Species richness in the open and under P. juliflora canopies at each site was compared using independent samples t-tests, one- way ANOVA or Mann-Whitney U tests [29]. No specific permits were required for the field studies carried out in Venezuela, India or Hawaii, USA; and field studies in these areas did not involve endangered or protected species. For the field studies carried out in Peru, permission was obtained from the Agriculture Ministry – Peru´ (Nu145-2008-INRENA- IFFS-DCB). The field study did not involve endangered or protected species. Effects of Prosopis Congeners on Species Richness We compared species richness beneath P. juliflora canopies to that beneath canopies of the native congener, P. cineraria, at Deer Park at Patiala, Punjab, India (N 30u179 00.20; E 076u239 07.50; 239 m). The area has a sub-tropical climate and total annual rainfall during the last 5 years of 703.6 mm. The selected site has been invaded by P. juliflora that reach heights of 12 to 14 m and densities of 1.1 trees per 25 m2. Prosopis cineraria occurs there as scattered individuals reaching heights of 4 to 9 m and densities of 2 less than 0.4 trees per 25 m . The canopy areas occupied by 2 individual P. juliflora and P. cineraria trees were 139.9 and 20.8 m , respectively. We used slightly smaller quadrats (464 m2) for measurements at this site so that a single quadrat represented a single tree either of P. juliflora or P. cineraria. In October 2010, we randomly selected 10 trees of P. juliflora or P. cineraria of similar sizes and laid a 16 m2 quadrat under the canopy of each tree keeping the trunk in the centre, and recorded the number of plant species in each quadrat. We also located 10 randomly located 16 m2 quadrats in open areas where neither P. juliflora nor P. cineraria was present. Differences in species richness in open, under P. juliflora and P. cineraria were tested using one-way ANOVA and post-hoc Tukey’s test [30]. Effects of Congeners on Soil Chemistry We also compared soils from the rhizospheres of P. juliflora and P. cineraria at Deer Park. We did sampling at this site during two time periods, November 2009 and March 2011. During Novem- ber 2009, soil was collected from rhizosphere of 4 individual trees of each Prosopis species and from 4 nearby open locations (n = 4 for each of the three treatments). Soil was collected near tree trunk to a depth of 30 cm and presence of a mesh of horizontal and vertical roots in the pit confirmed that it was rhizosphere soil. In March 2011, soil was collected from rhizosphere of 6 individual trees of each Prosopis species and from 6 nearby open locations (n = 6 for each of the three treatments). Soil was collected from 3 points around the tree trunk of each tree selected (no. of subsamples per tree = 3). The soil was then air-dried, sieved (2 mm), and stored in paper bags for later analyses. Five g soil from each sample was shaken with 25 mL water for 1 h followed by filtration through Whatman # 1 filter papers. pH and electrical conductivity were measured in soil extracts using a digital meter (Metrex Sci. Instr. Pvt. Ltd., India). Organic carbon was estimated following methods of Walkley and Black [31]. Total organic nitrogen was estimated using semi-micro Kjeldahl digestion [32]. We estimated PO43–P in 2.5% acetic acid soil extracts through molybdenum blue method [32]. Total phenolic content of soils was measured with the Folin- Ciocalteu method [33]. Data from collection during two time periods were combined (n = 10 for each treatment) and, tested for the differences among open, P. juliflora and P. cineraria soils for each soil chemical property using one-way ANOVA and post-hoc Tukey’s tests [29]. Invasion of Prosopis juliflora Effects of Leaf Litter from Prosopis Congeners on Other Species We first tested the effects of leaf litter at The University of Montana in a greenhouse experiment. We compared the effects of litter of P. juliflora and P. cineraria on the mortality of six native Indian species (Acacia nilotica, Brassica campestris, Brassica juncea, Chloris dolichostachya, Dalbergia sissoo and Prosopis cineraria). Litter from the Prosopis congeners were collected from three different trees in Aravalli Biodiversity Park, Delhi, India (N 28u 339 18.90; E 077u 089 56.80; 247 m), mixed, and then air dried. Rocket pots (200 cm3, 3 cm diam) were filled with 20/30 grit silica sand, and 5–10 seeds were planted 5 mm below the surface of the sand in each pot. We put 1.0 gm of litter from either P. juliflora or P. cineraria on the surface of the substrate, so that the seeds would have to germinate and grow through the leaves as they would through litter in the field. As seeds germinated they were thinned continually so as to keep only the largest seedling in each pot. The experiment started on 8 January 2010 and ended on 9 February 2010. We summed the total final mortality for all of the remaining largest seedlings in all pots for each species, and then compared treatments using the means of mortality for the six species as replicates in an ANOVA testing treatments (control, P. juliflora, P. cineraria) and followed by Tukey HSD tests [29]. Effects of Leaf Leachate from Prosopis Congeners on Other Species We also tested the effects of soil amended with leachate from leaves of the two congeners on three crop species, B. campestris, B. juncea and Sorghum bicolor, at the University of Delhi. We used these three crop species because these are common crop species in north-west India where P. juliflora has invaded agricultural fields or is present at the boundaries. Prosopis cineraria is often present in crop fields (Figure 1F). Leaves of the two Prosopis species were collected from the same sites as in the previous experiment and air dried prior to use. Ten g of air dried leaves of each were soaked separately in 100 mL distilled water for 14 h in the dark followed by filtration through Whatman # 1 filter paper. Soil (sandy loam) for the bioassay was collected from an area not occupied by P. juliflora, and at the same site where leaves were collected. 50g air- dried soil was weighed into a 9 cm Petri dish and treated with 15 mL distilled water, 5x, 2x, or 1x diluted P. juliflora or P. cineraria leaf leachate which yielded concentrations of leaf leachate in soil at 0, 60, 150 or 300 mL of leaf leachate/g soil. Brassica campestris and B. juncea seeds were washed with distilled water before use. Sorghum bicolor seeds were surface-sterilized by soaking in 1% sodium hypochlorite for 10 min and then rinsing in distilled water 20 times before use. For each of the seven treatments and three test species, we used six Petri dishes. Ten seeds of B. campestris, B. juncea, or S. bicolor were placed in each Petri dish and kept at 20–23uC. Seven days after treatment application we measured the shoot and root lengths of the germinants. Two-way ANOVA was used to test the effect of leachate source and concentration as fixed factors on root length. We first compared the effect of P. juliflora leachate or P. cineraria leachate treatment (each concentration) with control and then the effect of P. cineraria leachate with P. juliflora leachate applied at identical concentrations, using independent samples t- tests [30].We also correlated leachate concentration with root length for each bioassay species [30]. Effects of Microbes in Litter of Congeners on Other Species The biochemical effects of litter from different species or different regions could be confounded by different effects of the PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44966