Everything About Wood

The primarily tropical Zingiberales are phylogenetically embedded within the derived eumonocots (Arecales, Commelinales, Poales) and include many conspicuous taxa, such as the bananas (Musaceae), birds of paradise (Strelitziaceae), heliconias (Heliconiaceae), and gingers (Zingiberaceae). With 53 genera and over 1200 species, the Zingiberaceae is the largest of the eight families of the order (Kress, 1990⇓). In earlier classifications (e.g., Petersen, 1889⇓; Schumann, 1904⇓) the family Costaceae (Fig. 1) was included in the Zingiberaceae, but with a number of distinctive characters (e.g., lack of aromatic oils, branched aerial stems, and spiral monostichous phyllotaxy; Specht et al., 2001)⇓ it is now accepted as the sister clade to the gingers (Kress, 1990⇓, 1995⇓; Kress et al., 2001⇓). The Zingiberaceae is distributed pantropically with one genus (Renealmia) found in the Neotropics, four genera (Aframomum, Aulotandra, Siphonochilus, and Renealmia) found in Africa, and the rest of the genera distributed in east Asia and the Pacific Islands. The family is still poorly known taxonomically with many species (Theilade and Mood, 1997⇓; Sakai and Nagamasu, 1998⇓; Poulsen, Mood, and Ibrahim, 1999⇓; Williams, Kress, and Thet Tun, 2002⇓) and even genera (Newman, 1995⇓; Mood and Larsen, 1997⇓; Larsen and Mood, 1998⇓; Sakai and Nagamasu, 2000⇓; Kress and Larsen, 2001⇓; Larsen and Jenjittikul, 2001⇓) newly described in the last several years.

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Figs. 1–6. Representatives of the major groups of the family Zingiberaceae and the sister family Costaceae. 1. Costaceae (Monocostus). 2. Hedychieae (Siphonochilus). 3. Hedychieae (Hedychium). 4. Alpinieae (Alpinia). 5. Zingibereae (Zingiber). 6.Globbeae (Mantisia)

The currently accepted classification of the Zingiberaceae (Petersen, 1889⇓; Schumann, 1904⇓; Holttum, 1950⇓; Burtt and Smith, 1972⇓; Larsen et al., 1998⇓) includes four tribes (Hedychieae: 22 genera, Figs. 2–3; Alpinieae: 25 genera, Fig. 4; Zingibereae: one genus, Fig. 5; and Globbeae: four genera, Fig. 6) and is based on both vegetative and floral characteristics (Table 1). Although a number of morphological features have been used to distinguish the four tribes, in most cases the defining characters are either not unique to any one tribe or are not universal for all taxa within any tribe (Table 1). For example, the plane of distichy of the leaves is perpendicular to the rhizome in the Alpinieae (except in the genera Rhynchanthus and Pommereschea) and is parallel to the rhizome in the other three tribes (except for Siliquamomum in the Hedychieae). Similarly, the ovary is unilocular in all members of the Globbeae, but trilocular in the other three tribes with some exceptions, e.g., unilocular in Tamijia and partially unilocular in Caulokaempferia, Paracautleya, Scaphochlamys, Siphonochilus, and Riedelia (Larsen et al., 1998⇓; Sakai and Nagamasu, 2000⇓). The lateral staminodes are well developed in Hedychieae, Globbeae, and Zingibereae (Figs. 2–3, 5–7) and generally absent in the Alpinieae (Fig. 4; but all five staminodes are absent in Rhynchanthus). The fusion of the lateral staminodes to the central labellum (Fig. 7) in Siphonochilus (Hedychieae; Fig. 2) and Zingiber (Zingibereae; Fig. 5) has now been reported in the narrow endemic Tamijia(Alpinieae) from Borneo (Sakai and Nagamasu, 2000)⇓. Most of the characters that are used to define the tribes are often inconsistent and variable.

