Mesorhizobium calcicola sp. nov., Mesorhizobium waitakense sp. nov., Mesorhizobium sophorae sp. nov., Mesorhizobium newzealandense sp. nov. and Mesorhizobium kowhaii sp. nov. isolated from Sophora root nodules.

In total, 31 strains of Gram-stain-negative, rod-shaped bacteria were isolated from Sophora root nodules and authenticated as rhizobia on this host. Based on 16S rRNA gene phylogeny, they were shown to belong to the genus Mesorhizobium, with the representative strains ICMP 19560T, ICMP 19523T, ICMP 19535T, ICMP 19545T and ICMP 19512T being related most closely to Mesorhizobium sangaii SCAU7T (99.9-99.6 % similarity), Mesorhizobium cantuariense ICMP 19515T (99.7-99.6 %) and Mesorhizobium ciceri UMP-CA7T (99.7-99.5 %). Additionally, the novel strains formed distinct groups based on housekeeping gene sequence analysis and were closely related to Mesorhizobium waimense ICMP 19557T (93.5-94.9, 92.5-95.6 and 94.2-96.0 %), M. cantuariense ICMP 19515T (93.1-97.7, 93.5-95.4 and 94.8-96.8 %) and M. ciceri UMP-CA7T (93.2-97.2, 94.6-96.8 and 95.5-97.3 %) for glnII, recA and rpoB, respectively. Chemotaxonomic data supported the assignment of the strains to the genus Mesorhizobium, and DNA-DNA hybridizations, matrix-assisted laser desorption/ionization time-of-flight MS analysis, enterobacterial repetitive intergenic consensus PCR, physiological and biochemical tests allowed the genotypic and phenotypic differentiation from their nearest neighbouring species. Therefore, these strains represent five novel species for which the names Mesorhizobium calcicola sp. nov. (type strain ICMP 19560T = LMG 28224T = HAMBI 3609T), Mesorhizobium waitakense sp. nov. (type strain ICMP 19523T = LMG 28227T = HAMBI 3605T), Mesorhizobium sophorae sp. nov. (type strain ICMP 19535T = LMG 28223T = HAMBI 3606T), Mesorhizobium newzealandense sp. nov. (type strain ICMP 19545T = LMG 28226T = HAMBI 3607T) and Mesorhizobium kowhaii sp. nov. (type strain ICMP 19512T = LMG 28222T = HAMBI 3603T) are proposed.

Alkaloid variation in New Zealand kowhai, Sophora species.

Alkaloid contents of leaf and seed samples of eight species of Sophora native to New Zealand, plus Sophora cassioides from Chile are reported. Fifty-six leaf and forty-two seed samples were analysed for alkaloid content by proton nuclear magnetic resonance spectroscopy, which showed major alkaloids as cytisine, N-methyl cytisine and matrine. GC analyses quantified these and identified further alkaloid components. The alkaloids identified were cytisine, sparteine, and matrine-types common to Sophora from other regions of the world. Cytisine, N-methyl cytisine, and matrine were generally the most abundant alkaloids across all species with seeds containing the highest concentrations of alkaloids. However, there was no clear taxonomic grouping based on alkaloid composition. A quantitative analysis of various parts of two Sophora microphylla trees showed that the seeds were the richest source of alkaloids (total 0.4-0.5% DM), followed by leaf and twig (0.1-0.3%) and then bark (0.04-0.06%), with only low amounts (<0.02%) found in the roots. This study represents the most comprehensive phytochemical investigation of New Zealand Sophora species to date and presents data for three species of Sophora for which no prior chemistry has been reported.

Mesorhizobium waimense sp. nov. isolated from Sophora longicarinata root nodules and Mesorhizobium cantuariense sp. nov. isolated from Sophora microphylla root nodules.

