Rapid Identification of Acacia Species With Potential Salt Tolerance by Using Nuclear Ribosomal DNA Markers

Use of saline lands for agroforestry relies primarily on plant species that have the trait of salinity tolerance, and also other economic and agronomic benefits. The selection of species, however, also needs to consider other key factors such as compatibility with existing flora, and potential for environmental benefits such as improved soil fertility or lowering of the water table in the case of dryland salinity. The testing of candidate species in particular environments needs substantial investments of costs and time. In this paper, a novel approach is presented for the rapid identification of potentially salt tolerant Acacia species, based on molecular phylogenetic analysis. The approach has been applied to four species groups, Acacia pendula, A. salicina, A. victoriae and A. stenophylla. The nuclear-encoded ribosomal DNA internal and external transcribed spacer (ITS and ETS) regions were used as markers, and phylogenetic analyses undertaken to identify closely related species that may share the salt tolerance traits. Such a methodology could be used to more rapidly identify candidate native species for agroforestry in salinity-affected regions and for preventing further expansion of salinity, thus assisting in biodiversity conservation.


Introduction
The environmental challenge of dryland salinity currently affects approximately 3.3 million hectares of arable lands in Australia, and could further expand to 5.7 million hectares if left unchecked (Department of Sustainability, Environment, Water, Population and Communities; http://www.environment.gov.au/land/pressures/salinity/).The negative impact of salinity has prompted serious action by the Australian government to implement programs such as the National Action Plan for Salinity and Water Quality (Pannell & Roberts, 2010).The Australian landscape has vast reserves of salt beneath the land surface.Species of perennial Australian native vegetation are well-adapted to these harsh conditions, utilizing available water, also maintaining the water table and immobilizing the salt.However, dramatic changes in land use since the European settlement have led to replacement of the deep-rooted native perennials with shallow-rooted cereal crops and pastures.This has resulted in mobilization of salt to the soil surface, causing toxicity to plants and significant loss of crop land and native vegetation.One of the key strategies for reclamation of salinity-degraded lands is revegetation with well-adapted native species, some of which maybe additionally useful as sources of food, fodder, fuel, fiber, resins, essential oils or pharmaceutical products (Maslin & McDonald, 2004).
The genus Acacia (wattle), first described in 1754 by Philip Miller, belongs to the tribe Acacieae and is the largest genus within the legume subfamily Mimosoideae (Murphy, 2008).The name Acacia is now restricted to the subgenus formerly known as Phyllodineae (synonym Racosperma), confining it mainly to Australia (Maslin, 2006), where it is the largest genus of vascular plants and widespread in dry and semi-arid regions.Acacia species thrive in a diverse range of environments and many species tolerate high salinity, sodicity, high pH and waterlogged soils (Niknam & McComb, 2000).Additionally, several salt-tolerant acacias, such as A. saligna, A. stenophylla, A. salicina and A. ampliceps, have the potential to provide forage and fodder (Vercoe, 1987).Acacia species harbor nitrogen-fixing rhizobia that can improve soil fertility (Hoque, Broadhurst, & Thrall, 2011) and are in use for reclamation of severely degraded lands in Brazil (Chaer, Resende, Campello, De Faria, & Boddey, 2011).Some species provide timber and edible fruit and seeds.However, despite the benefits, Acacia species are currently under-investigated and underutilized, and information on salinity tolerant species with agroforestry potential is especially limited.Four species (A. pendula, A. salicina, A. stenophylla, A. victoriae), were found to be salt tolerant (among other vegetation) in a unique project on reclamation of saline lands in Victoria (Australia), [Phil Dyson and Ian Rankin, Northern United Forestry Group (NUFG) Bendigo, Australia; personal communications 2009] areas of which show high surface soil salinity [12-14 deci-Siemens/metre (dS/m)] and sub-soil salinity (4 dS/m) (NUFG Kamarooka Project; http://www.nufg.org.au/Kamarooka%20Project.htm).This study aims to use molecular phylogenetics to rapidly identify the close relatives of these species in order to exploit their potential for agroforestry on saline lands.
Nuclear ribosomal DNA (rDNA) markers such as the internal transcribed spacer (ITS) and the external transcribed spacer (ETS) (Baldwin & Markos, 1998;Mort et al., 2007) and chloroplast loci (e.g. atpF-H, matK, psbK-I, rbcL, rpoB, rpoC1 and trnH-psbA) (Dong, Liu, Yu, Wang, & Zhou, 2012) have been commonly used in plant molecular systematics.The ITS1 and ITS2 regions of plant rRNA genes are used most frequently to study phylogeny at genus and species levels due to advantages such as high sequence variability, copy numbers and ease of amplification (Kay, Whittall, & Hodges, 2006;Mort et al., 2007).ITS is adjacent to the conserved 5.8S rRNA gene region and is flanked by the conserved 18S and 26S rRNA genes, and this entire region is tandemly repeated thousands of times to make up the rDNA.The ETS of 18S-26S rDNA belongs to the same transcriptional unit.It may have evolved under similar functional constraints and complements the ITS data to yield more characters for significant phylogenetic inferences in angiosperms (Baldwin & Markos, 1998).The ITS and ETS are currently the most commonly sequenced loci for Acacia and provide the best available comparative dataset.
Comparative biology is based on the expectation that closely related organisms share traits, such as salinity tolerance, that are less common in more distantly related organisms (Cracraft, 2002).Therefore, clarifying molecular phylogenetic relationships can aid in selecting candidate species for a particular trait.Miller, Murphy, Brown, Richardson, and González-Orozco (2011) used plastid and nuclear rDNA data to test for invasiveness of species across a broad framework of 110 acacias.Although the invasive species did not form a monophyletic group, some evidence for phylogenetic grouping of invasive species was found.The study also identified sister species of the known invasive species that may have increased potential for invasiveness.The present study takes a similar approach, to rapidly identify sister species of known salt tolerant taxa.ITS and ETS sequence data were obtained for species in morphological groups closely related to the four salt-tolerant species, A. pendula, A. salicina, A. stenophylla and A. victoriae, mentioned above.These data, combined with an extensive Acacia dataset of ITS and ETS markers, was used to generate phylogenetic relationships and identify further species with a potential for salt tolerance.

