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. 2010 Oct 7;106(5):775–790. doi: 10.1093/aob/mcq167

Molecular phylogenetics of Ruscaceae sensu lato and related families (Asparagales) based on plastid and nuclear DNA sequences

Joo-Hwan Kim 1,2,†, Dong-Kap Kim 1,†, Felix Forest 2, Michael F Fay 2, Mark W Chase 2,*
PMCID: PMC2958784  PMID: 20929900

Abstract

Background

Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae.

Methods

A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4·8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades.

Key Results

The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae–Agapanthaceae–Alliaceae); (2) Asparagaceae–Laxmanniaceae–Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae–Behniaceae–Herreriaceae–Agavaceae (clades 2–5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum.

Conclusions

The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (= Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.

Keywords: Aphyllanthes, Asparagaceae, Convallariaceae, Dracaenaceae, Eriospermum, monocot phylogenetics, Nolinaceae, Nolinoideae

INTRODUCTION

Asparagales are the largest order among the five orders of Lilianae (= Liliiflorae) sensu Dahlgren et al. (1985), who followed the concepts of Huber (1969). There are up to 29 families [APG (Angiosperm Phylogeny Group), 1998] in the order, which has been considered monophyletic on the basis of their phytomelan-containing seed coat and several other characteristics (Huber, 1969; Rudall et al., 2000; Chase et al., 2006). Chase et al. (1995a) performed the first extensively sampled phylogenetic analysis to examine their circumscription. This analysis led to the recircumscription of Asparagales to include Orchidaceae (including the former Apostasiaceae and Cypripediaceae) and Iridaceae (including the former Geosiridaceae), both families formerly Liliales/Orchidales, and to exclude Dasypogonaceae s.l., Hanguanaceae, Luzuriagaceae and Philesiaceae. The boundary between Asparagales and Liliales can be difficult to define with morphological data alone because several characters are shared by some lilioids and asparagoids, especially net-veined taxa (Conran, 1989; Rudall et al., 2000). The combined molecular–morphology analysis (Chase et al., 1995b) indicated that although the lilioid monocots were monophyletic, several asparagoid families were paraphyletic or polyphyletic (Chase et al., 1995a, 2006). Within Asparagales there was a paraphyletic grade (predominantly characterized by simultaneous microsporogenesis and inferior ovaries) and a ‘core asparagoid’ clade, uniformly characterized by successive microsporogenesis and mostly superior ovaries (Rudall et al., 1997; Furness and Rudall, 1999). The combined plastid DNA (including rbcL, atpB, trnL intron, and trnL-F intergenic spacer) analyses by Fay et al. (2000) and additional DNA sequences by Pires et al. (2006) further resolved phylogenetic relationships within Asparagales. To accord with the molecular and morphological studies (Chase et al., 1995b, b; Fay and Chase, 1996; Rudall et al., 1997, 2000; Fay et al., 2000), many families in Asparagales have been recircumscribed (APG, 1998; APG II, 2003), and several new families have been erected (Chase et al., 1996, 1997; Conran et al., 1997; Fay and Chase, 1996; Rudall and Chase, 1996).

Ruscaceae sensu lato are a recently recognized family in the broad sense (Chase et al., 1995a; Rudall et al., 2000; APG, 1998); they include Ruscaceae s.s., Convallariaceae, Nolinaceae, Dracaenaceae, Eriospermaceae and Comospermum (the last of highly speculative placement in Dahlgren et al., 1985). Ruscaceae s.l. can be distinguished from other higher asparagoid groups by usually possessing berries or other indehiscent fruit types and absence of phytomelan in the seed coat. One might suppose that indehiscent fruits and absence of phytomelan could be correlated characters, but in Asparagus (Asparagaceae s.s.) berries and phytomelan co-occur. The combined analysis of rbcL and morphology (Chase et al., 1995b; Rudall et al., 1997) indicated that several genera that had been included in Convallariaceae were members of other families or were embedded within a larger clade; this larger clade was recognized as the newly circumscribed broad-sense Convallariaceae (APG, 1998; Fay et al., 2000). Rudall et al. (2000) suggested Ruscaceae Sprengel (1826) had priority over Convallariaceae Horaninow (1834), and they are now generally referred to as Ruscaceae s.l. (Jang and Pfosser, 2002; APG II, 2003), which could also be included in a much-expanded circumscription of Asparagaceae. The latter was presented as an alternative classification in APG II. In APG III (2009), the broadly circumscribed families (including Asparagaceae s.l.) were accepted as the only circumscription in accord with APG, in which case this clade would be referred to as subfamily Nolinoideae.

Ruscaceae s.s., comprising three genera (Ruscus, Danae and Semele), are distributed in the Mediterranean–Macronesian area; they have woody stems, scale-like leaves, berries, and a basic chromosome number of x = 20. Dahlgren et al. (1985) and Takhtajan (1997) regarded Ruscaceae s.s. as the most closely related group to Asparagaceae s.s., but there has been no clear evidence on relationships of these families. The two families have several similarities including phylloclades (but even for this character there are questions about homology; Arber, 1924; Cooney-Sovetts and Sattler, 1986), baccate fruits and similar karyotypes (Sato, 1942; Tamura, 1995), and they have differences in the position of inflorescences and seed coat (Conran and Tamura, 1998). Rudall et al. (1998) recognized that the karyotype of Ruscaceae (x = 20) is more similar to Convallariaceae (usually x = 19, rarely 18, 20) than to that of Asparagaceae s.s. (mostly x = 10). Serological analyses and lack of phytomelan in the seed coat indicated a closer relationship between Ruscaceae s.s. and Convallariaceae than either to Asparagaceae s.s. (Chupov and Cutjavina, 1980).

Convallariaceae are rhizomatous perennial herbs distributed in the Northern Hemisphere; they are abundant in eastern and southeastern Asia and comprise four tribes: Polygonateae, Ophiopogoneae, Convallarieae and Aspidistreae (Dahlgren et al., 1985; Tamura, 1995). They share calcium oxalate crystals and two ovules (rarely or over) per locule, but it is not so easy to identify distinguishing morphological characters for the tribes in Convallariaceae, and Dahlgren et al. (1985) used plesiomorphic characters for the taxonomic key, including baccate fruits, non-phytomelaniferous seeds and nuclear endosperm formation. Polygonateae share a sympodial rhizome, an elongated aerial stem and berries, and the position and shape of inflorescences (axillary in Polygonatum and Disporopsis, terminal in Smilacina and Maianthemum, and axillary and terminal in Heteropolygonatum) are variable in the tribe. Ophiopogoneae have a sympodial rhizome, fruits that rupture at an early stage, seeds with sarcotesta, and basic chromosome number x = 18; this tribe comprises three genera (Liriope, Ophiopogon and Peliosanthes) distributed in eastern and southeastern Asia. Convallarieae and Aspidistreae have a monopodial rhizome and a short stem, usually berries (except drupes in Tricalistra), and basic chromosome number usually of x = 19, rarely 20 (Theropogon) or 18 (some Aspidistra). Conran and Tamura (1998) merged Aspidistreae with Convallarieae. The plastid trnK sequence analysis of Yamashita and Tamura (2000) supported the treatment of Conran and Tamura (1998).

Nolinaceae are arborescent, anomalously woody plants with terminal rosette leaves and indehiscent nutlets, and they comprise four genera, Nolina, Dasylirion, Calibanus and Beaucarnea, found in warm, dry regions of North America. Nolinaceae were often previously included in a broadly defined family Liliaceae near Dracaena, and they had been treated in the tribe Dracaeneae (Bentham and Hooker, 1883) or Nolineae (Krause, 1930). Hutchinson (1934) included Nolinaceae, Yuccoideae and Dracaenae in Agavaceae because of their anomalous woody growth (via a secondary thickening meristem) and fibrous leaves, but this treatment was not supported by other morphological characters (flowers, fruits and seeds) and karyology (Sharma and Chaudhuri, 1964). Nolinaceae were excluded from Agavaceae and arranged near Dracaenaceae in Dahlgren et al. (1985).