In total 14 strains of Gram-stain-negative, rod-shaped bacteria were isolated from Sophora longicarinata and Sophora microphylla root nodules and authenticated as rhizobia on these hosts. Based on the 16S rRNA gene phylogeny, they were shown to belong to the genus Mesorhizobium, and the strains from S. longicarinata were most closely related to Mesorhizobium amorphae ACCC 19665T (99.8­99.9 %), Mesorhizobium huakuii IAM 14158T (99.8­99.9 %), Mesorhizobium loti USDA 3471T (99.5­99.9 %) and Mesorhizobium septentrionale SDW 014T (99.6­99.8 %), whilst the strains from S. microphylla were most closely related to Mesorhizobium ciceri UPM-Ca7T (99.8­99.9 %), Mesorhizobium qingshengii CCBAU 33460T (99.7 %) and Mesorhizobium shangrilense CCBAU 65327T (99.6 %). Additionally, these strains formed two distinct groups in phylogenetic trees of the housekeeping genes glnII, recA and rpoB. Chemotaxonomic data, including fatty acid profiles, supported the assignment of the strains to the genus Mesorhizobium and allowed differentiation from the closest neighbours. Results of DNA­DNA hybridizations, MALDI-TOF MS analysis, ERIC-PCR, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from their closest neighbouring species. Therefore, the strains isolated from S. longicarinata and S. microphylla represent two novel species for which the names Mesorhizobium waimense sp. nov. (ICMP 19557T = LMG 28228T = HAMBI 3608T) and Mesorhizobium cantuariense sp. nov. (ICMP 19515T = LMG 28225T = HAMBI 3604T), are proposed respectively.

When do plant radiations influence community assembly? The importance of historical contingency in the race for niche space.

Plant radiations are widespread but their influence on community assembly has rarely been investigated. Theory and some evidence suggest that radiations can allow lineages to monopolize niche space when founding species arrive early into new bioclimatic regions and exploit ecological opportunities. These early radiations may subsequently reduce niche availability and dampen diversification of later arrivals. We tested this hypothesis of time-dependent lineage diversification and community dominance using the alpine flora of New Zealand. We estimated ages of 16 genera from published phylogenies and determined their relative occurrence across climatic and physical gradients in the alpine zone. We used these data to reconstruct occupancy of environmental space through time, integrating palaeoclimatic and palaeogeological changes. Our analysis suggested that earlier-colonizing lineages encountered a greater availability of environmental space, which promoted greater species diversity and occupancy of niche space. Genera that occupied broader niches were subsequently more dominant in local communities. An earlier time of arrival also contributed to greater diversity independently of its influence in accessing niche space. We suggest that plant radiations influence community assembly when they arise early in the occupancy of environmental space, allowing them to exclude later-arriving colonists from ecological communities by niche preemption.

Diverse novel mesorhizobia nodulate New Zealand native Sophora species.

Forty eight rhizobial isolates from New Zealand (NZ) native Sophora spp. growing in natural ecosystems were characterised. Thirty eight isolates across five groups showed greatest similarity to Mesorhizobium ciceri LMG 14989(T) with respect to their 16S rRNA and concatenated recA, glnll and rpoB sequences. Seven isolates had a 16S rRNA sequence identical to M. amorphae ATCC 19665(T) but showed greatest similarity to M. septentrionale LMG 23930(T) on their concatenated recA, glnll and rpoB sequences. All isolates grouped closely together for their nifH, nodA and nodC sequences, clearly separate from all other rhizobia in the GenBank database. None of the type strains closest to the Sophora isolates based on 16S rRNA sequence similarity nodulated Sophora microphylla but they all nodulated their original host. Twenty one Sophora isolates selected from the different 16S rRNA groupings produced N2-fixing nodules on three Sophora spp. but none nodulated any host of the type strains for the related species. DNA hybridisations indicated that these isolates belong to novel Mesorhizobium spp. that nodulate NZ native Sophora species.

Sophora microphylla (Fabaceae) microsatellite markers and their utility across the genus.