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Table 1. Characteristics and genera of the previously recognized tribes of the Zingiberaceae (after Schumann, 1904; Holttum, 1950; Burtt and Smith, 1972; Larsen et al., 1998). Authorities follow Reveal (2002)

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Fig. 7. Floral diagram of the Zingiberaceae with perianth whorls, fertile stamen, lateral staminodes, and labellum indicated. (Not drawn to scale; based on Kress, 1990⇓)

The circumscription of various genera of the Zingiberaceae has also been debated by investigators (e.g., Etlingera: Burtt and Smith, 1986⇓; Curcumorpha: Larsen, 1997⇓; Paramomum: Wu, 1997⇓), and a consensus has not been reached on the actual number of genera in the family. Nearly one-quarter of the genera in the Zingiberaceae are monospecific, but no criteria for the recognition of these species at the generic level have been established. One important question in the classification of the family is how to determine the taxonomic status of these often morphologically very distinctive monospecific genera.

Recently several papers have used molecular data to explore the phylogenetic relationships within the family Zingiberaceae (Searle and Hedderson, 2000⇓; Wood, Whitten, and Williams, 2000)⇓ as well as within several genera (Hedychium: Wood, Whitten, and Williams, 2000⇓; Alpinia: Rangsiruji, Newman, and Cronk, 2000a⇓, b⇓; Roscoea: Ngamriabsakul, Newman, and Cronk, 2000⇓; Aframomum: Harris et al., 2000)⇓. These analyses have succeeded in clarifying the patterns of evolutionary relationships to varying degrees, but in general have been limited in breadth of taxon sampling as well as resolution.

Our goal in the present study is to use molecular sequence data to generate hypotheses on the phylogenetic relationships among the genera of the Zingiberaceae in order to evaluate past classifications of the family, to identify morphological features that characterize the various clades detected by the molecular analyses, to evaluate the status of monotypic genera where possible, and to construct a new phylogenetic classification of the family.

MATERIALS AND METHODS

Taxa

We sampled 104 species in 41 genera representing all four tribes of the Zingiberaceae (http://ajbsupp.botany.org/v89/). In the larger genera (e.g., Alpinia, Curcuma, Hedychium, Globba, Zingiber) several species representing infrageneric morphological diversity were included. Unfortunately, for several important genera (e.g., Elettaria, Geocharis, Geostachys) we either did not have adequate tissue samples or we were not able to amplify DNA. We also lacked adequate samples of a number of rare monospecific genera (e.g., Leptosolena, Parakaempferia, Paracautleya, Nanochilus, and Cyphostigma).

Outgroup

We initially used representative taxa of the seven other families of the Zingiberales as outgroups in our molecular analyses (http://ajbsupp.botany.org/v89/). However, because of the difficulty of aligning the sequences of the internal transcribed spacer (ITS) loci and matK-trnK flanking intergenic spacer regions in these other families with our ingroup taxa, we first conducted more focused analyses of the coding region of matK, which is easier to align among all of the families. Our results provide strong support for the placement of the African genus Siphonochilus as the basal lineage to the remaining Zingiberaceae. In addition, all of the other members of the Zingiberaceae we sequenced except Siphonochilus share a 12-base deletion at the 3′-end of matK; this deletion is absent in all other Zingiberales. We interpret this deletion as a synapomorphy of the Zingiberaceae excluding Siphonochilus, which retains this 12-base sequence as a symplesiomorphy with the rest of the Zingiberales. We therefore used Siphonochilus as the outgroup for the rest of the Zingiberaceae in the subsequent analyses of the complete ITS and matK regions in order to facilitate sequence alignment within the family and provide maximum resolution among genera.