Dracaenaceae include perennial plants with a more or less woody trunk, but many do not have a trunk; they comprise two genera, Dracaena and Sansevieria (perhaps best combined into one genus), which occur in subtropical to tropical regions of the Old World. Dracaenaceae are distinguished from Nolinaceae in having berries, no oils in guard cells and mucilage-filled cells with crystal raphides in vegetative parts.

Eriospermaceae are perennial herbs with various types of tubers and free perianth parts. They comprise a single genus (Eriospermum) distributed in southern parts of Africa. This family shows seasonal developmental differences between leaves and inflorescences. Because they have extraordinary characters such as leaf appendages, epidermal hairs on the seeds and embryological attributes but have successive microsporogenesis and thin testa, Dahlgren et al. (1995) suggested that treatment as a family separate from related groups was probably best. The taxonomic position of Eriospermaceae has been controversial whether included (Rudall et al., 2000) or not (Jang and Pfosser, 2002) in Ruscaceae s.l.

Ruscaceae s.l. have no distinguishable synapomorphic characters from the other higher asparagoids except the absence of phytomelan in the seed coat, but analysis of the combined molecular and morphology matrix (Chase et al., 1995b) indicated that Ruscaceae s.l. was a well-supported clade that was largely unresolved relative to the related families and genera. This grouping of Ruscaceae s.l. (= Convallariaceae s.l.) was supported by plastid DNA restriction-site analyses of some taxa, although Bogler and Simpson (1995) lacked some of the core taxa such as Ruscaceae s.s. and Comospermum. Several molecular studies supported monophyly of Ruscaceae s.l. (Rudall et al., 1997, 2000). Yamashita and Tamura (2000) sequenced the plastid trnK region (including the matK exon) for 39 Convallariaceae species and related families, which indicated that there were six major clades; Convallariaceae s.s. were paraphyletic in this analysis. They compared the trnK tree with the rbcL tree and looked at basic chromosome numbers, but they occasionally had unresolved relationships due to a lack of informative characters and sampling of potential sister groups; they nonetheless found evidence to support the tribal limits in Convallariaceae of Conran and Tamura (1998). Jang and Pfosser (2002) performed a phylogenetic analysis based on rbcL and trnL-F intron/spacer sequences, but there were no improved assessments of relationships because of poor sampling of taxa in Ruscaceae s.l.

Asparagaceae s.s. have been usually considered sister to Ruscaceae s.l. due to their cytological and morphological similarities (Tamura, 1995). Aphyllanthes (Aphyllanthaceae) was also indicated as a possible sister group to Ruscaceae (Conran, 1998; Yamashita and Tamura, 2000), but Fay et al. (2000) made a cautious accessment of Aphyllanthes, a taxonomically isolated Mediterranean genus, because of its labile phylogenetic position. Laxmanniaceae were sister to Ruscaceae s.l. plus Asparagaceae (Rudall et al., 1997; Fay et al., 2000; Bogler et al., 2006; Givnish et al., 2006; Graham et al., 2006; Pires et al., 2006). APG II (2003) and APG III (2009) suggested a broader circumscription of Asparagaceae based largely on results of analysis for four plastid DNA regions (Fay et al., 2000); Ruscaceae s.l. was treated as an optional circumscription along with Agavaceae s.l. (including Anemarrhenaceae, Anthericaceae, Behniaceae, Herreriaceae and Hesperocallidaceae) and related families such as Aphyllanthaceae, Hyacinthaceae, Laxmanniaceae and Themidaceae.

A molecular phylogenetic study was conducted to re-evaluate delimitation of Ruscaceae s.l. of Rudall et al. (2000) and related families (APG, 1998; APG II, 2003; APG III, 2009; Chase et al., 2006), especially to assess their possible sister groups in Asparagales and evaluate phylogenetic relationships with the related families in the core asparagoids. The aim was to investigate relationships in Asparagales by sequencing three genes, 18S nuclear ribosomal DNA and plastid rbcL and matK, for 121 taxa of Asparagales. These genes were chosen because of their use in recent studies of familial and higher-level phylogenetics (Chase et al., 1995a, 2006; Soltis et al., 1997, 2000; Fay et al., 2000; Yamashita and Tamura, 2000; Hilu et al., 2003; Devey et al., 2006). The impact of these data on the classification of Ruscaceae s.l. and related families was also evaluated. New sequences from mostly new accessions of the sampled taxa were produced for this study; this was done to avoid possibile misidentification of taxa in the earlier published studies or sequences with errors due to the prelatively primitive techniques used to produce rbcL and 18S rDNA sequences in the early period of DNA sequencing.

MATERIALS AND METHODS

Plant materials

The taxa used for this study included all genera (except Heteropolygonatum) in Ruscaceae s.l. sensu Rudall et al. (2000) and representatives of all families of Asparagales (APG). The plant material used was either fresh, collected from the field and dried, taken from specimens in the herbarium, or was a DNA sample borrowed from the Royal Botanic Gardens, Kew, DNA Bank (http://data.kew.org/dnabank/DnaBankForm.html). Voucher specimens of the taxa were prepared; source, voucher information and database accession numbers are listed in the Appendix. Provenance and distributions were also prepared from voucher specimens and the World Checklist of Selected Plant Families (http://apps.kew.org/wcsp/home.do). For one taxon (Bulbine sp.), sequences from different species (B. succulenta and B. frutescens) in GenBank were used, and several sequences (six for 18S rDNA, nine for rbcL and ten for matK) were from GenBank and previous papers (Chase et al., 2006). Otherwise, new sequences were prepared.

DNA extraction, PCR, sequencing and alignment

Total genomic DNA was extracted from 0·5–1·0 g of fresh or silica gel-dried leaves using the 2× CTAB buffer method (Doyle and Doyle, 1987). Lipids were removed with SEVAG solution (24 : 1 chloroform : isoamyl alcohol), and DNA was precipitated with isopropanol at –20 °C. Total extracted DNA was dissolved in 1× TE buffer and stored at –70 °C, and the concentration of DNA was determined with GeneQuant pro (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA) before use.

The 18S rDNA gene was amplified using the primers and protocols of White et al. (1990), Nickrent and Soltis (1995), and Soltis and Soltis (1998); matK was amplified with primers and protocols of Johnson and Soltis (1995) and Hilu et al. (2003), and the rbcL gene was amplified with primers and protocols of Omstead et al. (1992), Shinwari et al. (1994) and Fay and Chase (1996). Amplifications were carried out in 50-μL reactions, containing 2 units Taq DNA polymerase, 5 µL 10× reaction buffer (100 mm Tris–HCl, 500 mm KCl, 15 mm MgCl2), 2·5 mm dNTPs, 5 pmol μL−1 forward and reverse primers, using Perkin-Elmer 9700 machine (Applied Biosystems, Inc., Beverly, MA, USA). DMSO (2 %) was added to reduce the secondary structure in PCR. PCR conditions were premelt of 94 °C for 2 min, followed by 30–35 cycles of denaturation at 94 °C for 1 min, annealing at 50–55 °C for 1 min, extension at 72 °C for 3 min, followed by a final extension of 7 min at 72 °C.

All PCR products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH, USA) according to the manufacturer's protocols. Dideoxy cycle sequencing was performed using the chain-termination method and the ABI prism big dye reaction kit (ver. 3·1) following the manufacturer's protocols. Products were run on an ABI 3700 genetic analyser or MegaBace1000 (Amersham Pharmacia Biotech, Inc.) using the manufacturers' protocols. Sequence editing and assembly of contigs were carried out using Sequence Navigator and AutoAssembler software (ABI).

