DOI: 10.5061/dryad.m86s6
title: Data from: Invasive legumes can associate with many mutualists of native legumes, but usually do not

description: Mutualistic interactions can strongly influence species invasions, as the
 inability to form successful mutualisms in an exotic range could hamper a
 host’s invasion success. This barrier to invasion may be overcome if an
 invader either forms novel mutualistic associations or finds and
 associates with familiar mutualists in the exotic range. Here we ask (1)
 does the community of rhizobial mutualists associated with invasive
 legumes in their exotic range overlap with that of local native legumes
 and (2) can any differences be explained by fundamental incompatibilities
 with particular rhizobial genotypes? To address these questions, we first
 characterized the rhizobial communities naturally associating with three
 invasive and six native legumes growing in the San Francisco Bay Area. We
 then conducted a greenhouse experiment to test whether the invasive legume
 could nodulate with any of a broad array of rhizobia found in their exotic
 range. There was little overlap between the Bradyrhizobium communities
 associated with wild-grown invasive and native legumes, yet the invasive
 legumes could nodulate with a broad range of rhizobial strains under
 greenhouse conditions. These observations suggest that under field
 conditions in their exotic range, these invasive legumes are not currently
 associating with the mutualists of local native legumes, despite their
 potential to form such associations. However, the promiscuity with which
 these invading legumes can form mutualistic associations could be an
 important factor early in the invasion process if mutualist scarcity
 limits range expansion. Overall, the observation that invasive legumes
 have a community of rhizobia distinct from that of native legumes despite
 their ability to associate with many rhizobial strains, challenges
 existing assumptions about how invading species obtain their mutualists.
 These results can therefore inform current and future efforts to prevent
 and remove invasive species.
description: La Pierre_invasive
 shrub_nodulation assay_dryad submissionData addressing the
 breadth of rhizobial isolates that three invasive and two native legumes
 from the San Francisco Bay Area, California, USA could nodulate with in
 single-isolate inoculations under greenhouse conditions. We assessed the
 promiscuity of the three invasive plants in their invasive range by
 determining their ability to associate with a broad range of 117 rhizobial
 isolates originally collected from 12 different leguminous hosts (both
 native and invasive, including hosts not studied here, but all growing in
 the SF Bay Area; Table S2) in a greenhouse-based nodulation assay. Seeds
 of each legume species were surface sterilized in full-strength commercial
 bleach (3% sodium hydroxide) for 30 sec, rinsed five times with sterile
 water, scarified with sulfuric acid for either 10 min (S. junceum), 30 min
 (U. europaeus), or 40 min (G. monspessulana), neutralized with a sterile
 20% sodium bicarbonate solution and thoroughly rinsed using sterile water.
 Scarified seeds were germinated in the dark at room temperature in
 individual wells of 96-well plates filled with 100 µl sterile water. Two
 weeks later, germinated seedlings were individually planted into 22 mm
 diameter, 20 cm tall sterile glass 75 ml culture tubes filled with 25 ml
 sterile vermiculite moistened with sterile water. Tubes were plugged with
 sterile cotton and kept under shade cloth, which provided indirect natural
 light, and were provided supplemental artificial light in the Jane Gray
 Research Greenhouse at the University of California, Berkeley. Twelve days
 following planting, 1 ml sterile Jensen’s fertilizer (Somasegaran and
 Hoben 1994) containing 7 ppm nitrogen was added to each tube. The 117
 rhizobial isolates used in the nodulation assay were obtained from two
 sources: (1) many isolates were obtained from the collection described
 above prior to genotyping (99 isolates); (2) several isolates were
 obtained from the investigators’ additional research collections to
 represent strains associated with other native and invasive legumes common
 in the San Francisco Bay Area (18 isolates; see Table S2 for a list of the
 isolates used in the nodulation assay and their sources). Isolates were
 chosen to span a broad range of host species and collection sites.
 Inoculum from each isolate was prepared from 50 µl of -80°C glycerol stock
 prepared from field-collected nodules (see above), grown in YM broth at
 25°C shaken at 120 rpm to a density of per ml, as measured by optical
 density at 600 nm. Each rhizobial isolate was inoculated onto one seedling
 of each legume species. Seedlings were randomly assigned rhizobial
 isolates and inoculated 17 days after planting by adding 1 ml of the
 appropriate inoculum to the base of the plant stem in each tube. An
 additional ten plants per legume species were inoculated with sterile YM
 broth as negative controls; none of the control plants were nodulated at
 harvest. Plants were harvested 47 days after planting (30 days after
 inoculation), the roots thoroughly washed, and the presence of nodules
 recorded. Successful association was defined as the formation of at least
 one robust nodule that appeared to be effectual (i.e., not &lt;1 mm and/or
 white or clear). Reanalysis of our results increasing the cutoff for
 defining successful nodulation to two nodules did not qualitatively alter
 our findings.