

to eastern coastal Africa and introduced to all most countries in Africa (e.g., Ethiopia,

Ghana, Kenya, Ivory Coast, Morocco, Mozambique, Somalia, and Tanzania), China (e.g.,

Hongkong, Macao, Guangdong, Hainan, Guangxi and Yunnan Provinces) (Yan et al.,

2001), South East Asia (such as Vietnam), India, Hawaii, Australia, Pacific Islands, and

Brazil (Raut & Barker, 2002).


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WJLS is active at night but hide itself underground during the day light. The individuals

of this species are voracious and omnivorous herbivores and feed on a wide variety of

plants such as grass, crops, fruits and vegetables. voracious and omnivorous can lay 5 – 6

clutches of eggs a year with 50 – 250 eggs per clutch. Their life expectancies are 4- 6

years in captivity, but they may live up to ten years in well feeding conditions.

Furthermore, WJLS is a vector to transmit pathogens or diseases to human, animal and

plant. Hence, this species causes great threats to native plant, agricultural crops,

horticultural and also industrial plants, and is considered as one of the top 100 invasive

species in the world (Lowe et al., 2000).

Fifteen - month old WJLS were fed with leaves of Pakchoi (Brassica chinensis) and

Lettuce (Lactuca sativa) at room temperature(about 20℃). Forty of the most voracious

snails were selected and starved for 24h prior to our experiment with leaves of S.

vulgaris, B. chinensis and L. sativa in white plastic boxes (17.5 cm × 14.5 cm × 11 cm).

2.2 Plant resource

Seeds of S. vulgaris were collected from the plants in the native range (Europe) and

invasive range (China) in 2012 and 2013 (Table 1). To minimize maternal effects, plants

from the seeds collected from field were grown in a climate room (12h/12h dark/light at

24oC, humidity 80%), and resulting seeds of the first flower head of each plant were used

in this study. Plants of six invasive populations and six native populations of S. vulgaris

were used in this study. Each population had 3materanal families.

2.3 Plant growth and harvest

2.3.1 Plant material

Twenty-four seeds of each maternal family were sown in planting boxes that have 12

cells and every cell received four seeds. Each cell (3.7 cm × 3.7 cm × 5 cm) filled with a

substrate of coconut peat (Zhenjiang Jingkou Green Island Horticultural Development

Center, Zhenjiang, Jiangsu, China) mixed with sand (collected from Wuhan, Hubei,

China) (1:1 by volume). The sown seeds were watered once per day using a small

sprayer, and the boxes were placed in a climate chamber (12h dark/12h light at 24oC

temperature, relative humidity 80%). The first seedlings appeared at the 2nd day after


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sowing. Four days later, they were transferred to a greenhouse (at Hubei Academy of

Forestry, Wuhan, Hubei, China). Finally, the seedlings were placed in the greenhouse for

one week then each seedling was transplanted to a 0.5-liter pot (8 cm × 8 cm × 9 cm)

filled with substrate prepared as described above. The pots containing seedlings were

randomly placed in blocks under a roof of net in order to protect seedlings from being

devastated by insects. They were watered once every two days.

2.3.2 Procedure of experiment

When the S. vulgaris seedlings had 5-8 leaves at the age of five weeks, three seedlings

were chosen from each mother plant to form a group of 108 seedlings that have same

size. The seedlings were grown in safe condition covered by a white sheet of net in the

greenhouse to prepare plant materials for a bioassay. When the plants started to have the

first flower, the feeding experiments with WJLS were conducted.

To test the resistance of native and invasive plants of S. vulgaris to WJLS, the snails were

fed on leaves of B. chinensis, L. sativa, native and invasive plants of S. vulgaris in plastic

white boxes (17 cm × 12 cm × 5 cm). The feeding experiment was conducted in 12

consecutive days which was divided into 4 waves. Each wave included 3 replicated times

conducted in 3 consecutive days. In each day of the 1st and 3rd waves of the snail feeding

experiment (hereafter, 1st wave and 3rd wave, respectively), each snail was fed on 3 leaves

of S. vulgaris from 3 plants of the same materanal family. The leaves used in the 1st wave

were the 4th, 5th 6th oldest leaf from a plant and the 7th, 8th and 9th oldest leaf were used in

the 3rd waves. WJLS were fed on leaves of B. chinensis and L. sativa in the 2nd and 4th

waves, respectively. Between each replicated time, all snails were starved for 12h. The

consumption of fresh biomass in each replicated time was assessed by fresh biomass

weighted before feeding minus the weight of fresh biomass remained. See a summary of

this experimental design in Table 2.