All sequences were aligned initially in ClustalX (ver. 1·83; Thompson et al., 1997) and MacClade (ver. 4·0; Maddison and Maddison, 2000) and then manually adjusted following the guidelines of Kelchner (2000). Alignment of sequences for these coding genes was easily performed because there were no insertions/deletions (indels) among the sequences of Ruscaceae s.l., but there were indels in the sequences of other Asparagales and outgroups: three in 18S rDNA and nine in matK; the aligned matrix is available from kimjh@dju.ac.kr or m.chase@kew.org. The three indels in 18S rDNA correspond to positions 496–501, 666–672 and 1363–1369 on the reference sequence of Glycine max (L.) Merr. (Soltis et al., 1997, 2000; Soltis and Soltis, 1998).

Parsimony analysis

Two separate sets of analyses were carried out. The first (analysis A) comprised the plastid sequences of 121 taxa representing all 29 families of Asparagales, and the second (analysis B) comprised the combined 18S rDNA and plastid DNA sequences for the same taxa. Orchidaceae were designated as the outgroup for both analyses based on previous results (Chase et al., 1995a, 2000b; Fay et al., 2000). PAUP* (ver. 4·10b; Swofford, 2007) was used for parsimony analysis and followed the widely used parsimony analysis with successive approximations weighting and bootstrapping (Fay et al., 2000; Clarkson et al., 2004; the bootstrap did not use the relative weights). In analyses A and B, tree searches were performed under the Fitch (equal weight, EW; Fitch, 1971) criterion with 1000 random sequence additions and tree–bisection–reconnection (TBR) branch swapping, permitting ten trees to be held at each step (Multrees on) to reduce time searching suboptimal ‘islands’ of trees (Chase et al., 2006). All shortest trees collected in the 1000 replicates were swapped on to completion without a tree limit. Successive approximation weighting (SW; Farris, 1989) was carried out to select the most stable trees (Carpenter, 1988) according to the rescaled consistency index, using the maximum value (best fit) criterion and a base weight of 1·0, followed by 100 replicates of heuristic search with random sequence additions and subtree pruning-regrafting (SPR) swapping. All shortest trees from these 100 replicates were then swapped to completion, after which another round of weighting was implemented. This process was repeated until the same tree length was obtained twice in succession. DELTRAN character optimization was used to illustrate branch length throughout. To evaluate internal support, 1000 bootstrap replicates were carried out with equal weights, TBR branch swapping with five trees held at each step and simple taxon addition (Felsenstein, 1985). The following descriptions for categories of bootstrap support were used: weak, 50–74; moderate; 75–84; well supported, 85–100 % (Chase et al., 2000a).

Bayesian analysis

Further phylogenetic analyses were performed using Bayesian inference as implemented in MrBayes (ver. 3·12; Ronquist et al., 2005). MrModeltest (ver. 2·2; Nylander, 2005) was used to determine the best model of DNA substitution for each partition, evaluating all models against defaults of the program. The GTR + I + G model (a general time reversible model with a proportion of invariable sites and a gamma-shaped distribution of rates across sites) was chosen for the three genes as the best-fitting among the 24 models compared. Thus, all three genes were assigned a model of six substitution types (n = 6) with a proportion of invariable sites. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 5 × 106 generations and sampled every 100 generations, and the first 1 × 105 trees were excluded (‘burn-in’). Post-burn-in samples of trees drawn from the posterior probability distribution were summarized, and this tree is illustrated (see Fig. 3). Bayesian analysis was performed three times to ensure convergence of results.

Fig. 3.

Fig. 3.

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Bayesian tree from combined DNA analysis (analysis C) for 121 taxa of Asparagales. The numbers above branches are posterior probabilities from 5 × 106 generations with the GTR + I + G model. A, Asparagaceae; B, Laxmanniaceae.

RESULTS

A summary of characteristics of the DNA data is presented in Table 1. The aligned number of characters was 4802, but 71 positions for 18S rDNA were excluded from phylogenetic analyses as in previous studies due to ambiguous alignments in these short sections of the matrix (Soltis et al., 1997, 2000; Soltis and Soltis, 1998). The total number of included bases was 4731 of which 1851 were variable (39·1 %) and 1301 (27·5 %) were potentially parsimony informative. The number of positions in the matrix included 1338 for rbcL, 1668 for matK and 1725 for 18S rDNA. The matK gene was the most variable among the three genes and gave the greatest number of parsimony informative sites; 18S rDNA showed the lowest variation. The number of parsimony-informative characters was 327 (25·1 %) for rbcL, 784 (60·3 %) for matK and 190 (14·6 %) for 18S rDNA.

Table 1.

Statistics for the three genes analysed in this study

Characters rbcL (I) matK (II) 18S rDNA (III) Plastid data (I + II) Combined (I + II + III)
Aligned 1338 1668 1796 3006 4802
Included 1338 1668 1725 3006 4731
Parsimony informative 327 784 190 1111 1301
Variable 462 1072 317 1534 1851
Constant 876 596 1408 1472 2880
Transition/transversion 877/456 (1·87) 1868/1189 (1·52) 507/179 (2·52)
G + C (%) 43·45 31·37 50·43
Tree length (EW/SW) (1572/407·660) (3744/1156·147) (902/290·889) (5435/1537·590) (6442/1811·619)
CI (EW/SW) (0·40/0·71) (0·44/0·70) (0·46/0·80) (0·42/0·70) (0·42/0·71)
RI (EW/SW) (0·69/0·84) (0·74/0·86) (0·65/0·82) (0·72/0·85) (0·70/0·84)
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EW, Equally weighted; SW, successive weighted; CI, consistence index; RI, retention index.

Parsimony analysis based on plastid DNA (analysis A)

The final alignment of the combined (rbcL and matK) plastid DNA matrix comprised 3006 positions, of which 1534 were variable (51·0 %) and 1111 (37·0 %) were potentially parsimony informative. Fitch analysis (EW; Table 1) produced 5760 equally most-parsimonious trees [length = 5435 steps; CI (consistency index, including autapomorphies) = 0·42; RI (retention index) = 0·72]. Successive weighting (SW) identified one shortest tree as optimal with an SW score of 1537·59 (5435 Fitch length; CI = 0·70, RI = 0·85). The SW tree is therefore one of the trees found with equal weights; it is shown with its Fitch branch lengths (DELTRAN optimization) in Fig. 1. Groups (nodes) not found in the consensus tree of Fitch analysis are marked with triangles. Bootstrap percentages (BP) consistent with the strict consensus tree are shown below each branch; groups with BP < 50 are not indicated.

Fig. 1.

Fig. 1.

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The single shortest tree from successive weighting of plastid rbcL and matK (analysis A) for Ruscaceae s.l. and related groups of Asparagales. Numbers of substitutions are indicated below each branch (DELTRAN optimization), and bootstrap percentages >50 % are given above each branch. Triangles indicate branches not present in the strict consensus tree of 5760 equally MP trees by Fitch analysis (equal weight). Tree length is 5435 steps with CI = 0·70 and RI = 0·85. The dashed line in the lower left-hand corner marks the point where the non-core asparagoids are attached to this part of the tree (non-core taxa are not shown; this part of the tree is identical to that show in Fig. 2).

In this study, only the core asparagoids are presented for the plastid DNA tree (Fig. 1) since it showed a topology similar to that of the combined DNA tree except for relationships among Ruscaceae s.l. and related families. The core asparagoids formed a strongly supported group (BP 100), and the other asparagoids were paraphyletic (not shown). The core asparagoids fell into two clades, one moderately (BP 84) and the other well supported (BP 90). The first consisted of four families including Agavaceae s.l. sensu APG I (BP 96), Hyacinthaceae (BP 100) and Themidaceae (BP 100), as well as Aphyllanthaceae. The second consisted of Ruscaceae s.l., Asparagaceae, Laxmanniaceae, Alliaceae, Agapanthaceae and Amaryllidaceae.

