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Keywords:

  • Bayesian analyses;
  • chloroplast inverted repeat;
  • Cynomorium;
  • holoparasite;
  • maximum likelihood;
  • parsimony;
  • Rosales

Abstract

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References

Abstract Cynomorium is a herbaceous holoparasite that has been placed in Santalales, Saxifragales, Myrtales, or Sapindales. The inverted repeat (IR) region of the chloroplast genome region is slow evolving and, unlike mitochondrial genes, the chloroplast genome experiences few horizontal gene transfers between the host and parasite. Thus, in the present study, we used sequences of the IR region to test the phylogenetic placements of Cynomorium. Phylogenetic analyses of the chloroplast IR sequences generated largely congruent ordinal relationships with those from previous studies of angiosperm phylogeny based on single or multiple genes. Santalales was closely related to Caryophyllales and asterids. Saxifragales formed a clade where Peridiscus was sister to the remainder of the order, whereas Paeonia was sister to the woody clade of Saxifragales. Cynomorium is not closely related to Santalales, Saxifragales, Myrtales, or Sapindales; instead, it is included in Rosales and sister to Rosaceae. The various placements of the holoparasite on the basis of different regions of the mitochondrial genome may indicate the heterogeneous nature of the genome in the parasite. However, it is unlikely that the placement of Cynomorium in Rosales is the result of chloroplast gene transfer because Cynomorium does not parasitize on rosaceous plants and there is no chloroplast gene transfer between Cynomorium and Nitraria, a confirmed host of Cynomorium and a member of Sapindales.

Holoparasitic plants are difficult to classify morphologically because of convergence and reduction (Nickrent et al., 2005; Barkman et al., 2007). In recent years, molecular studies have provided new insights into the phylogenetic positions of parasitic plants (Nickrent et al., 1998, 2004; Nickrent, 2007). Nevertheless, several parasites, including Cynomorium L. (“Maltese Mushroom”), continue to have obscure and controversial phylogenetic positions within angiosperm phylogeny (Nickrent, 2002; Angiosperm Phylogeny Group (APG), 2003; Nickrent et al., 2005; Barkman et al., 2007; Jian et al., 2008). Sequences of 18S rDNA and the mitochondrial gene matR have placed Cynomorium in Saxifragales (Nickrent, 2002; Nickrent et al., 2005). However, other mitochondrial genes suggest placement of the parasite in Sapindales (Barkman et al., 2007). Furthermore, Cynomorium may be most closely related to Santalales (Jian et al., 2008).

Cynomorium is a holoparasitic herbaceous plant genus with two species that are distributed in the Mediterranean, northern Africa, and north-western Asia. The two species are closely related and have been considered as conspecific (Mabberley, 1997). The plant has been used as valuable dysentery cure in Malta and as food source and medicinal herb in China for many centuries (Wu et al., 2003). Modern phytochemical research has shown that Cynomorium contains triterpenes that can inhibit HIV (Ma et al., 1999; Nakamura, 2004). Because of its medicinal value and difficulties associated with artificial propagation, Cynomorium has been overexploited, resulting in a sharp decline in natural populations (Nickrent et al., 2005). Therefore, finding free-living relatives of Cynomorium has scientific, ecological, and economic significance (Nickrent et al., 2005).

The aim of the present study was to test previous hypotheses concerning the phylogenetic position of Cynomorium within the tricolpates (or eudicots) using chloroplast sequence data.

1 Material and methods

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References

Thirty-four species were included in the present study to represent Cynomoriaceae, Santalales, Saxifragales, and other major clades of the tricolpates, including Ranunculales, Proteales, Buxales, Vitaceae, Malvids, Fabids, Caryophyllales, Lamiids, and Campanulids, based on the most recent 81-gene angiosperm phylogeny (Jansen et al., 2007). Taxon sampling was extensive for Saxifragales because the study of Nickrent et al. (2005) has placed Cynomorium within Saxifragales, but support for the possible relationship of the parasite with Crassulaceae is weak. Ranunculales was used to root the trees because it has been shown to be sister to the remainder of the tricolpates (Jansen et al., 2007).