Molecular methods

Total genomic DNAs were extracted using a minor modification of the Doyle and Doyle (1987)⇓ hexadecyltrimethylammonium bromide (CTAB) method. The aqueous phase was extracted with 24 parts chloroform : 1 part isoamyl alcohol. DNA was resuspended in tris and ethylenediaminetetraacetic acid (TE) buffer following isopropyl alcohol precipitation. Amplification of ITS was accomplished using ITS4 and ITS5 primers (White et al., 1990⇓). The chloroplast matK region was amplified with trnK1F (Manos and Steele, 1997⇓) and trnK2R (Steele and Vilgalys, 1994⇓). All amplifications used Gibco BRL Taq DNA polymerase (Carlsbad, California, USA) according to the manufacturer's directions with annealing temperatures of 54–58°C. Amplified products were purified using the polyethylene glycol 8000 (PEG) precipitation protocol (Johnson and Soltis, 1995⇓) with the products sequenced directly using automated sequencing methodology of the ABI Prism™ Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Foster City, California, USA). Sequencing primers included the amplification primers plus ITS2 (White et al., 1990⇓) and ITS3G (5′-GCA TCG ATG AAG AAC GTA GT-3′; K. J. Williams) for the ITS region. Zingiberales-specific internal sequencing primers were designed by L. M. Prince for the matK region including mSP2F (5′-TGG GTT AGA GAC GAA TGT GT-3′), mIF (5′-GTT CAG TAC TTG TGA AAC GTT-3′), m5Fa (5′-CTC TAT GGG TCT TCA AGG AT-3′), m8Fa (5′-TAC TTC GAC TTT CCT GTG CC-3′), mSP2R (5′-TTT AAC GTC TAA TTA GAT CGG-3′), mIR (5′-CGT TTC ACA AGT ACT GAA CTA-3′), m5R (5′-AGG ATC CTT GAA AAT CCA TAG A-3′), and m8R (5′-AGC ACA AGA AAG TCG AAG-3′). Primers m5Fa, m8Fa, and m8R are modifications or complements of the primers of Steele and Vilgalys (1994)⇓. Products were cleaned in Sephadex G-50 (fine) Centri-Sep spin columns (Princeton Separations P/N 901, Adelphia, New Jersey, USA), dried under vacuum, and run on an ABI 377 Automated Sequencer (Applied Biosystems, Foster City, California, USA) at the Smithsonian Institution's Laboratory for Molecular Systematics. Raw sequences were assembled and edited using Sequencher 3.1.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and manually aligned in Se-Al 2.0a3 (Rambaut, 2000)⇓. Results of exploratory analyses (not shown) that excluded difficult-to-align regions (104 base pairs [bp] in ITS and 254 bp in matK) produced topologies very similar to those including the entire alignment. For this reason, we are presenting here only results from analyses of the full data set.

Phylogenetic analyses

Maximum parsimony analyses of the ITS and matK sequence data were conducted using PAUP*4.0 (Swofford, 1998⇓) with unweighted characters and 500 random-sequence-addition replicates, saving all shortest trees under tree bisection-reconnection (TBR) branch swapping, STEEPEST DESCENT off, MULTREES on, COLLAPSE branches if maximum length is zero. Multiple random-sequence additions were chosen to minimize the likelihood of being trapped on any particular tree island (Maddison, 1991⇓). Bootstrap analyses (Felsenstein, 1985⇓; Mort et al., 2000⇓) were conducted using PAUP*4.0 with ten random addition replicates, TBR branch swapping, for 100 bootstrap replicates. Bootstrap support was categorized as strong (>85%), moderate (70–85%), weak (50–70%), or poor (<50%). The data sets for each gene region were analyzed separately and then, following the total evidence approach for multiple data sets (de Queiroz, Donoghue, and Kim, 1995⇓; Nixon and Carpenter, 1996⇓), we combined the sequence data. For the combined analysis, all 104 species were included, although six species (Caulokaempferia coenobialis, Curcuma aeruginosa, Distichochlamys citrea, Kaempferia parviflora, K. pulchra, and Zingiber corallinum; http://ajbsupp.botany.org/v89/) lacked sequence data for either ITS or matK.

RESULTS

Internal transcribed spacer

The ITS-1 had a total aligned length of 277 bp (unaligned sequences ranged from 206 to 247 bp) with a mean guanine-cytosine (GC) content of 54.06%; the 5.8S region had an aligned length of 165 bp (range of 163–165 bp) and a GC content of 51.18%; and the ITS-2 aligned length was 372 bp (range of 242–292 bp) with a GC content of 59.53%.

The analysis of the ITS sequence data resulted in 48 equally parsimonious nearly fully resolved trees of 1603 steps (number of parsimony-informative characters = 323; consistency index [CI] = 0.366; retention index [RI] = 0.724; rescaled consistency index [RC] = 0.265; Fig. 8). Tamijia is strongly supported (bootstrap value = 90%) as a stem lineage one node above Siphonochilus. The remaining taxa comprise two major monophyletic lineages: one includes most of the Alpinieae (bootstrap value = 86%) and the other includes the Hedychieae plus the Globbeae and Zingibereae, although bootstrap support for the latter clade is weak (bootstrap value = 52%).