2.3.3 Data analysis

The mean consumed biomass of S. vulgaris, B. chinensis and L. sativa during the four

waves were compared using paired t-test to test whether B. chinensis and L. sativa were

more palatable than S. vulgaris. Paired t-test was also applied for biomass consumed per

day to test whether there was difference between the days within waves. (Table S1).


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The data on consumed biomass of S. vulgaris leaves were analyzed using a two - level

nested ANOVA with ranges, populations nested within ranges to test whether there was

significant difference between ranges and between populations within ranges. A one-way

ANOVA was applied to test whether there is any difference between the 12 populations in

relation to palatability of S. vulgaris leaves to WJLS.

A nested ANOVA was applied for the relative reductions (reduc1, reduc2, reduc3 and

reduc4) to test whether there were significant differences in relative reduction of the

biomass consumed by A. fulica between ranges, and between populations within ranges.

Reduc1, reduc2, reduc3 and reduc4 (%) were calculated as follows (feedday1-12:

represent for the mean of fresh biomass consumed by A. fulica from day 1 to day 12,

respectively):

Reduc1 = (feedday1 – feedday3) / (feedday1 + feedday2 + feedday3) 100




All analyses were performed with R, version 3.1.2 (R Core Team, 2014).

3. Results

We found that WJLS consumed significantly more fresh biomass of the leaves from L.

sativa than that of B. chinensis and S. vulgaris, and the biomass consumed of S. vulgaris

was significantly less than that of L. sativa and B. chinensis. Meanwhile, the biomass of

S. vulgaris plants consumed by WJLS in the 1st wave was significantly higher than that in

the 3rd wave (Figure. 1; Table S1). In both 1st and 3rd waves of the experiments when

WJLS were fed with leaves from S. vulgaris, there were no significant differences in

biomass consumed between plants from different range or populations (two – level nested

ANOVA, for the range and population in range, df = 1 and 11, P > 0.05; Figure 2).

In both 1st and 3rd waves, the S. vulgaris biomass consumed was significantly decreased

day by day. In the 2nd (day 4 - 6) wave, when WJLS was fed by leaves from B. chinensis,

the biomass consumed was significantly declined in day 4-5, and no significantly


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decrease was found between the day 5-6. The same pattern was found for the 4th wave

(day 10 - 12) when WJLS was fed with L. sativa (Figure 3, Table S1). Relative reduction

of S. vulgaris biomass consumed between day 8 - 9 was marginally different between

ranges; greater reduction was found for the invasive plants. Moreover, relative reduction

between day 1- 3 and day 2 -3 was significantly different between populations within the

invasive ranges. However, no significant difference was found between ranges or

population in relation to relative reduction between other days (Figure 4, Table 3).

4. Discussion

In snial feeding experiment, we found WJLS removed significantly less quantity of S.

vulgaris leaves than that from akchoi and Lettuce. This demonstrated that S. vulgaris less

palatable to WJLS than these two vegetables. Plants that contain toxin could be less

consumed by generalist herbivores than the food without or less toxicity (Speiser,

Harmatha & Rowell-Rahier, 1992; Van Dam et al., 1995). For example, snails (Helix

aspersa Muller) will prefer foods containing low PAs content than those contain more

PAs (Van Dam et al., 1995; Cano et al., 2009). Thus, if plants contain toxicity grow

together with other plants, herbivores tend to feed on the plants without or less toxicity.

The success of S. vulgaris to invade new areas could result in part from resistance or low

palatability to generalist herbivores which it possesses before its introduction and

maintains in introduced range.

The contious reduction of consumed biomass of S. vulgaris leaves in the 1st and 3rd waves

indicated postingestive effect of S. vulgaris leaves on WJLS. The consumed weight of B.

chinensis (or L. sativa) was significantly higher in day4 (or day10) compared to the day5

and day6 (or day11 and day 12) of the feeding experiment, while the biomass consumed

in day5 and day6 (or day11 and day 12) were the same (Table 3, 4). These higher biomass

consumed in day4 (or day10) expresses the compensatory feeding caused by the feeding

on S. vulgaris in the day before.

In snail feeding experiments, the amount of biomass consumed was determined by

palatability of plants, appetite of snails, and even environmental factors such as light and

humidity. The close correlation of biomass consumed between the days within the same


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wave attributed by the effect of plant and snail (see results of correlation test in Table S1).

When comparing the difference of biomass consumed between days or waves, we

removed the effect of snails by using pair -t test. When investigate whether the snail –

resistance was different between the plants from different ranges or populations, we used

relative reduction of biomass consumed which calculated with reduction of biomass

consumed between days divided by total amount biomass consumed for a wave. By using

both absolute amount and relative reduction biomass consumed, we found no difference

in palatability or resistance to WJLS between native and invasive plants, although

significant difference showed between populations. In another word, no evidence was

found for evolution of defense against generalist herbivores happened to invasive S.

vulgaris in China; which is contrast to our prediction.

