DOI: 10.5061/dryad.dr7sqvb39
title: Introduction pathways and evolutionary mechanisms of the spread of alien species of Lolium on sandy coasts in Japan

description: 1. Estimating the role of specific processes in the spread of alien
 species necessitates the determination of introduction pathways and source
 populations of invaded areas. Alien grasses in the genus Lolium that have
 become widely invasive in Japan provide provides a unique
 opportunity to estimate the expansion process through a direct comparison
 between source and naturalised populations because the introduction
 pathways, contaminants in grain commodities and commercial cultivars for
 fodder crops or revegetation materials are well-known. Therefore, by
 directly comparing between source and naturalised populations, we
 estimated the introduction pathways and whether adaptative evolution
 occurred in Lolium species on sandy coasts in Japan. 2. Lolium individuals
 sampled from naturalised populations in croplands, seaports and sandy
 coasts were compared with those from two introduction sources for
 morphological and genetic variations based on a genome-wide single
 nucleotide polymorphism analysis and a common garden experiment.
 Furthermore, we conducted a reciprocal sowing experiment between a
 cropland and sandy coast. 3. Populations naturalized in croplands were
 closely related to the cultivars, whereas those naturalized in seaports
 and sandy coasts were associated with contaminants. This pattern is common
 in western Japan. Therefore, these results indicate that the cropland and
 sandy coast populations are derived from cultivars and contaminants,
 respectively. The reciprocal transplant experiment clearly demonstrated
 the home site advantage; populations derived from croplands yielded higher
 floret numbers than those derived from other habitats at the cropland
 site; however, such an advantage was not detected with the coastal site
 population. Sandy coast populations had higher survival rates than those
 from croplands at coastal sites. Port populations exhibited a similar
 tendency as sandy coast populations, indicating that contaminants may be
 originally adapted to salty and dry environments, such as that in sandy
 coasts. 4. Synthesis. We demonstrated that two congeneric species with
 different ecological characteristics were introduced through multiple
 introduction pathways and spread into different habitats. A direct
 comparison between source and naturalised populations can greatly advance
 our understanding of the patterns and processes of biological invasions.
description: Plant materials For contaminant seeds from
 imported grains, we obtained three 20-kg samples of five wheat classes
 imported between 2006 and 2007: three classes of U.S. wheat (Hard Red
 Winter wheat: HRW, Western White wheat: WW and Dark Northern Spring wheat:
 DNS), one class of Canadian wheat (No.1 Canada Western Red Spring
 wheat:1CW), and one class of Australian wheat (Australian Standard White
 wheat: ASW) (Table S1). Furthermore, we obtained three types of fodder
 crops imported from Australia between 2016 and 2017: ground barley, wheat
 straw and oat hay (Table S1). The seeds of Lolium species were selected
 from each sample, according to the method described by Shimono et al.
 (2010). For cultivated varieties, we purchased 1 kg
 bags each of Italian ryegrass seeds and perennial ryegrass seeds from Snow
 Brand Seed Co., Ltd. (Hokkaido, Japan) and Takii Seed Co., Ltd. (Kyoto,
 Japan) in 2012. We selected the most currently available commodity,
 ‘Common’ (a generic term, not a cultivar name), for fodder crop or
 revegetation material (Japan Grassland Agriculture and Forage Seed
 Association, 2010). Additionally, eight Japanese cultivars of Italian
 ryegrass (Hanamiwase, Akatsuki, Waseyutaka, Inazuma, Tachimusya,
 Satsukibare, Mammos B and Jiant) were kindly provided by the Institute of
 Livestock and Grassland Science, National Agriculture and Food Research
 Organization in Japan. The following foreign cultivars were obtained from
 GenBank, National Agriculture and Food Research Organization (NARO): the
 United States cultivar of Italian ryegrass, Westerwold ryegrass (GULF);
 New Zealand cultivar of Italian ryegrass, Westerwold ryegrass (MOTO 191,
 TETRAPLOID); and Australian cultivar of annual ryegrass, Wimmera ryegrass
 (Table S2). Additionally, seeds were collected from
 naturalised populations in three habitat types–cropland, seaport and sandy
 coast–in each of the three geographic regions of Japan (Kanto, Kansai and
 Kyushu) (Table S3). All the selected seaports (Kobe, Kashima and Hakata
 ports) are major international trading ports where more than 1,000 kt of
 imported grain is unloaded every year. Matured seeds were individually
 collected from 10 plants at intervals of at least 2 m from each habitat
 from May to June, 2017. Common garden
 experiment On October 2017, 10–30 seeds were randomly
 selected from each sample and germinated on two pieces of moistened filter
 papers placed in 9-cm plastic petri dishes in an incubator set at 25°C for
 a 12-h light period and at 15°C for a 12-h dark period. One week after
 germination, the seedlings were individually transplanted into pots (10.5
 cm in diameter, 9 cm in depth) filled with potting soil (Tachikawa Heiwa
 Nouen Co., Tochigi, Japan) and placed in a glasshouse under natural
 sunlight at the experimental farm of Kyoto University, Kyoto, Japan
 (35.032121°N, 135.783453°E). The plants were regularly watered.
 To investigate whether phenological and morphological differences
 exist between individuals with different provenances and habitats, the
 heading  (ear emergence from the flag leaf sheath) date and the following
 morphological characteristics at 3 weeks after heading were measured:
 number of tillers, length and width of the longest culm, inflorescence
 length and width of the longest culm and number of spikelets of the
 longest culm. In addition, spikelet length, glume length, number of
 florets per spikelet, first floret length and awn length were measured
 using a spikelet placed in the middle of the inflorescence of the longest
 culm. Awn length was graded on a scale of 1 to 3 depending on the length
 (1: no awn, 2: &lt;5 mm and 3: ≥5 mm). The ratio of glume to spikelet
 length was subsequently calculated. Reciprocal transplant
 experiment Habitat-specific adaptations were examined in a reciprocal
 transplantation experiment. The experimental farms of Kyoto University
 (35.032121°N, 135.783453°E) and Shimazaki Sandy Coast (34.7319463°N, 136.5287767°E) were selected as
 representatives of the
 cropland and sandy
 coastal habitats,
 respectively, and were used in a reciprocal transplant experiment (Fig. S1). From May to
 June 2017, bulk seeds
 were harvested from more
 than 30 individuals in naturalised populations in three habitat types: cropland, seaport and sandy
 coast, in each of the three geographic regions of Japan (Kansai, Kanto and
 Kyushu). Seeds were stored in an airtight container
 containing silica gel at room temperature (20°C–28°C) until September
 2017.. Fifty seeds from each population were sown in
 plastic trays (length: 26.5 cm, width: 18.3 cm and depth:
 7.7 cm) filled with soil from bare land
 in each habitat. Eight trays were prepared for each population, three of
 which were set on croplands and five on the sandy coast on
 29
 September 2017 which is
 the typical germination period for <em>Lolium</em> species.
 The trays were covered with fine mesh nets to prevent further seed input
 and removal by wind or animal dispersal. The trays were buried at depths
 where the rims were at the same level as the soil surface. Seedling emergence, survival and flowering
 were recorded at 1-week intervals through July 2018, except for the period
 from December to March, during which observations were made every two
 weeks. Individuals were
 harvested three weeks after heading, and the number of culms,  the number
 of spikelets per culm and the number of florets per spikelet in the middle
 of the inflorescence were measured. The total number of florets produced by
 surviving individuals was calculated as follows: number of culms × number of spikelets per culm
 × number of florets per
 spikelet. Survival rate
 was defined as the ratio of the number of surviving individuals to the
 number of emerged seedlings.