Within the second group, Ruscaceae s.l. were well supported (BP 90; Fig. 1). Asparagaceae s.s. were strongly supported (BP 100) and sister to Ruscaceae s.l., but the two families together were weakly supported (BP 50); Laxmanniaceae were strongly supported (BP 96) as a member of the clade with Ruscaceae s.l. and Asparagaceae s.s. Alliaceae s.l. sensu APG (1998) including Alliaceae s.s., Agapanthaceae and Amaryllidaceae form a moderately supported clade (BP 75) as the sister of the rest (Fig. 1).

The tree topology of Ruscaceae s.l. in this study did not accord or was only partly congruent with previous studies (Rudall et al., 1997; Yamashita and Tamura, 2000; Jang and Pfosser, 2002). Ruscaceae s.l. were strongly supported (BP 90), and within this clade fell Ruscaceae s.s., Dracaenaceae, Convallariaceae, Nolinaceae and Eriospermaceae (Fig. 1). The combined Ruscaceae s.s. and Dracaenaceae clade was moderately supported (BP 75), and they were interdigitated within clades of Convallariaceae. Within Convallariaceae, Aspidistreae (BP 96; including Campylandra, Rohdea, Tupistra and Aspidistra) and Ophiopogoneae (BP 98; including Liriope, Ophiopogon and Peliosanthes) were strongly supported. Convallarieae were not monophyletic, and Polygonateae were only weakly supported as monophyletic (BP 64) and excluded Disporopsis (BP 100). Eriospermaceae (BP 100) were sister to highly supported Nolinaceae (BP 100).

Parsimony analysis based on combined DNA (analysis B)

The number of positions included in the combined analysis (18S rDNA, rbcL and matK) was 4731. The number of bases contributed by each individual gene was 1338 for rbcL, 1668 for matK and 1725 for 18S rDNA. The number of variable sites was 1851 (39·1 %), and 1301 (27·5 %) were potentially parsimony informative. Fitch analysis (EW), including 121 asparagoid monocots (Table 1), produced 5721 equally most-parsimonious trees of 6442 steps with CI (including autapomorphies) = 0·42 and RI = 0·70. Successive weighting (SW) identified one shortest tree as optimal with an SW score of 1811·62 (6442 Fitch length; CI = 0·71, RI = 0·85). The SW tree was one of the Fitch trees, and it is shown with its Fitch branch lengths (DELTRAN optimization) in Fig. 2. Groups not found in the strict consensus tree of the Fitch analysis are marked with triangles. Bootstrap percentages (BP; equal weights) consistent with the strict consensus tree are shown below each branch, but groups with BP < 50 are not indicated (Fig. 2).

Fig. 2.

Fig. 2.

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One MP tree from combined DNA data (analysis B) for 121 taxa of Asparagales. Numbers of substitutions are indicated below each branch, and bootstrap percentages >50 % are given above each branch. Triangles indicate groups not present in the strict consensus tree of the Fitch analysis. Bars show points at which changes in microsporogenesis have taken place. Tree length is 6442 steps with CI = 0·71 and RI = 0·85. A, Asparagaceae; B, Laxmanniaceae; C, Alliaceae s.l.; D, Agavaceae s.l.; E, Hyacinthaceae; F, Aphyllanthaceae; G, Themidaceae; H, Xanthorrhoeaceae s.l.

The topology of the combined DNA tree for Asparagales largely followed the previous analyses in the broad sense of the core asparagoids concept (Chase et al., 1995a; Fay et al., 2000; Pires et al., 2006). The core asparagoids formed a strongly supported group (BP 100) with the rest of the families of Asparagales forming a grade relative to the core group (Fig. 2). The core asparagoids fell into two big clades, one with strong support (BP 86; group B) and the other with weak support (BP 56; group A). The former consisted of four families including Agavaceae s.l. sensu APG II (BP 94), Hyacinthaceae (BP 100), Themidaceae (BP 100) and Aphyllanthaceae. The other consisted of Ruscaceae s.l., Asparagaceae s.s., Laxmanniaceae and Alliaceae s.l.

Within group A in the core asparagoids, Ruscaceae s.l. were well-supported (BP 88; Fig. 2). Asparagaceae s.s. were strongly supported (BP 100) as sister to Ruscaceae s.l., even though the clade of the two was weakly supported (BP 50), and Laxmanniaceae appeared as sister (BP 96) to Ruscaceae s.l. and Asparagaceae. Alliaceae s.l. sensu APG (1998) were weakly supported (BP 73) as sister to group A (Fig. 2).

The tree topology of Ruscaceae s.l. from the combined analysis did not accord with or was only partly congruent with previous plastid analyses (Rudall et al., 1997; Yamashita and Tamura, 2000; Jang and Pfosser, 2002). Ruscaceae s.l. were grouped together in one strongly supported clade (BP 88; Fig. 2), and Ruscaceae s.s., Dracaenaceae, Nolinaceae and Eriospermaceae received strong bootstrap support (BP > 99 %) even though Convallariaceae were polyphyletic (Fig. 2). For the tribes of non-monophyletic Convallariaceae, Aspidistreae (BP 64) and Ophiopogoneae (BP 99) were monophyletic, but Convallarieae were not monophyletic. Polygonateae were monophyletic but weakly supported (BP 60). Nolinaceae were sister to the rest of Ruscaceae s.l. minus Eriospermum (BP 99). Strongly supported Eriospermaceae (BP 100) were sister to the rest of Ruscaceae s.l.

Outside the core asparagoids, the tree topology from analysis of the combined DNA data was congruent with those from previous analyses (Fay et al., 2000; Pires et al., 2006). The major differences between combined and plastid results were mostly not in topology but rather in levels of support. The core asparagoids were sister to Xanthorrhoeaceae s.l. sensu APG III (2009) with strong support (BP 100). Xeronemataceae were sister to the large clade (core asparagoids and Xanthorrhoeaceae s.l.; BP 99), which was strongly supported. Iridaceae were strongly supported (BP 100) as sister to the above clade. The relationships among the next asparagoid families [(Ixioliriaceae + Tecophilaceae) Doryanthaceae] were strongly supported (BP < 94), and the sister to the above clade (BP 100) consisted of <Branfordiaceae {Boryaceae [Asteliaceae (Hypoxidaceae + Lanariaceae)]}>. The final clade in Asparagales was Orchidaceae, which was designated in this study as outgroup to the rest of the order (BP 100), following results of broader monocot analyses that demonstrated Orchidaceae to be sister to the rest of Asparagales (Chase et al., 2006).

Bayesian analysis of combined matrix (Analysis C)

The Bayesian tree (Fig. 3) shows the posterior probabilities summarized from the set of recovered post-burn-in trees; parameters of the GTR + I + G model used in this analysis are listed in Table 2. Although one node in the core asparagoids had low posterior probability (PP), 0·62, the majority of nodes in the tree are supported by PPs >0·95. Bayesian analysis produced a similar overall topology to that of the maximum parsimony analysis (Fig. 3), but it showed a few differences in the core asparagoids. The core asparagoids were strongly supported (1·00 PP; Fig. 3). Within the core asparagoids, a big clade consisting of Ruscaceae s.l. (PP 0·99), Asparagaceae (PP 1·00) and Laxmanniaceae (PP 1·00) was highly supported (PP 1·00). Among the taxa of Ruscaceae s.l., Dracaenaceae (PP 1·00), Ruscaceae s.s. (PP 1·00) and Eriospermaceae (PP 1·00) were strongly supported, but the former four tribes of Convallariaceae were not monophyletic except for Ophiopogoneae (PP 1·00). Agavaceae s.l. (sensu APG III) including Anemarrhenaceae, Anthericaceae, Behniaceae and Herreriaceae were weakly supported (PP 0·86). A combined clade with Agavaceae s.l. and Aphyllanthaceae showed low PP (PP 0·37), and the node with Themidaceae (PP 1·00) and Hyacinthaceae (PP 1·00) was highly supported (PP 0·92). Amaryllidaceae s.l. (sensu APG III) consisting of Alliaceae (PP 1·00), Amaryllidaceae s.s. (PP 1·00) and Agapanthaceae were strongly supported (PP 1·00).