Plant Tissue Sampling for Genomic DNA Extraction
The thirty species to be analysed were initially selected based on species groups morphologically related to the salt tolerant species described above (A. pendula, A. salicina, A. stenophylla and A. victoriae), as described in Flora of Australia (Orchard & Wilson, 2001a;b).The phyllode or leaf tissue (20mg) was removed from the herbarium sheets held at Royal Botanic Gardens, Melbourne (herbarium voucher numbers given in Table 1) and used for genomic DNA extraction using the DNeasy Plant Mini Kit (Qiagen Australia).A dataset of ITS and ETS sequences was constructed using data from Brown, Murphy, Kidman, and Ladiges (2012).Paraserianthes lophantha (voucher MEL2057862; GenBank accessions: ITS: EF638203; ETS: EF638105.1)was used as the out-group, based on Brown, Murphy, Miller, and Ladiges (2008) who concluded that it is sister to Acacia.

Phylogenetic Analysis
The sequences generated in this study were edited using Sequencher v3.0 (Gene Codes Corporation) and concatenated manually using BioEdit v7.0.0 (Hall, 2007), followed by alignment using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The sequence dataset obtained for 19 species was combined with previous data (Brown et al., 2012) and subjected to Bayesian analysis using MrBayes v3.2.1 (Ronquist & Huelsenbeck, 2003).For analysis, the combined ITS and ETS data were divided into six partitions: ITS1, 5.8S, ITS2, LSU, SSU and ETS.Insertion/deletion (indel) events were scored as multistate characters.An evolutionary model, GTR (Generalised Time Reversible) substitution model with gamma-distributed rate variation, was applied to each partition.A Markov Chain Monte Carlo (MCMC) search was run for 8 million generations, with trees sampled every 100 generations.Starting from different random trees, the analyses were performed twice simultaneously (Nruns = 2) with four Markov chains (N chains = 4) for each tree.Burn-in was set to 25001 (i.e. the first 25001 trees were discarded from each run).A Bayesian consensus phylogram was generated and for each node posterior probability (PP) values were calculated.The phylogenetic tree was visualised and coded for display using FigTree (Rambaut & Drummond, 2008).

Sequence Data
Of the 30 species of interest sampled based on morphological relatedness, 19 yielded good quality sequence data for both ITS and ETS markers.For five species (A. victoriae, A. alexandri, A. aphanoclada, A. cuspidifolia, A. dempsteri) that did not produce high quality sequences, the ITS and ETS data available in GenBank as part of the dataset of Brown et al. (2012) were used for further analysis.Six species (A. ampliceps, A. bivenosa, A. didyma, A. sclerosperma, A. startii, A. telmica) could not be analysed due to poor amplification results or poor quality sequences, and there were no corresponding sequences in GenBank; hence these were excluded from further analysis.The length of the individual regions, including the aligned versus the unaligned (raw sequence) lengths, for the 24 species is given in Table 2.The partial length of ITS1 (196 base pairs; bp) (Table 2) appears somewhat shorter than previously reported lengths for Acacia ITS1 (Murphy et al., 2003;2010), due to some unresolved bases occurring close to the primer binding site near the 5' end of the ITS.The sequence data for 5.8S subunit and ITS2 region were complete.All 5.8S subunit sequences were 159 bp long, as found previously (Murphy et al., 2003;2010).The ITS2 ranged from 149 bp (A.pickardii) to 215 bp (A.xanthina).The ETS region varied from 393 to 407 bp.Numbers indicate length in bp.The aligned length indicates an overall range of lengths, based on the alignment of all individual raw sequences and including gaps.The unaligned length denotes the lengths of raw sequences from individual species without gaps.