Fresh leaf tissues of Buckleya henryi Diels and cortex tissues in the inflorescence of Cynomorium songaricum Rupr. were collected in the field. Both tissue types were immediately dried after collection using silica gel. For Cynomorium, we sampled two individuals to represent two natural populations approximately 100 km apart (Table 1) and the extractions and polymerase chain reaction (PCR) amplification of these samples were performed at different times to avoid cross-contamination. In addition, we obtained sequences of mitochondrial matR and coxI genes from our samples and incorporated them into the published data sets containing Cynomorium coccineum L. from Spain (Nickrent et al., 2005; Barkman et al., 2007). The three samples of Cynomorium formed a robust clade in both matR and coxI phylogenies (data not shown), verifying that our samples have the authentic genomes of Cynomorium.

Table 1.  Species used in the present study (new sequences are in bold)
SpeciesSource and GenBank Accession no.
Aethionema grandflorum Boiss. & Hohen.AP009367
Buckleya henryi DielsJLI 6001, Baotianman, Henan, China; FJ895899 – FJ895903
Buxus microphylla Sieblold & Zucc.NC_009599
Cercidiphyllum japonicum Sieblold & Zucc.EF207443
Citrus sinensis Pers.NC_008334
Cucumis sativus L.DQ119058
Cynomorium songaricum Rupr.JLI 5941, Liangucheng, Minqin, Gansu, China; FJ895885 - FJ895893
Cynomorium songaricumJLI 5958, border of Gansu and Inner Mongolia, China; FJ895894 - FJ895898
Daphniphyllum sp.EF207444
Eucalyptus globules Lobill.NC_008115
Fragaria ananassa DuchesneDQ768221
Glycine max Merr.DQ317523
Gossypium hirsutum L.DQ345959
Hamamelis japonica Siebold & Zucc.EF207445
Heuchera micrantha Dougl.EF207446
Itea virginica L.EF207447
Jasminum nudiflorum Lindl.NC_008407
Kalanchoe daigremontiana Raym.-Hamet & H. PerrierEF207448
Liquidambar styraciflua L.EF207449
Morus indica L.NC008359
Myriophyllum spicatum L.EF207450
Nandina domestica Thunb.NC_008336
Paeonia brownii Douglas ex Hook.EF207451
Panax ginseng C. A. Mey.NC_006290
Penthorum chinense PurshEF207452
Peridiscus lucidus Benth.EF205453
Platanus occidentalis L.NC_008335
Populus trichocarpa Torr. & A. GrayNC_009143
Prunus persica (L.) BatschDQ768222
Pterostemon rotundifolius RamirezEF207455
Rhodoleia championii Hook.EF207455
Ribes americanum Mill.EF207456
Saxifraga stolonifera W. CurtisEF207457
Spinacea oleracea L.NC_002202
Vitis vinifera L.NC_007957

Parasitic plants generally have elevated substitution rates in both nuclear and chloroplast genomes (Nickrent & Starr, 1994; Li et al., 2001), which may hamper unambiguous sequence alignment across tricolpates taxa. Thus, in the present study, we used the inverted repeat (IR) region, which has slower rates of substitutions than both the large and single copy regions of the chloroplast genome (Bortiri et al., 2008). Although many chloroplast genes associated with photosynthesis may frequently be lost in holoparasitic plants (dePamphilis & Palmer, 1990), the IR regions continue to exist (Machado & Zetsche, 1990; Colwell, 1994). Our amplifications of the entire chloroplast genome using angiosperm universal primers (Grivet et al., 2001; Dhingra & Folta, 2005) have been successful for photosynthetic plants, including Nitraria L., the host of Cynomorium (Z-H Zhang & J-L Li, unpubl. data, 2009), but failed to amplify photosynthetic genes (e.g. rbcL) in our Cynomorium samples. However, amplifications were largely successful for the IR region in Cynomorium, indicating that the conserved IR region has been retained in the holoparasite.

Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen Germantown, MD, USA) according to the manufacturer's instructions. The PCR of the entire IR region of the chloroplast genome was conducted using primers described by Dhingra and Folta (2005). The PCR products were purified using a Qiagen Gel Purification Kit and sequenced directly using the PCR primers and BigDye Terminator Chemistry Technology (Applied Biosystems, Foster City, CA, USA). Sequences were edited and assembled using Sequencher (version 4.0; Gene Codes, Ann Arbor, MI, USA). Cloning was conducted for some regions to ensure complete assembly of the IR region using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA).