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Fig. 8. One of the 48 equally parsimonious trees of the Zingiberaceae in the analysis of the ITS sequence data (length = 1603; consistency index = 0.366 excluding uninformative characters; retention index = 0.724; and rescaled consistency index = 0.265) showing branch lengths (above the line) and bootstrap values (below the line if ≥50%; values for branches within genera are excluded). Asterisks indicate nodes that collapse in the strict consensus tree. Polyphyletic species groups in the genera Alpinia(Alp), Amomum (Amo), Etlingera (Etl), and Curcuma (Cur) are indicated

Within the Alpinieae clade, Pleuranthodium + Riedelia + Burbidgea + Siamanthus are strongly supported as a monophyletic group (bootstrap value = 98%). Among the larger genera, Aframomum and Renealmia are strongly supported as monophyletic. However, several genera are not monophyletic: Alpinia, with four nonmonophyletic groups of species referred to as Alpinia I (A. conchigera and A. galanga), Alpinia II (A. elegans, A. luteocarpa, and A. vittata), Alpinia III (A. blepharocalyx, A. intermedia, A. pumila, A. calcarata, A. officinarum, and A. foxworthyi), and Alpinia IV (A. carolinensis); Amomum, with two groups of species referred to as Amomum I (A. glabrum and A. longipetiolatum) and Amomum II (A. villosum and A. sp.); and Etlingera, with two groups of species referred to as Etlingera I (E. elatior and E. yunnanensis) and Etlingera II (E. littoralis).

Within the Hedychieae clade the Globbeae are monophyletic and sister to the remaining taxa, but with poor bootstrap support (<50%). Globba + Mantisia and Hemiorchis + Gagnepainia are strongly supported as sister genera (bootstrap value = 100%). The Zingibereae (the genus Zingiber) are placed as a crown clade within the Hedychieae. Although the topology within this clade is fully resolved there is only poor support for all nodes connecting more than four genera. Most branches on the tree are short. A clade including Stahlianthus, Hitchenia, Smithatris, and Curcuma is strongly supported (bootstrap value = 98%), and the latter genus is paraphyletic with three groups of species referred to as Curcuma I (C. comosa, C. aeruginosa, and C. roscoeana), Curcuma II (C. thorelii), and Curcuma III (C. bicolor and C. sp.). Boesenbergia is also paraphyletic with two groups of species referred to as Boesenbergia I (B. pulcherrima) and Boesenbergia II (B. rotunda). All of the remaining genera in the Hedychieae are strongly supported as monophyletic. Pommerescheaand Rhynchanthus of the Alpinieae are embedded within the Hedychieae clade and Siliquamomum of the traditional Hedychieae is placed with other Alpinieae.

matK

The 5′ trnK-matK intergenic spacer region had a total aligned length of 1101 bp (unaligned sequences ranged from 767 to 884 bp) with a mean GC content of 30.01%; the matK coding region had an aligned length of 1641 bp (range of 1542–1572 bp) and a GC content of 29.69%; and the matK-3′ trnK intergenic spacer region aligned length was 507 bp (range of 279–316 bp) with a GC content of 29.98%.

The analysis of the matK region (coding and noncoding) resulted in more than 40 000 equally parsimonious trees (at which point the analysis was stopped) of 1104 steps (number of parsimony-informative characters = 482; CI = 0.630; RI = 0.893; RC = 0.562; Fig. 9). A strict consensus of these shortest trees (Fig. 9) provides strong support (bootstrap values >99%) for the same two major clades as defined by the ITS data (the traditional Alpinieae and the traditional Hedychieae plus Globbeae and Zingibereae), but does not resolve the position of the genus Tamijia with respect to these two clades. There is little resolution among genera in the latter clade. Globba + Mantisia and Hemiorchis + Gagnepainia are strongly supported as sister genera (bootstrap values = 100%), but there is only poor support for uniting these four genera of the traditional Globbeae into a single clade. The paraphyletic nature of Globba with Mantisia is strongly supported. As in the ITS tree, a “Curcuma clade,” including Hitchenia, Stahlianthus, Smithatris, and a paraphyletic Curcuma, is moderately supported (bootstrap value = 74%). The matK data weakly support (bootstrap value = 54%) a “Kaempferia clade” consisting of Zingiber, Boesenbergia, Curcumorpha, Kaempferia, Scaphochlamys, Distichochlamys, Cornukaempferia, and Haniffia.