PREMISE OF THE STUDY: Genus-specific microsatellite markers were developed for Sophora for population genetic and systematic studies of the group in New Zealand, and potentially elsewhere in the geographic range. • METHODS AND RESULTS: From sequencing a total genomic DNA library (using Roche 454), we identified and developed 29 polymorphic microsatellite markers for S. microphylla and S. chathamica. We tested 12 of these markers on 14 S. chathamica individuals and four S. microphylla populations. All loci amplified in both species and species-specific alleles occurred at seven loci. In S. microphylla populations, the observed and expected heterozygosities ranged from 0.000-0.960 and 0.000-0.908, respectively, with alleles per locus ranging from seven to 23. • CONCLUSIONS: The developed markers will be valuable in studies of phylogenetics, population structure, mating system, and selection of provenances for restoration projects.

Species radiation by niche shifts in New Zealand's rockcresses (Pachycladon, Brassicaceae).

Adaptive radiations such as the Darwin finches in the Galapagos or the cichlid fishes from the Eastern African Great Lakes have been a constant source of inspiration for biologists and a stimulus for evolutionary thinking. A central concept behind adaptive radiation is that of evolution by niche shifts, or ecological speciation. Evidence for adaptive radiations generally requires a strong correlation between phenotypic traits and the environment. But adaptive traits are often cryptic, hence making this phenotype-environment approach difficult to implement. Here we propose a procedure for detecting adaptive radiation that focuses on species' ecological niche comparisons. It evaluates whether past ecological disparity in a group fits better a neutral Brownian motion model of ecological divergence or a niche shift model. We have evaluated this approach on New Zealand rockcresses (Pachycladon) that recently radiated in the New Zealand Alps. We show that the pattern of ecological divergence rejects the neutral model and is consistent with that of a niche shift model. Our approach to detect adaptive radiation has the advantage over alternative approaches that it focuses on ecological niches, a key concept behind adaptive radiation. It also provides a way to evaluate the importance of ecological speciation in adaptive radiations and will have general application in evolutionary studies. In the case of Pachycladon, the high estimated diversification rate, the distinctive ecological niches of species, and the evidence for ecological speciation suggest a remarkable example of adaptive radiation.

Chemosystematic analyses of Gingidia volatiles.

The leave volatiles of six Gingidia species from New Zealand and Australia and the seed volatiles of G. grisea were characterized by solid-phase microextraction (SPME)-GC/MS analysis. This technique, using a small quantity of samples and automated extraction, gave repeatable results, with maximum sensitivity for medium volatility compounds. The major monoterpenes among the volatiles, i.e., ß-phellandrene (4), limonene (6), and γ-terpinene (5), and phenylpropanoids, i.e., estragole (3), (E)-anethole (7), and myristicin (1), showed to be useful chemotaxonomic markers. For G. grisea leaves and seeds, similar compositions were detected, characterized by high contents of 4. As leaves were more readily available for study than seeds, they were used for further investigations. The G. grisea leaf volatiles showed infraspecific variation in the ratio of 4/5 between and within sites of collection. The G. montana leaf volatiles also showed infraspecific variation, with high contents of 3 at one site and high contents of 7 at another. The SPME-GC/MS analysis of G. montana herbarium voucher specimens resulted in the identification of further chemotypes for this species. The volatiles of the G. amphistoma samples were all dominated by 7 and those of the G. haematitica samples were rich in 5. Moreover, single plants of two Australian Gingidia species were analyzed; the volatiles of G. harveyana showed high concentrations of 5 and 7, whereas those of G. rupicola were dominated by 5 and 1.

Rhizobia with 16S rRNA and nifH similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC genes are symbionts of New Zealand Carmichaelinae.

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

Plant radiation history affects community assembly: evidence from the New Zealand alpine.

The hypothesis that early plant radiations on islands dampen diversification and reduce habitat occupancy of later radiations via niche pre-emption has never, to our knowledge, been tested. We investigated clade-level dynamics in plant radiations in the alpine zone, New Zealand. Our aim was to determine whether radiations from older colonizations influenced diversification and community dominance of species from later colonizations within a common bioclimatic zone over the past ca 10 Myr. We used stem ages derived from the phylogenies of 17 genera represented in alpine plant communities in the Murchison Mountains, Fiordland, and assessed their presence and cover in 262 (5 × 5 m) vegetation plots. Our results show clear age-related community assembly effects, whereby congenerics from older colonizing genera co-occur more frequently and with greater cover per unit area than those from younger colonizing genera. However, we find no evidence of increased species richness with age of colonization in the alpine zone. The data support priority effects via niche pre-emption among plant radiations influencing community assembly.