Table 2.

Parameters of models for each gene as estimated by MrModeltest 2·1

Parameters* rbcL matK 18S rDNA
r(G ↔ T) 1 1 1
r(C ↔ T) 4·0874 2·9017 11·9400
r(C ↔ G) 1·1394 0·9554 0·4848
r(A ↔ T) 0·4645 0·2741 2·1859
r(A ↔ G) 2·6483 3·2593 1·9161
r(A ↔ C) 0·8335 1·7435 0·9482
freqA 0·2854 0·3060 0·2573
freqC 0·1900 0·1488 0·2134
freqG 0·2278 0·1425 0·2724
freqT 0·2968 0·4062 0·2569
Shape 0·7818 1·1016 0·5748
Pinvar 0·5254 0·0879 0·6916
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* r(N ↔ N), Substitution rates for each nucleotide pair; freqA, freqC, freqG, freqT, empirical base frequency; Shape, gamma distribution shape parameter; Pinvar, proportion of invariable sites.

The spine of the tree among the non-core asparagoids was nearly congruent to that of the maximum-parsimony (MP) tree with high PP (1·00; Fig. 3). All nodes were strongly supported (PP > 0·89) with only one exceptional branch (PP 0·30), that linking Doryanthaceae (PP 1·00), Ixiolirionaceae and Tecophilaceae (PP 1·00). Also Xanthorrhoaceae s.l. (PP 1·00) were sister to the core asparagoids (Fig. 3); Xeronemataceae were sister to Xanthorrhoaceae s.l. plus core asparagoids.

DISCUSSION

The tree topology in Asparagales from analysis of three genes is nearly congruent with those of previous analyses, although this study used Orchidaceae as the only outgroup (Chase et al., 1995a, b; Rudall et al., 2000; Fay et al., 2000; Pires et al., 2006). The overall results produced here, with different accesissions of species and a different set of taxa, indicate that the tree topologies from the previous studies are robust with respect to the samples used to represent genera and the taxa sampled. The core asparagoid clade was strongly supported, and the tree topology of the asparagoids characterized by simultaneous microsporogenesis and inferior ovaries, is congruent with the previous analyses and has strong support (Figs 2 and 3; Chase et al., 1995a; Fay et al, 2000; Pires et al., 2006). The family composition of the core asparagoids is the same as that in APG (1998) and characterized by a reversal to successive microsporogenesis, although there are a few parallel occurrences in Xanthorrhoeaceae and Hypoxidaceae (Rudall et al., 1997). In this study, the core asparagoids was split into two subclades: (1) Ruscaceae s.l. + Asparagaceae s.s. + Laxmanniaceae + Alliaceae s.l. sensu APG II; and (2) Agavaceae s.l. sensu APG II + Hesperocallidaceae + Hyacinthaceae + Themidaceae with Aphyllanthaceae. These two major clades differ from the two identified in the study by Pires et al. (2006), upon which the APG III set of families was based (see below for more discussion). The present study also supports Xanthorrhoeaceae s.l. sensu APG III as sister to all core asparagoids.

The most variable gene was matK, and 18S rDNA exhibited the lowest level of variation. The variable positions in the two plastid DNA genes changed twice as fast as those in 18S rDNA. The topologies exhibited similar patterns in the asparagoids for each analysis from three genes separately (not shown) as in the previous combined analyses (Rudall et al., 2000; Fay et al., 2000).

Phylogenetics of Ruscaceae s.l. and related families

Ruscaceae s.l. are a recently recognized family (APG, 1998; Rudall et al, 2000), which can be distinguished by the absence of phytomelan in the seed coat and indehiscent or berry-like fruits (Rudall et al., 2000). Ruscaceae s.l. represent a well-supported clade in DNA alone (Fay et al., 2000; Pires et al., 2006) and combined DNA–morphological analyses (Rudall et al., 2000). This study strongly supports monophyly of Ruscaceae s.l. (BP 90 from plastid DNA alone, and BP 88 from the combined data). Asparagaceae s.s. were monophyletic (BP 100, plastid; BP 100, combined data), and the sister group to Ruscaceae s.l. (BP 90, plastid; BP 93, combined) was Laxmanniaceae, as in previous analyses (Fay et al., 2000; Pires et al., 2006). The clade with Ruscaceae s.l., Asparagaceae and Laxmanniaceae was sister to Amaryllidaceae s.l. (APG III, 2009), including Alliaceae, Amaryllidaceae s.s. and Agapanthaceae. This set of relationships, particularly with respect to the position of Amaryllidaceae s.l., was a little different from previous results. However, the relationships identified here were only moderately supported and contradicted by Pires et al. (2006), who found Asparagaceae s.l. (sensu APG II) to be sister to Amaryllidaceae s.l.; the core asparagoids were thus composed of three clades in the strict consensus tree (not shown) of the equally weighted analysis of the plastid DNA data. Yamashita and Tamura (2000) suggested that the outgroups for Convallariaceae were Eriospermum, Aphyllanthes and former Anthericaceae genera in their trnK region analyses, but the present study shows that Aphyllanthes and Anthericaceae have a more remote relationship to that family than Asparagaceae s.s.

Aphyllanthes has been a problem taxon in core asparagoid phylogenetics. In this study Aphyllanthes was found to be sister to Themidaceae (BP 100) in both MP analyses, and this combined clade (77/81 BP) of Aphyllanthes and Themidaceae was the sister to Hyacinthaceae (100/100 BP) and Agavaceae s.l. (94/94 BP). Also, in the present MP analyses that excluded Aphyllanthes (results not shown) there was no change in tree topology and a small increase in internal support, but in the Bayesian tree (Fig. 3) Aphyllanthes was sister to Anemarrhenaceae in Agavaceae s.l., although this result was weakly supported (37 PP). Further detailed studies are required to establish the phylogenetic relationships of Aphyllanthes. If Asparagaceae s.l. is recognized as in APG III (2009), then at least the problem becomes one of within-family phylogenetics.

Phylogenetics within Ruscaceae s.l.

Although Asparagales were established with phytomelaneous seeds as the synapomorphic character by Huber (1969), Ruscaceae s.l., which have non-phytomelaneous seeds, were controversially included within the core asparagoids that exhibit successive microsporogenesis. Most taxa in Ruscaceae s.l. have several additional synapomorphies, such as articulate pedicels, septal nectaries and berries. Chase et al. (1995a) first mentioned the expanded range of taxa in Ruscaceae s.l. including Convallariaceae s.s., Ruscaceae s.s., Nolinaceae, Dracaenaceae, Eriospermaceae and Comospermum, and this group of taxa was treated as Convallariaceae s.l. in some papers (Rudall et al., 1997; APG, 1998; Fay et al., 2000), but Ruscaceae has priority (Rudall et al., 2000).

This study confirmed the monophyly of Ruscaceae s.l. with strong support (BP 90, Fig. 1), including Eriospermaceae (BP 100, Figs 1 and 2; Rudall et al., 2000). Based on the combined three-gene analyses, Ruscaceae s.l. consist of six subclades: (1) Polygonateae (excluding Disporopsis), (2) Ophiopogoneae, (3) Convallarieae (excluding Theropogon), (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum, (5) Nolinaceae and (6) Eriospermum. This result corresponds with that of Rudall et al. (2000): (1) Eriospermum, (2) Comospermum, (3) nolinoids (Nolinaceae, Ophiopogoneae except Peliosanthes), (4) dracaenoids (Dracaenaceae), (5) Polygonateae, (6) Convallarieae with ruscoids (Ruscaceae s.s.) and Peliosanthes. Yamashita et al. (2000) also found six groups: (1) Polygonateae, (2) Ophiopogoneae, (3) Convallarieae, (4) Nolinaceae, (5) Ruscaceae (with Dracaenaceae) and (6) Comospermum. Only Eriospermum and Polygonateae were consistent in the results from all three sets of analyses.