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Fig. 9. One of over 40 000 equally parsimonious trees of the Zingiberaceae in the analysis of the matK region (coding and noncoding) sequence data (length = 1104; consistency index = 0.630 excluding uninformative characters; retention index = 0.893; and rescaled consistency index = 0.562) showing branch lengths (above the line) and bootstrap values (below the line if ≥50%; values for branches within genera are excluded). Asterisks indicate nodes that collapse in the strict consensus tree. Polyphyletic species groups in the genera Alpinia (Alp), Amomum (Amo), Etlingera (Etl), and Curcuma (Cur) are indicated

Within the Alpinieae, four major clades are resolved. Siamanthus and Siliquamomumare weakly supported as sister genera (bootstrap value = 53%), but unresolved with the other Alpinieae. Pleuranthodium + Riedelia + Burbidgea are strongly supported as a clade (bootstrap value = 96%; hereafter referred to as the “Riedelia clade”), but likewise unresolved in the Alpinieae. Among the remaining Alpinieae, two primary and almost fully resolved clades are strongly supported (bootstrap value ≥87%). Renealmia + Aframomum + Elettariopsis + Paramomum + Amomum I + Alpinia I form one monophyletic group (hereafter referred to as the “Renealmia clade”) and Alpinia II + Vanoverberghia + Etlingera I + Hornstedtia + Etlingera II + Amomum II + Alpinia III + Plagiostachys form the second (hereafter referred to as the “Etlingera clade”). As in the ITS results, Pommereschea and Rhynchanthus of the Alpinieae are embedded within the Hedychieae, and Siliquamomum of the Hedychieae is placed within the Alpinieae.

Combined data set

The analysis of the combined ITS and matK sequence data resulted in 980 equally parsimonious trees of 2647 steps (number of parsimony-informative characters = 806; CI = 0.450; RI = 0.805; RC = 0.362; Figs. 10–11). A strict consensus of these 980 shortest trees (Figs. 10–11) provides strong support (bootstrap values >99%) for the two major clades identified by the ITS and matK data analyzed separately: (1) the Hedychieae, Globbeae, and Zingibereae, and (2) the traditional Alpinieae. Pommereschea and Rhynchanthus are embedded within the former, and Siliquamomum is placed in the latter. Tamijia is strongly supported (bootstrap value = 100%) as sister to these two major clades. There is only weak resolution and support among genera in the Hedychieae clade, except for Globba + Mantisia, Hemiorchis + Gagnepainia, Cautleya + Roscoea, and Distichochlamys + Scaphochlamys (bootstrap values = 95–100%). The Curcuma clade, including Hitchenia, Stahlianthus, Smithatris, and a paraphyletic Curcuma, is strongly supported (bootstrap value = 100%), whereas the Kaempferia clade is weakly supported (bootstrap value = 54%).

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Fig. 10. One of 980 equally parsimonious trees of the Zingiberaceae in the analysis of the combined ITS and matK region sequence data (length = 2647; consistency index = 0.450 excluding uninformative characters; retention index = 0.805; and rescaled consistency index = 0.362) showing branch lengths (above the line) and bootstrap values (below the line if ≥50%; values for branches within genera are excluded). Asterisks indicate nodes that collapse in the strict consensus tree. Polyphyletic species groups in the genera Alpinia (Alp), Amomum (Amo), Etlingera (Etl), and Curcuma (Cur) are indicated

Excluding Tamijia, three of the four major clades in Alpinieae are resolved as in the matK analysis. Siliquamomum is unresolved and Siamanthus is strongly supported (bootstrap value = 88%) as part of the Riedelia clade (bootstrap value = 100%). The remaining Alpinieae are moderately supported as a monophyletic group (bootstrap value = 85%) with strong support for the Renealmia clade and Etlingera clade (bootstrap values = 86 and 91%, respectively) within it.