Island species radiation and karyotypic stasis in Pachycladon allopolyploids.

BACKGROUND: Pachycladon (Brassicaceae, tribe Camelineae) is a monophyletic genus of ten morphologically and ecogeographically differentiated, and presumably allopolyploid species occurring in the South Island of New Zealand and in Tasmania. All Pachycladon species possess ten chromosome pairs (2n = 20). The feasibility of comparative chromosome painting (CCP) in crucifer species allows the origin and genome evolution in this genus to be elucidated. We focus on the origin and genome evolution of Pachycladon as well as on its genomic relationship to other crucifer species, particularly to the allopolyploid Australian Camelineae taxa. As species radiation on islands is usually characterized by chromosomal stasis, i.e. uniformity of chromosome numbers/ploidy levels, the role of major karyotypic reshuffling during the island adaptive and species radiation in Pachycladon is investigated through whole-genome CCP analysis. RESULTS: The four analyzed Pachycladon species possess an identical karyotype structure. The consensual ancestral karyotype is most likely common to all Pachycladon species and corroborates the monophyletic origin of the genus evidenced by previous phylogenetic analyses. The ancestral Pachycladon karyotype (n = 10) originated through an allopolyploidization event between two genomes structurally resembling the Ancestral Crucifer Karyotype (ACK, n = 8). The primary allopolyploid (apparently with n = 16) has undergone genome reshuffling by descending dysploidy toward n = 10. Chromosome "fusions" were mediated by inversions, translocations and centromere inactivation/loss. Pachycladon chromosome 3 (PC3) resulted from insertional fusion, described in grasses. The allopolyploid ancestor originated in Australia, from the same or closely related ACK-like parental species as the Australian Camelineae allopolyploids. However, the two whole-genome duplication (WGD) events were independent, with the Pachycladon WGD being significantly younger. The long-distance dispersal of the diploidized Pachycladon ancestor to New Zealand was followed by the Pleistocene species radiation in alpine habitats and characterized by karyotypic stasis. CONCLUSIONS: Karyotypic stasis in Pachycladon suggests that the insular species radiation in this genus proceeded through homoploid divergence rather than through species-specific gross chromosomal repatterning. The ancestral Pachycladon genome originated in Australia through an allopolyploidization event involving two closely related parental genomes, and spread to New Zealand by a long-distance dispersal. We argue that the chromosome number decrease mediated by inter-genomic reshuffling (diploidization) could provide the Pachycladon allopolyploid founder with an adaptive advantage to colonize montane/alpine habitats. The ancestral Pachycladon karyotype remained stable during the Pleistocene adaptive radiation into ten different species.

Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon.

BACKGROUND: Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species. RESULTS: Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not. CONCLUSIONS: We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.

A Pleistocene inter-tribal allopolyploidization event precedes the species radiation of Pachycladon (Brassicaceae) in New Zealand.

The Southern Alps in New Zealand contain many herbaceous plant groups that have radiated during the Plicoene-Pleistocene. The species in these genera tend to be polyploid relative to their overseas close relatives, an observation of much interest given that hybridization and allopolyploidy have recently been suggested as a possible stimulus for adaptive radiation. We were interested to determine whether or not allopollyploidy was a feature of Pachycladon, a genus which is hypothesised to have adaptively diversified onto different geological substrates in the mountains of the South Island of New Zealand. Phylogenetic analyses of five single-copy nuclear genes show that Pachycladon species have two copies of each gene representing two highly diverged evolutionary lineages from the Brassicaceae. Molecular clock analyses of all loci suggest that the two genome copies in Pachycladon diverged 8 million years ago, and that the allopolyploid origin of the genus occurred during the Pleistocene between 1.6 and 0.8 million years ago. This hybridization event at the origin of the Pachycladon radiation is perhaps the most extreme example yet reported of successful hybridization between distantly related parents.