Cross-continental variation of herbivore resistance in a global plant invader

While successful plant invasions often occur in novel environments, invasive species usually occupy broad niches within their native and introduced ranges. A better understanding of the process of invasion therefore requires a wide sampling of ranges, and a good knowledge of introduction history. We tested for differentiation in herbivore resistance among 128 introduced (European, North American) and native (Chinese, Japanese) populations of the invasive Japanese knotweed (Reynoutria japonica) in two common gardens in the native range: one in Shanghai and the other in Yunnan. In both common gardens, we found that herbivore resistance of plants from introduced populations differed from that from native populations in China but not from native populations in Japan, the putative source of introduction. Compared to native Chinese populations, plants from native Japanese populations and introduced European and North American populations had thicker leaves in both common gardens, and a lower C: N ratio but higher flavonoids content in the Shanghai garden. Variation in herbivore resistance was more strongly associated with climate of collecting sites for populations from the native range than for those from introduced ranges. Our results support the hypothesis that introduction of particularly resistant plants from Japan may have played a key role in driving biogeographic variation in herbivore resistance. Our study demonstrates the importance of understanding introduction history to interpret the biogeographic divergence of global plant invaders.

Methods
In the middle of the growing season, we estimated herbivore damage—separately for beetle and caterpillar damage—as the percentage of leaf area eaten on each plant. Two days later, we sampled five fresh, fully developed leaves (the 1st, 2nd, 4th, 5th, and 6th leaves from the top) from the tallest shoot of each plant and measured the thickness of each leaf with a digital micrometer (Digimatic Outside Micrometer, Mitutoyo, Japan) in both gardens, and its toughness with a penetrometer (FA10, SAUTER, Balingen, Germany) in the Shanghai garden and with a mechanical testing machine (ZQ990A, Dongguan Zhiqu Precision Instrument Co., Ltd, China) in the Xishuangbanna garden. We then estimated the leaf thickness and toughness for each plant as the averages of the five measurements. We measured all plant traits in the Xishuangbanna and Shanghai common gardens from July 14 to 21, 2022, and July 24 to 31, 2022, respectively.

Finally, we dried all leaves at 60 °C for 72 h to analyze for leaf chemistry. After grinding samples to the required particle size with a ball mill (MM400, Retsch, Germany), we measured total C and N with an organic elemental analyzer (FlashSmart™ Elemental Analyzer, Thermo-Fisher Scientific, USA) via thermal combustion and TCD/IR detection of CO2/N2. Then, we measured leaf lignin, alkaloids, and total flavonoids using the MZS-1-G, SWJ-1-Y, and LHT-1-G test kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China), respectively. After acetylation, the phenolic hydroxyl group in lignin had a characteristic absorption peak at 280nm. Alkaloids can react with bromocresol green indicator to generate green compounds with a maximum absorption peak at 416nm. In alkaline nitrite solution, flavonoids can form red complexes with aluminum ions with a characteristic absorption peak at 510nm. Then, we used the photometric method to measure the absorbance of the sample solution at each wavelength using an enzyme-labeled instrument (Synergy2, Biotek Instrument Co., Ltd, Winooski, USA), and calculated the content of each chemical compound group.