Within the Ruscaceae s.l. clade, Eriospermaceae (BP 100/PP 1·00), Nolinaceae (BP 100/PP 1·00), Ruscaceae s.s. (BP 100/PP 1·00) and Dracaenaceae (BP 100/PP 1·00) were well supported. However, Convallariaceae are paraphyletic (Figs 1 and 2) as in previous studies (Rudall et al., 2000; Yamashita and Tamura, 2000; Jang and Pfosser, 2002). If Convallariaceae is to be recognized, it should be recircumscribed; this result has been well supported by the results of molecular and combined molecular and morphological data (Chase et al., 1995b; Rudall et al., 1997; Fay et al., 2000; Rudall et al., 2000; Yamashita et al., 2000; Tamura and Yamashita, 2004).

Relationships of Ophiopogoneae

In this study, Ophiopogoneae (BP 98/PP 100) were the only monophyletic tribe among the four previously recognized in Convallariaceae. Ophiopogoneae share hypodermal fibres and well-developed fruits with a thin, papery pericarp and fleshy seeds (Conran and Tamura, 1998). The leaf epidemal cells are ridged and sculptured with the subsidary cells surrounding the guard cells in Liliope and Ophiopogon, and flowers are perigynous in Ophiopogon and Peliosanthes (Cutler, 1992).

Polygonateae

Monophyly of Polygonateae has been supported in previous studies (Rudall et al., 2000; Yamashita et al., 2000; Tamura and Yamashita, 2004). Polygonateae share sympodial rhizomes, elongate stems and broad leaves relative to those of Ophiopogoneae. Their chromosome numbers and karyotypes are diverse: Polygonatum, x = 9–15; Heteropolygonatum, x = 16; Maianthemum (including Smilacina, x = 18); Disporopsis, x = 20. It was reported recently that variation in chromosome numbers of Polygonateae was derived from an ancestral basic one (x = 19) in Ruscaceae s.l. (Yamashita and Tamura, 2004). Polygonateae including Disporopsis was strongly supported as monophyletic in Bayesian tree (PP 0·95; Fig. 3).

Smilacina was treated within Maianthemum by LaFrankie (1985a, b; 1986), and many studies have agreed with combining these two genera (Conran and Tamura, 1998; Yamashita et al, 2000; Rudall et al., 1997; 2000; Shinwari, 2000). The two genera exhibit several distinguishing characters. For example, Smilacina has trimerous flowers, multiple (>6) leaves, and adventitious roots from both nodes and internodes of the rhizome, whereas Maianthemum has dimerous flowers, 2–5 leaves and adventitious roots only from the internodes of the rhizome. Kim and Lee (2007) also proposed to merge the two genera based on analyses of the trnK data (including matK). We also agree with the previous studies that proposed their merger (LaFrankie, 1985a; Conran and Tamura, 1998; Yamashita et al, 2000; Rudall et al., 1997, 2000; Shinwari, 2000), but more intensive studies including distributional diversity and more samples are needed to elucidate this relationship more clearly.

Convallarieae clade

Dahlgren et al. (1985) divided Convallariaceae into four tribes, Polygonateae, Ophiopogoneae, Convallarieae and Aspidistreae, but they did not suggest any obvious characteristics to delimit Convallarieae relative to Aspidistreae. After Dahlgren et al. (1985), most of studies treated Convallariaceae as composed of three tribes and merged Aspidistreae with Convallarieae (Conran and Tamura, 1998; Yamashita et al. 2000; Rudall et al, 2000), which was supported here. Theropogon differs from Convallarieae in anatomical features (Utech, 1979), basic chromosome number and floral morphology. Rudall et al. (2000) mentioned close relationships of Convallarieae, Ruscaceae s.s. and Peliosanthes, but Ruscaceae s.s. and Peliosanthes are different in their basic chromosome numbers (x = 20 and x = 18, respectively) and septal nectaries from Convallarieae. Also, Peliosanthes is included in Ophiopogoneae, which have some special fruit features and perigynous flowers. Convallarieae/Aspidistreae have several synapomorphies such as basic chromosome numbers (x = 19), monopodial rhizomes and shoots and non-septal nectaries (Dahlgren et al., 1985; Tamura, 1995). In the Convallariae/Aspidistreae clade, Campylandra, Rohdea, Tupistra, Aspidistra, Convallaria and Speirantha formed a group (BP 96/PP 0·96), but the genera are not monophyletic.

Ruscaceae s.s. + Dracaenaceae + Theropogon + Comospermum clade

The close relationships of Ruscaceae s.s., Dracaenaceae and Comospermum have been found in previous studies (Tamura, 1995; Rudall et al., 1997), which all have tenuinucellate parietal cells and the same basic chromosome number (x = 20). The basic chromosome numbers of Theropogon (x = 19) differs from Convallarieae and Polygonateae, and it has septal nectaries, otherwise found only in Convallarieae. Additional molecular and morphological studies should be pursued to resolve the phylogenetic problems and controversies concerning relationships of Theropogon.

Nolinaceae clade

It has been previously reported that Nolinaceae have a close relationship with Dracaenaceae. They were often treated in tribe Dracaeneae (Bentham and Hooker, 1883) or Nolineae (Krause, 1930). Recently several studies suggested that they are close to Dracaenaceae and Convallariaceae (Bogler and Simpson, 1995, 1996), particularly Ophiopogoneae, even though there are no obvious morphological characters to support this (Rudall et al., 2000). Nolinaceae are sister to Convallariaceae–Ruscaceae s.s.–Dracaenaceae (BP 100) in the MP tree but sister to Convallariae/Aspidistreae alone with BA (PP 0·96).

Eriospermum clade

Eriospermum, endemic to southern Africa, is strongly supported (BP 100/PP 1·00) as sister to Ruscaceae s.l. (BP 100/PP 0·99; Figs 2 and 3). In previous studies, Eriospermum with Aphyllanthes were close to Ruscaceae s.l. (Rudall et al., 1997; Fay et al., 2000) or proposed to be included in Convallariaceae (Yamashita and Tamura, 2000). However, Jang and Pfosser (2002) suggested Aphyllanthes should go with Anthericaceae and Eriospermum should be included in Ruscaceae s.l.; Eriospermum and Ruscaceae s.l. share many characters such as seeds without phytomelan, articulate peduncles and septal nectaries, but Eriospermum differs in its seed trichomes, special leaf appendages, large ovules and oily perisperm (Dahlgren et al., 1985; Lu, 1985). The phylogenetic position of Eriospermum seems secure; it shares many of the traits of Ruscaceae s.l. Little is gained by recognizing it as a family on its own.

Conclusions

This study with different taxon sampling and different species representing genera than in previous phylogenetic studies documents the stability of relationships within Asparagales. Moreover, a better-supported topology for relationships within Ruscaceae (Nolinoideae of Asparagaceae sensu APG III, 2009) than in any previous study is provided here, and it is documented that there are still subjects for more detailed future studies of genera and tribes in this clade. The higher-level relationships (interfamilial) found in this study are not totally in agreement with other broad studies (e.g. Pires et al., 2006), in particular the parsimony analysis in this study does not find support for the broader circumscription of Asparagaceae sensu APG III. Amaryllidaceae s.l. are supported, but in this study Asparagaceae s.l. are paraphyletic to Amaryllidaceae s.l. However, this set of relationships is not strongly supported. In contrast, the Bayesian analysis found that Asparagaceae s.l. were sister to Amaryllidaceae s.l. but with PP < 95. All other aspects of the higher-level relationships within Asparagales are similar to those found previously. We intend to collect more data to evaluate this disagreement in greater detail and also to investigate relationships in Ruscaceae further by increasing both taxa and numbers of loci.