Our finding is contrary to results of most studies that the invasive plants are less preferred

by generalist herbivores. For instance, protection against generalist herbivores increased

in invasive populations of S. jacobaea (Joshi & Vrieling, 2005), Jacobaea vulgaris (Rapo

et al., 2010) and Brassica nigra (Oduor et al., 2011) compared to their native populations.

However, this finding is similar to those found by Alba et al., (2011) who stated that no

evidence for evolution of defense happened to introduced populations of Verbascum

thapsus while those populations performed better than the native ones (Alba et al., 2011).

Parker & Hay (2005) also found that invasive generalist herbivores exhibited no

preference for native vs. exotic species Parker, Hay (2005).

Natural enemies in new ranges could influence on defense level in invasive plants. If

invasive plants undergo less or higher pressure of herbivores than they were in native

range, the plants will reduce or enhance their defense level in introduced ranges. If the

pressure of natural enemies on invasive No difference was found in palatability or

resistance to A. fulica between native and invasive plants that may indicate the

maintainance of defense level in invasive plants. This could suggest that the invasive

plants in China suffered from the same herbivory pressure in the introduced range as they

were in the native range.

Achatina fulica mainly distributes in Africa and Asia (Lowe et al., 2004), hence the

generalist herbivory of snails did not probably share evolutionary history with the native


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S. vulgaris in Europe. In contrast, invasive S. vulgaris was first recorded in China in 19th

century (Xu et al., 2012), and A. fulica was first reported in China in 1931 (Yan et al.,

2001) suggesting that the snails could share evolutionary history with the invasive S.

vulgaris in China. For this logic, A. fulica should prefer native populations of S. vulgaris

vs. invasive ones.

5. Conclusion

In this study we found that native and invasive plants of S. vulgaris were equally resistant

and less palatable to the generalist herbivores. If generalist herbivores consume this

species, the toxicity will cause aversion feeding and be accumulated in their bodies.

Hence, if the S. vulgaris grows together with other species, they will not be consumed by

generalist herbivores, which contribute to the success of this species to invade new areas.

Furthermore, no difference in resistance or palatability to WJLS between ranges indicates

that there might be no evolution of defense against generalist herbivores happened to S.

vulgaris in China. Invasive plants of S. vulgaris possesses traits that make it successful to

be an invader before it was introduced to new ranges; and the defense traits are still

maintained in introduced ranges. Those findings could be considered as good evidences

to explain why S. vulgaris becomes a strong invader in all over the world(Cano et al.,

2009).

Acknowledgements

Colleagues are thanked for their help during the experiment carried out at Hubei

Academy of Forestry, Wuhan city, Hubei province, China. Colleagues and students in the

School of Environmental Studies in CUG and Institution of Biology in Leiden University

are thanked for their help in seed collecting, plant rearing and harvesting.

Funding

This work is supported by the Fundamental Research Funds for the Central Universities

and National Natural Science Foundation of China (31570537 and 31200425) granted to

Dandan Cheng. Viet Thang Nguyen and Noel Ndihokubwayo thank the China

Scholarship Council (CSC) of the Ministry of Education for the study in China.


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Table 1. Resource of Senecio vulgaris seeds for this study

Range Country Population ID. Collection site Latitude Longitude Collected time

Native

Spain 1 Barcelona 41.67 2.73 06/2012

Switzerland 2 Fribourg 46.79 7.15 07/2012

The Netherlands 3 Leiden 52.17 4.48 10/2013

Germany 4 Potsdam 52.40 13.07 06/2012

Poland 5 Puławy 51.40 21.96 07/2012

Scotland 6 St. Andrews 56.33 -2.78 05/2012

Invasive China

7 Fuyuan, Heilongjiang 48.37 134.28 06/2013

8 Hegang, Heihongjiang 47.33 130.30 06/2013

9 Siping, Jilin 43.17 124.38 072013

10 Tongjiang, Heihongjiang 47.98 133.17 06/2013

11 Yichun, Heihongjiang 47.72 128.79 07/2013

12 Lijiang, Yunnan 26.89 100.23 09/2013


https://en.wikipedia.org/wiki/Pu%C5%82awy_County

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Table 2. white Jade land snails (WJLD, Achatina fulica) feeding experiment design in 12 consecutive days divided into 4 waves (3 days per

wave).