Sequences were aligned using ClustalX (Jeanmougin et al., 1998) and adjusted manually in MacClade (Maddison & Maddison, 2000). Phylogenetic analyses were conducted using the maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) methods. Characteristics were weighted equally and their states unordered. Gaps were treated as missing data. Our MP analyses used the heuristic tree search in PAUP* with the following options: TBR branch swapping, Multrees on, deepest descent off, and random sequence addition with 1000 replicates and one tree held each replicate. Bootstrap analyses of 500 replicates were conducted to estimate support for clades (Felsenstein, 1985) and the heuristic search options were similar to the parsimony analyses except for the simple sequence addition. The MODELTEST was used to select the optimal model for the sequences with the Akaike criterion (Posada & Crandall, 1998). Maximum likelihood analyses were conducted using the optimal evolutionary model and GARLI (version 0.96b8; Zwickl, 2006). Bayesian inference was done with the selected model and four chains were run for 2 000 000 generations. Trees were sampled every 1000 generations and those before the maximum likelihood scores have reached stationarity were discarded as burn-in. The remaining trees were used to calculate posterior probabilities (pp) for individual clades. Both ML and BI analyses were performed using the Odyssey computer clusters of Harvard University. Templeton (Templeton, 1983) and Shimodaira-Hasegawa (Shimodaira & Hasegawa, 1999) tests were implemented in PAUP* to compare opposing phylogenetic positions of Cynomorium.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References

The entire IR region of Buckleya henryi and Cynomorium songaricum was amplified using the primers described by Dhingra and Folta (2005). Sequences of the other species were available from GenBank (Table 1). The entire IR region was sequenced for one sample of Cynomorium (JLI 5958; Table 1), whereas only one-third of the IR region (7971 bp) was sequenced for the second sample of Cynomorium (JLI 5941; Table 1) because there was little sequence variation between the two samples. Phylogenetic analyses based on individual gene regions did not generate conflicting relationships (trees not shown); therefore, we combined sequences from all regions. The combined data set had 31 292 sites, 475 of which were excluded from phylogenetic analyses due to the difficulty of unambiguous alignment. The MP analyses produced two trees of 12 804 steps, a consistency index of 0.77, and retention index of 0.51 (Fig. 1). When rooted with Ranunculales, the tree had Proteales and Buxales as basal branches, with Buxales being sister to the core tricolpates (bootstrap (bs) = 100%). Caryophyllales and Santalales formed a clade (bs = 96%), which was sister to euasterids (bs = 100%). Vitaceae was sister to the clade consisting of Saxifragales and eurosids; however, the support was weak (<50%). Saxifragales formed a clade (bs = 96%), excluding Peridiscaceae, which was sister to eurosids with moderate support (bs = 73%). Within eurosids (bs = 98%), Malphigiales was sister to the rest of eurosds (bs = 93%); fabids (bs = 96%) and malvids (bs = 98%) were sister clades. Within fabids, the two samples of Cynomorium as a clade (bs = 100%) were included within Rosales (bs = 99%), which was sister to the clade of Curcurbitales and Fabales (bs = 97%). Forcing Cynomorium to form a clade with Santalales or Saxifragales required 145 and 178 extra steps, respectively. In the malvids clade, Myrtales was sister to the rest (bs = 99%) and Sapindales was sister to the clade of Brassicales and Malvales (bs = 100%). Within Saxifragales there were three clades: (i) woody (bs = 76%); (ii) Saxifragaceae and alliance (bs = 100%); and (iii) Crassulaceae and Haloragaceae (bs = 100%). The position of Paeonia was not resolved. The second MP tree (not shown) differed from the first in showing a sister relationship of Daphniphyllum with the remainder of the woody clade in Saxifragales.

image

Figure 1. One of two parsimonious trees based on sequences of the chloroplast inverted repeat region. Consistency index = 0.75; retention index = 0.49. Numbers along the branches are bootstrap percentages.