ACKNOWLEDGEMENTS

This research was supported by the Mid-career Reseacrher Program through an NRF grant funded by MEST to J.-H. Kim (No. R01-2008-000-11910-0) and by the Korean Research Foundation Grant Fund (MOEHRD, Basic Research Promotion Fund; KRF-2007-C00036).

APPENDIX

Voucher data and GenBank accession numbers for Ruscaceae s.l. and related groups in the order Asparagales. Order and family circumscriptions are as in APG (1988) with slight modification (Chase et al., 2000). Names with asterisks are the family circumscriptions of Dahlgren et al. (1985).

Family/Tribe Taxon Source/voucher Origin/distribution 18S matK rbcL
Agapanthaceae Agapanthus africanus Chase 627 (K) SW Cape, S Africa HM640715 HM640599 HM640485
Agavaceae Agave ghiesbreghtii Chase 3467 (K) Mexico, N and C America HM640709 HM640592 HM640478
Yucca filamentosa DK Kim 06-077 (TUT) E USA, N America HM640713 HM640596 HM640482
Leucocrinum montanum Chase 795 (K) S USA. N America HM640712 HM640595 HM640481
Hosta plantaginea JX Feng s.n. (HZU) Hangzhou, China, E Asia HM640711 HM640594 HM640480
Alliaceae Allium victorialis var. platyphyllum DK Kim 04-142 (TUT) Korea, E Asia HM640714 HM640597 HM640483
Ipheion uniflorum Murakami 631 (KYO) N Argentina, S America – HM640598 HM640484
Amaryllidaceae Lycoris uydoensis DK Kim 05-102 (TUT) Korea, E Asia HM640716 HM640600 HM640486
Narcissus tazetta var. chinensis DK Kim 06–167 (TUT) W Mediterranean HM640717 HM640601 HM640487
Crinum asiaticum var. japonicum GH Tae s.n. (TUT) Korea, E Asia HM640718 HM640602 HM640488
Clivia nobilis Chase 3080 (K) E Cape, S Africa AF206889 HM640603 Chase et al., 2006
Anemarrhenaceae* Anemarrhena asphodeloides TCMK 312 (K) Korea, NE Asia HM640719 HM640604 HM640489
Anthericaceae* Anthericum liliago Chase 515 (K) N, C and S Europe HM640720 HM640605 HM640490
Chlorophytum minor BY Ding s.n. (KUN) Zambia, Africa HM640721 HM640606 HM640491
Chlorophytum suffruticosum Chase 1043 (K) E Africa HM640723 HM640608 HM640493
Chlorophytum orchidastrum Chase 2155 (K) W and C Africa HM640722 HM640607 HM640492
Chlorophytum tetraphyllum Chase 1044 (K) Ethiopia, N Africa HM640724 HM640609 L05031
Comospermum yedoense Chase 833 (K) Japan, E Asia HM640725 HM640610 HM640494
Echeandia sp. Chase 602 (K) S and C America HM640727 HM640612 HM640495
Paradisea liliastrum Chase 826 (K) Pyrenees, Alps, S Europe HM640728 HM640613 HM640496
Aphyllanthaceae Aphyllanthes monspeliensis Chase 614 (K) W and C Mediterranean HM640729 HM640614 Z77259
Asparagaceae Asparagus cochinchinensis DK Kim 04–122 (TUT) Korea, E and SE Asia HM640730 HM640615 HM640497
Asparagus schoberioides DK Kim 04–165 (TUT) Korea, NE Asia HM640731 HM640616 HM640498
Hemiphylacus latifolius Chase 668 (K) Mexico, N America HM640732 HM640617 HM640499
Behniaceae Behnia reticulata Goldbladtt 9273 (MO) S and E Africa HM640733 HM640618 HM640500
Convallariaceae*
  Convallarieae Convallaria majalis DK Kim 04–082 (TUT) Korea, NE Asia, Europe HM640672 HM640557 HM640443
Reineckea carnea DK Kim 05–008 (TUT) Korea, E Asia HM640673 HM640558 HM640444
Speirantha gardenii Chase 495 (K) SE China HM640674 HM640559 HM640445
Theropogon pallidus Chase 2933 (K) SW China, Himalaya HM640675 HM640560 HM640446
  Aspidistreae Aspidistra elatior DK Kim 05-013 (TUT) Korea, E Asia HM640676 HM640561 HM640447
Campylandra fimbriata Liu Yang 484 (KUN) Himalaya, NW China HM640677 HM640562 HM640448
Rohdea japonica DK Kim 05-005 (TUT) Korea, E Asia HM640678 HM640563 HM640449
Tupistra aurantiaca Chase 1100 (K) Yunnan, SW China, E Asia HM640679 HM640564 HM640450
  Polygonateae Disporopsis pernyi Chase 493 (K) S China, E Asia HM640681 HM640566 HM640452
Disporopsis sp. DK Kim 05-136 (TUT) Sichuan, China, E Asia HM640680 HM640565 HM640451
Maianthemum bifolium DK Kim 04-182 (TUT) Korea, temperate Eurasia HM640682 HM640567 HM640453
Maianthemum dilatatum DK Kim 04-165 (TUT) Korea, NE Asia HM640683 HM640568 HM640454
Polygonatum humile DK Kim 04-029 (TUT) Korea, C and E Asia HM640684 HM640569 HM640455
Polygonatum inflatum DK Kim 04-043 (TUT) Korea, NE Asia HM640685 HM640570 HM640456
Polygonatum involucratum DK Kim 04-059 (TUT) Korea, NE Asia HM640686 HM640571 HM640457
Polygonatum lasianthum var. coreanum DK Kim 04-046 (TUT) Korea, NE Asia HM640687 HM640572 HM640458
Polygonatum odoratum var. pluriflorum DK Kim 04-067 (TUT) Korea, NE Asia HM640688 HM640573 HM640459
Smilacina bicolor DK Kim 04-077 (TUT) Korea, E Asia HM640689 HM640574 HM640460
Smilacina dahurica DK Kim 04-082 (TUT) Korea, NE Asia HM640690 HM640575 HM640461
Smilacina japonica DK Kim 04-039 (TUT) Korea, NE Asia HM640691 HM640576 HM640462
  Ophiopogoneae Liriope platyphylla DK Kim 07-001 (TUT) Korea, E Asia HM640692 HM640577 HM640463
Liriope spicata DK Kim 07-002 (TUT) Japan, E Asia HM640693 HM640578 HM640464
Ophiopogon jaburan DK Kim 07-004 (TUT) Korea, E Asia HM640694 HM640579 HM640465
Ophiopogon japonicus DK Kim 07-003 (TUT) Korea, E Asia HM640695 HM640580 HM640466
Peliosanthes macrostegia G Murata 44832 (KYO) S China, E Asia HM640696 HM640581 HM640467
Dracaenaceae* Dracaena schizantha Chase 21514 (K) Ethiopia, NE Africa HM640698 HM640582 HM640469
Dracaena aubryana Chase 1102 (K) Uganda, WC Africa HM640699 HM640583 HM640470
Sansevieria trifasciata DK Kim 07-05 (TUT) Nigeria, WC Africa HM640700 HM640584 HM640471
Eriospermaceae* Eriospermum abyssinicum Chase 2051 (K) S Africa HM640706 HM640589 HM640475
Eriospermum natalense Chase 2052 (K) S Africa HM640707 HM640590 HM640476
Eriospermum parvifolium Chase 2053 (K) W Cape, S Africa HM640708 HM640591 HM640477
Herreriaceae* Herreria salsaparilha Chase 2154 (K) Brazil, S America HM640734 HM640619 HM640501
Hesperocallidaceae Hesperocallis undulata Cranfill & Schmid s.n. (JEPS) SW USA, N America HM640735 HM640620 HM640502