Days Wave of

experiments

Plant

materials

Order of leave used from

the oldest leave of plants

Total leaves used per

one snail per day Note

1

1st S. vulgaris

4th

3

Leaves fed one snail

from plants of one

maternal family

2 5th

3 6th

4

2nd B.

chinensis Randomly 1 5

6

7

3rd S. vulgaris

7th

3 8 8th

9 9th

10

4th L. sativa Randomly 1 11

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Table 3. F value of ANOVA tests for relative reduction of fresh biomass of Senecio vulgaris leaves consumed by white Jade land snails (WJLD,

Achatina fulica) in the 1st and 3rd wave (day 1-3 and day 10 - 12). Two -level nested ANOVA was used to test difference between the

ranges, and one -way ANOVA was used to test difference between the populations within each range.

Source of variance df Reduc1 Reduc2 Reduc3 Reduc4

Populations from China 5 3.48* 5.62 ** 1.20 ns. 0.45 ns.

Populations from Europe 5 1.00 ns. 2.67 . 1.45 ns. 1.18 ns.

Ranges 10 0.03 ns. 0.34 ns. 1.17 ns. 3.14 . Level of significance: p< 0.1, *p<0.05, **p<0.01, ***p<0.001; marginally significant are shown in italic.

reduc1 = (feedday1 – feedday3)/(feedday1 + feedday2 + feedday3)×100;




feedday1-12: represent for the mean of fresh biomass consumed from day 1 to day 12, respectively.


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Table S1. Results of correlation test and paired t-test for comparison between fresh biomass of leaves of Senecio vulgaris, Brassica chinensis

and Lactuca sativa consumed by white Jade land snails (WJLD, Achatina fulica) per day, and comparison between total biomass consumed.

feedday1-12: represent for the mean of fresh biomass consumed from day 1 to day 12, respectively.

ID

Mean SE Pairs of

the varaibles

Correlation pair-t-test

Variables of the

varaibles

of the

varaibles r p

Mean of the

differences t p

1 feedday1 0.27 0.03 feedday1 & feedday2 0.467 0.004 0.050 1.739 0.091











12 feedday12 2.28 0.15 feedday11 & feedday12 0.931 0.000 -0.041 -0.759 0.453

13 feedmean.S1 0.20 0.02 feedmean.B & feedmean.S1 0.172 0.316 0.314 6.641 0.000

14 feedmean.B 0.52 0.05 feedmean.B & feedmean.S2 -0.117 0.497 0.189 3.301 0.002

15 feedmean.S2 0.33 0.03 feedmean.L& feedmean.S2 0.479 0.003 2.085 19.929 0.000

16 feedmean.L 2.41 0.12 Feedmean.S1&feedmean.S2 -0.016 0.927 -0.125 -3.484 0.001

Level of significance: p< 0.1, *p<0.05, **p<0.01, ***p<0.001, ns. not significant.


15

Figure 1 Fresh biomass of leaves consumed by white Jade land snails (WJLD, Achatina fulica) in 12 consecutive days divided into 4 waves

(3 days per wave). Leaves from Senecio vulgaris were used in the 1st and 3rd wave, Pakchoi (Brassica chinensis) and Lettuce (Lactuca

were used in the 2nd and 4th, respectively. Biomass consumed between the four waves are significantly different (See statistic details

in Table S1).


16

Figure. 2 Fresh biomass of Senecio vulgaris leaves consumed by white Jade land snails (WJLD, Achatina fulica) in the 1st and 3rd wave (day

1-3 and day 10 - 12). Populations are listed as the name of places where the seed collected; six populations from each of the native and

invasive range. (a): biomass consumed the 1st wave ; (b): biomass consumed in the 3rd wave. There were no significant differences in

biomass consumed between plants from different range or populations (two – level nested ANOVA, for the range and population in

range, df = 1 and 11, P > 0.05)


17

Figure 3 Fresh biomass of leaves consumed by white Jade land snails (WJLD, Achatina fulica) in 12 consecutive days divided into 4 waves

(3 days per wave). Leaves from Senecio vulgaris were used in the 1st and 3rd wave, Pakchoi (Brassica chinensis) and Lettuce

(Lactuca were used in the 2nd and 4th, respectively. In both 1st and 3rd waves, the biomass consumed was significantly decreased day

by day. In the 2nd (day 4 - 6) and the 4th wave (day 10 – 12), biomass consumed was significantly declined from the first (day 4 and

day 10) to the second day (day 5 and day 11), and no significantly decrease was found between the second day and the third day (day

6 and day 12). (See statistic details in Table S1).


18

Figure. 4 Relative reduction of fresh biomass of Senecio vulgaris leaves consumed by white Jade land snails (WJLD, Achatina fulica) in the

1st and 3rd wave (day 1-3 and day 10 - 12). Populations are listed as the name of places where the seed collected; six populations from

each of the native and invasive range. See details about variable and statistic results in Table 3.


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