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The optimal model of evolution for the IR region sequences was the GTR+Γ+I model, as selected by the MODELTEST. The estimated parameters were as follows: Base = (0.29, 0.2, 0.22, 0.29), Rmat = (1.1, 1.86, 0.4, 0.66, 2.24), Gamma shape = 0.93, and Pinvar = 0.28. The first 60 000 generations of the Bayesian analyses were discarded as burn-in because the likelihood scores have since reached stationarity. The ML and BI phylogenies were identical with each other (Fig. 2) and showed congruent relationships to the MP trees, except for a few minor discrepancies. Santalales was sister to the clade containing Caryophyllales and asterids (pp = 1; bs = 88%). Within Saxifragales, Peridiscus was sister to the remainder of the order (pp = 1; bs = 72%), whereas Paeonia formed a clade with the woody clade (pp = 1; bs = 96%). The Saxifragaceae and alliance was sister to the Crassulaceae plus Haloragaceae (pp = 1; bs = 97%). As in the MP trees, Cynomorium was included within Rosales (pp = 1; bs = 100%).

image

Figure 2. Majority consensus of 3940 Bayesian trees based on sequences of the chloroplast inverted repeat region. Numbers above and below the branches are posterior probabilities and maximum likelihood bootstrap percentages, respectively.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References

Tricolpates are a well-supported clade with a synapomorphy of the tricolpate pollen (Wodehouse, 1935, 1936; Bailey & Nast, 1943; Hu, 1950; Walker & Doyle, 1975; Donoghue & Doyle, 1989; Doyle & Hotton, 1991; Chase et al., 1993), and the pollen grains of Cynomorium are tricolporate (Wu et al., 2003; Nickrent et al., 2005). Therefore, Cynomorium belongs to tricolpates.

Over the past two decades, many studies have been performed to estimate the relationships of angiosperms based on DNA sequence data (Chase et al., 1993; Chase & Cox, 1998; Qiu et al., 2000; Soltis et al., 2005) and several major clades have been recognized within the tricolpates, including caryophyllids, fabids, malvids, lamiids, and campanulids (Judd & Olmstead, 2004). A recent phylogenetic analysis based on the sequences of 81 chloroplast genes has provided high resolution for the ordinal relationships of angiosperms (Jansen et al., 2007). Our IR trees (Figs. 1, 2) show largely congruent relationships with the 81-gene trees, which did not include Santalales, Saxifragales, or Cynomorium.

Santalales may be sister to Caryophyllales (Soltis et al., 2000; Worberg et al., 2007) or sister to Caryophyllales plus asterids (Hilu et al., 2003). However, neither position is strongly supported. In our MP trees (Fig. 1), Santalales is sister to Caryophyllales (bs = 93%), whereas in the BI and ML trees (Fig. 2) it is sister to the clade of Caryophyllales and asterids (pp = 1; bs = 88%). Long branch attraction may be responsible for the sister relationship of Santalales and Caryophyllales. Nonetheless, our data support the close relationship between Santalales and Caryophyllales plus asterids (Soltis et al., 2000; Hilu et al., 2003; Worberg et al., 2007). Some authors have placed Cynomorium close to Santalales based on non-molecular data (Bentham & Hooker, 1880; Hutchinson, 1964; Cronquist, 1981; Heywood, 1993), whereas Engler (1904) placed Cynomoriaceae in Myrtiflorae. Recently, Jian et al. (2008) generated molecular data suggesting that Cynomorium belongs to Santalales. However, our IR sequence data do not support the placement of Cynomorium in either Santalales or Myrtales (Figs. 1, 2). Forcing Cynomorium to form a clade with Santalales, Myrtales, and Saxifragales requires more than 140 extra steps in the MP trees and produces lower likelihood scores. The differences are statistically significant, as indicated by both the Templeton and SH tests (P < 0.001).