Hyacinthaceae* Bowiea volubilis Chase 176 (K) Uganda, C and S Africa HM640736 HM640621 HM640503
Camassia cusickii Cronquist 6549 (RSA) C USA, N America HM640710 HM640593 HM640479
Dipcardi filifolium Chase 1783 (K) C Asia, Africa, India HM640737 HM640622 HM640504
Drimia altissima Chase 1870 (K) C and S Africa HM640738 HM640623 HM640505
Eucomis humilis Chase 1847 (K) Lesotho, S Africa HM640739 HM640624 HM640506
Lachenalia carnosa Chase 2261 (K) W Cape, S Africa HM640740 HM640625 HM640507
Ledebouria cooperi Chase 1786 (K) S Africa HM640741 HM640626 HM640508
Massonia angustifolia Chase 5666 (K) Cape, S Africa HM640742 HM640627 HM640509
Muscari aucheri Chase 21845 (K) Turkey, Med. to Caucasus HM640743 HM640628 HM640510
Ornithogalum armeniacum Chase 1682 (K) Turkey to Macedonia HM640744 HM640629 HM640511
Ornithogalum shawii Chase 1012 (K) S Africa HM640745 HM640630 HM640512
Rhadamanthus convallarioides Goldblatt 10852 (A) Cape, S Africa HM640746 HM640631 HM640513
Scilla scilloides DK Kim 05-039 (TUT) Korea, E Asia HM640747 HM640632 HM640514
Urginea epigea Chase 2055 (K) S Africa HM640748 HM640633 HM640515
Laxmanniaceae Arthropodium cirrhatum Chase 651 (NCU) New Zealand, Australia HM640749 HM640634 HM640516
Laxmannia squarrosa Chase 2214 (K) W and S Australia HM640751 HM640636 HM640518
Lomandra hastilis Brummitt et al. 21239 (K) W and SW Australia HM640750 HM640635 HM640517
Nolinaceae* Calibanus hookeri Chase 1006 (K) Mexico, N America HM640702 HM640585 HM640472
Dasylirion serratifolium Abisai et al., s.n. (RSA) Mexico, N America HM640704 HM640587 AB029847
Dasylirion wheeleri Chase 3469 (K) Texas, S USA, N America HM640705 HM640588 HM640474
Nolina recurvata Chase 3466 (K) Mexico, N America HM640703 HM640586 HM640473
Ruscaceae* Danae racemosa Chase 121 (K) Turkey, Syria, Iran, Caucasus HM640668 HM640553 HM640439
Ruscus aculeatus Bohuslavek 1348 (RSA) W and C Europe, Medit. HM640669 HM640554 HM640440
Ruscus streptophyllus Chase 21990 (K) Madeira HM640670 HM640555 HM640441
Semele androgyna Chase 997 (K) Canary Is., Madeira HM640671 HM640556 HM640442
Themidaceae Bessera elegans Chase 626 (K) Mexico, N America HM640752 HM640637 HM640519
Bloomeria aurea Chase 1010 (K) SW USA, N America HM640753 HM640638 HM640520
Dandya thadhowardii Chase s.n. (K) Mexico, N America HM640754 HM640639 HM640521
Dichelostemma multiflorum Chase 1830 (K) SW USA, N America HM640755 HM640640 HM640522
Muilla maritime Chase 779 (K) SW USA to Mexico, N America HM640757 HM640642 HM640524
Triteleia peduncularis Chase 1860 (K) California, W USA, N America HM640758 HM640643 HM640525
Asphodelaceae Eremurus chinensis Qing 00317 (KUN) Tibet to S Gansu, W China HM640759 HM640644 HM640526
Asphodelus aestivus Chase 482 (K) Portugal, Spain, SW Europe HM640760 HM640645 HM640527
Bulbine semibarbata K Dixon, s.n. (KPBG) S and E Australia HM640761 HM640646 HM640528
Bulbine succulenta Chase 5518 (K) Cape, S Africa AF206876 Z73684
Bulbine frutescens Chase 9215 (K) S Africa AJ511414
Asteliaceae Astelia alpina Chase 1103 (K) NSW to Tasmania, S Australia HM640762 HM640648 HM640530
Milligania stylosa Chase 511 (K) Tasmania, S Australia HM640763 HM640649 HM640531
Brandfordiaceae Brandfordia punicea MRK Rambert 787 (K) Tasmania, S Australia HM640764 HM640650 HM640532
Boryaceae Borya septentrionalis Chase 2205 (K) Perth, W Australia HM640765 HM640651 HM640533
Doryanthaceae Doryanthes excelsa Chase 188 (K) NSW, SE Australia HM640766 HM640652 HM640534
Doryanthes palmeri Chase 19153 (K) Queensland, SE Australia HM640767 HM640653 HM640535
Hemerocallidaceae Dianella ensifolia Nakai 5510 (KYO) Taiwan, SE and Tropical Asia HM640768 HM640654 HM640536
Hemerocallis minor DK Kim 05-091 (TUT) Korea, NE Asia HM640769 HM640655 HM640537
Hemerocallis littorea Chase 3833 (K) Korea, Japan, E Asia Chase et al., 2006 AJ581422 AY149364
Hypoxidaceae Curculigo capitulata SW Lee 05-001 (TUT) Yunnan, S Asia to N Australia HM640770 HM640656 HM640538
Rhodohypoxis milloides Chase 479 (K) E Cape, S Africa AF207008 AY368377 Z77280
Rhodohypoxis baurii Chase 16460 (K) Cape, S Africa HM640772 HM640658 HM640540
Hypoxis leptocarpa Chase 108 (NCU) Duke, SE USA, N America AF135209 AY368375 Z73702
Hypoxis hemerocallidea Chase 1045 (K) Tropical and S Africa HM640771 HM640657 HM640539
Iridaceae Iris rossii DK Kim 05-048 (TUT) Korea, NE Asia HM640773 HM640659 HM640541
Gladiolus illyricus Chase 9907 (K) Portugal, SW Europe HM640774 HM640542
Gladiolus papilio Goldblatt & Manning 9841 (MO) S Africa AJ579956
Ixioliriaceae Ixiolirion tataricum Chase 489B (K) E Turkey to Kashmir, W Asia HM640775 HM640660 HM640543
Lanariaceae Lanaria lanata Goldblatt 9410 (MO) Cape, S Africa Chase et al., 2006 AY368376 Z77313
Orchidaceae Calanthe discolor DK Kim 05-035 (TUT) Korea, E Asia HM640776 HM640665 HM640548
Cephalanthera longibracteata DK Kim 05-016 (TUT) Korea, NE Asia HM640777 HM640666 HM640549
Cypripedium calceolus Chase 9484 (K) Estonia, Europe to Asia HM640778 HM640667 HM640550
Oncidium ensatum Chase 9671 (K) Tropical C and S America HM640779 AY368423 HM640551
Apostasia wallichii Chase 15744 (K) Sri Lanka, S Asia to N Australia HM640780 AY557212 HM640552
Tecophilaceae Tecophilaea cyanocrocus Chase 447 (K) Chile, S America HM640781 HM640661 HM640544
Cyanella orchidiformis Chase 5896 (K) Cape, S Africa HM640782 HM640662 HM640545
Xanthorrhoeaceae Xanthorrhoea resinosa Chase 192 (NCU) NSW, Australia HM640783 HM640663 HM640546
Xanthorrhoea quadrangulata Hahn 6978 (WIS) S Australia U42064
Qiu 97039 (NID) DQ401345
Unvouchered Z73710
Xeronemataceae Xeronema callistemon Chase 653 (K) Poor Night Is., New Zealand HM640784 HM640664 HM640547
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