Bayesian Analysis of the Combined ITS and ETS Regions Incorporated Into the Larger Dataset
The sequence data generated for the 24 taxa altogether were incorporated into the larger dataset of Brown et al. (2012) for a total of 178 sequences (including the out-group P. lophantha) and used for Bayesian analysis.The length of the concatenated ITS and ETS sequences was 1290 bp.A total of 8 indel characters (6 from ITS and 2 from ETS regions) were scored.The average standard deviation of split frequencies was 0.010 and the average potential scale reduction factor was 1.001.The consistency index was 0.146 and retention index was 0.695.Incorporation of the new dataset showed that some of the species fall into previously recognised clades (Murphy et al., 2010;Miller et al., 2011;Brown et al., 2012), e.g., the A. aulacocarpa group, A. auriculiformis group, and the A. victoriae and A. pyrifolia group (Figure 2).

Figure 2. Phylogenetic tree of Acacia species
The A. victoriae group is a well-characterised monophyletic clade (Ariati et al., 2006).Maslin (1992) had revised this group and included ten species (nine included in the present analysis) found mainly in arid parts of Australia, i.e., A. victoriae Benth., A. alexandri Maslin, A. aphanoclada Maslin, A. chartacea Maslin, A. cuspidifolia Maslin, A. dempsteri F. Muell., A. glaucocaesia Domin, A. pickardii Tind., A. ryaniana Maslin and A. synchronicia Maslin.Most of these are characterised by spinose stipules, similar phyllode characteristics and a minute gland near the apical mucro (Ariati et al., 2006;Orchard & Wilson, 2001a;b).A. victoriae was found to be sister to a clade comprising A. synchronicia, and in turn to A. chartacea and A. ryaniana.The relationship of A. chartacea and A. ryaniana is strongly supported (PP =1.0).No data exists yet on the salinity tolerance of species closely related to A. victoriae.A. victoriae is of interests also due to its seed pods being a source of Avicins, which have a strong potential as anti-tumor drugs (Lemeshko, Haridas, Quijano-Pérez, & Gutterman, 2006).
A.ligulata is a prospective candidate for revegetation of areas with slight to moderate salinity, A. xanthina is recorded to grow on arid lands and limestone, while A. tysonii is a species with hard wood, and is advantageous for soil stabilisation in saline sites (World Wide Wattle; http://www.worldwidewattle.com/).Thus a number of species in the A. salicina clade may be suitable for revegetation purposes.
Ascertaining the relationships of the four target species and their closest relatives based on phylogenetics has helped in narrowing the identification process of putative salt tolerant species.This is significant since there is such limited data on salinity tolerance for the vast number (> 1000) of Australian Acacia species, as large scale testing of salinity tolerance parameters is time consuming and prohibitively expensive.As such, a rapid and inexpensive methodology to flag candidate species as described here, is highly significant.The utilisation of DNA markers such as ITS and ETS, is highly informative due to the large amount of already available sequence data.The molecular phylogenetic screening could be followed by testing the select species for physiological markers such as biomass and ion accumulations in laboratory and eventually field conditions.The selected molecular phylogeny approach could be extended to other taxa, or tolerance to other traits such as frost, drought, alkalinity or water logging often associated with salinity tolerance, or phytochemical composition, weediness potential, forage potential, and other characteristics important for utilisation.Thus the results presented here may help in rational selection of candidate plants that not only provide a 'green cover' for the landscape but also contribute to its productivity.Bui (2013) provide strong evidence that salt stress may have been a crucial element in the adaptation and evolution of Australian vegetation and propose that it may also have been involved in the speciation of acacias. A. harpophylla, A. cambagei and A. argyrodendron belong to the same clade and all can grow on alkaline and saline soils; however, not all species related to these can grow on saline lands.Species that grew on saline as well as alkaline soils were found to form distinct groups within the clade (Bui & Henderson, 2003;Bui, 2013).The reports show that molecular phylogeny can serve as a useful tool for envisaging plant growth characteristics based on edaphic, climatic and biogeographical factors.The method could also provide candidates from unrelated genera, based on species richness of a specific landscape.

Conclusion
Many Acacia species are capable of withstanding abiotic stress conditions.The preliminary molecular selection process developed here can assist in more rapid selection of native germplasm for field testing, to identify suitable candidates for agroforestry, land reclamation and biodiversity conservation, without the need for transgenic technologies.

Figure 1 .
Figure 1.Structure of the ribosomal DNA cistron S3 denotes the forward primer and 26SE denotes the reverse primer used to amplify the ITS region.AcR2 denotes the forward primer and 18S-IGS denotes the reverse primer used to amplify the ETS region (figure not drawn to scale).