Saxifragales form a robust clade excluding Peridiscus (Fig. 1) and, within Saxifragales, there are three major clades: (i) the Woody taxa; (ii) Crassulaceae + Haloragaceae; and (iii) Saxifragaceae and alliance (Figs. 1, 2). Therefore, our results largely agree with those of Jian et al. (2008). Paeonia and Peridiscus form a clade that is sister to the remainder of Saxifragales (Soltis et al., 2007; Jian et al., 2008). In our MP trees (Fig. 1), although Paeonia is included in the clade of the core Saxifragale and weakly allied with Haloragaceae plus Crassulaceae, Peridiscus is sister to rosids with moderate support (bs = 73%). Rosids were not included in the two studies of Saxifragales (Soltis et al., 2007; Jian et al., 2008). However, in the BI and ML trees (Fig. 2), Peridiscus is sister to the rest of Saxifragales (pp = 1; bs = 72%), whereas Paeonia forms a robust group with the woody clade (pp = 1; bs = 96%). Phylogenetic relationships between Paeonia and Peridiscus have been difficult to resolve due to long-branch attraction (Soltis et al., 2007). Additional data from slow evolving nuclear genes are needed to further resolve the issue.

Saxifragales is sister to the eurosids (Soltis et al., 2000) or the caryophyllids (Soltis et al., 2003; Kim et al., 2004). In our trees (Figs. 1, 2), Saxifragales is more closely related to Vitaceae plus eurosids than to the caryophyllids.

Phylogenetic analyses based on nuclear 18S rDNA and the mitochondrial gene matR suggest the placement of Cynomorium in Saxifragales and further indicate the close relationship with Crassulaceae within Saxifragales (Nickrent et al., 2005). However, other mitochondrial genes (atp1 and coxI) suggest the placement of Cynomorium in Sapindales (Barkman et al., 2007). In our trees (Figs. 1, 2), Cynomorium is not in the Saxifragales or Sapindales. Instead, it is included in the Rosales and sister to Rosaceae. Forcing Cynomorium to form a clade with Saxifragales and Sapindales entails 178 and 131 extra steps, respectively, which are significantly less parsimonious (P < 0.001). One potential synapomorphy for Cynomorium and Rosales is the presence of hypanthium (Wu et al., 2003; Judd & Olmstead, 2004). Nevertheless, loss of hypanthium occurs in a derived clade of Cannabaceae, Moraceae, and Urticaceae (Judd & Olmstead, 2004). Rosales lacks endosperm (Judd & Olmstead, 2004), which is present in Cynomorium (Wu et al., 2003). If Cynomorium is correctly positioned in Rosales, then a more extensive sampling of the families in Rosales is needed to determine the closest free-living relative of the parasite, which is important because wild resources of Cynomorium have declined owing to overexploitation and it is difficult to cultivate the holoparasite (Nickrent et al., 2005).

Various phylogenetic placements of Cynomorium by different genes (Nickrent, 2002; Nickrent et al., 2005; Barkman et al., 2007; Jian et al., 2008, present study) suggest that Cynomorium may have acquired genes from different hosts during its evolutionary history via horizontal gene transfers. The horizontal transfer of mitochondrial genes (e.g. nad1, atp1) has been reported in angiosperms (Bergthorsson et al., 2004; Davis & Wordack, 2004; Barkman et al., 2007). However, chloroplast gene transfer between angiosperm host and parasite has never been reported. Our comparison of the nuclear rDNA internal transcribed spacer (ITS) sequences from the host root in which Cynomorium grows and from neighboring photosynthetic plants confirmed field observations that one of the hosts of Cynomorium songaricum is Nitraria (Wu et al., 2003; J Yang & J Li, unpubl. obs., 2008), a member of Sapindales (APG, 2003). However, in the IR phylogenies, Cynomorium is not closely related to Sapindales (Figs. 1, 2). In addition, we have successfully amplified the rbcL gene from Nitraria, but failed to get any PCR products from Cynomorium genomes despite repeated attempts. In the field, we observed that there were no rosaceous plants in the areas surrounding Cynomorium songaricum. Therefore, the chloroplast genomes in Cynomorium are apparently native and unlikely to have been transferred from the host. Nevertheless, more data from nuclear genes are needed to further test the phylogenetic position of Cynomorium and gain insights into the nature of horizontal gene transfer between host and parasite.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References

Acknowledgements  The authors thank Jian-Quan LIU, Qiu-Shi WEI, and Xin-Min TIAN for field assistance, and Emily DITTMAR and Jeanwon YANG for laboratory support. This study was supported, in part, by a scholarship from the China Scholarship Council to ZZ.

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  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgments
  7. References
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