DISCUSSION

Molecular evidence and the current classification

The two molecular data sets used in our analysis of the evolutionary relationships of the genera in the Zingiberaceae resulted in generally congruent and compatible trees. The ITS sequence data produced an almost fully resolved tree, but in general lacked even moderate bootstrap support for many of the main clades, especially among the Hedychieae-Globbeae-Zingibereae taxa. The phylogenetic analysis of the matKcoding and noncoding regions resulted in stronger bootstrap support for relationships among the genera of the Alpinieae, but support within the Hedychieae was weak. The combined data sets produced a more fully resolved tree with strong support for many, but not all, groups of genera.

The taxonomic concepts of earlier workers (e.g., Schumann, 1904⇓; Holttum, 1950⇓; Burtt and Smith, 1972⇓; Larsen et al., 1998⇓) on the classification of the Zingiberaceae are partially congruent with the results of our phylogenetic analyses of molecular sequence data. Members of the two largest tribes, Alpinieae and Hedychieae, for the most part form well-supported monophyletic groups with the major rearrangements being the basal position of Siphonochilus and Tamijia in the family, the inclusion of Pommereschea and Rhynchanthus in the Hedychieae clade, and the position of Siliquamomum in the Alpinieae clade. We have confirmed that the former Globbeae are closely allied with the Hedychieae, but we have not resolved whether the four genera of this tribe constitute a monophyletic or paraphyletic group. It is clear that the genus Zingiber is a derived taxon within the Hedychieae and should not be recognized at the tribal level.

Our results provide some insight into the long-standing taxonomic problems that have been encountered in some of the largest genera of the family, e.g., Alpinia, Amomum, Etlingera, Curcuma, and Globba. Even though we only included a modest sampling of each genus in our analysis, the polyphyletic status of each of these five genera is clear. Other large genera such as Hedychium, Kaempferia, Zingiber, Aframomum, and Renealmia are strongly supported as monophyletic.

The present study is the most exhaustive investigation to date of the evolutionary relationships within the family Zingiberaceae. Searle and Hedderson (2000)⇓analyzed the relationships among 12 genera (including Siphonochilus) in the traditional Hedychieae plus Zingiber and five genera of the Alpinieae using ITS sequence data; Globba was used to root the tree. Their results were congruent with our analyses in uniting 11 of the 12 genera of the Hedychieae in a single clade and recognizing a “Kaempferia group” consisting of Kaempferia, Boesenbergia, Zingiber, Cornukaempferia, Scaphochlamys, and Distichochlamys. However, only weak jackknife support was present for any branches connecting more than two genera. They also noted that Siphonochilus occupied an isolated position exterior to the Hedychieae and sister to the traditional Alpinieae. This position sister to the Alpineae was clearly an anomaly resulting from selection of Globba as the outgroup in their study. Wood, Whitten, and Williams (2000)⇓ focused on the genus Hedychium in their analysis and included 17 other genera primarily in the traditional Hedychieae. Their results were similar to Searle and Hedderson's in defining a Kaempferia group (without Scaphochlamys, but with Zingiber) and placing Siphonochilus near Alpinia. As resolved by our data, the analysis by Wood, Whitten, and Williams provided support for a Curcuma clade (including the genera Curcuma, Hitchenia, and Stahlianthus) and the position of Pommereschea and Rhynchanthus in the Hedychieae.

The investigations by Rangsiruji, Newman, and Cronk (2000a⇓, b⇓) and Harris et al. (2000)⇓ concentrated on the traditional Alpinieae, especially infrageneric relationships within Alpinia and Aframomum, respectively, using ITS sequence data and included several additional genera as outgroups. Our analyses confirmed the close relationship of Pleuranthodium and Burbidgea, the polyphyly of Amomum, and the monophyly of Aframomum as shown in their studies. Rangsiruji, Newman, and Cronk suggested that Alpinia may be paraphyletic with Renealmia, but because of their limited outgroup selection they did not have sufficient data to support any major taxonomic changes in these taxa (see discussion below under Alpinioideae).

A new classification of the Zingiberaceae

Although some congruence exists between the former classifications of the Zingiberaceae and our current phylogenetic results (Fig. 11), we believe that a revised classification is needed that better represents the new data on evolutionary relationships. We accept that our results do not fully resolve all of the relationships among genera, that some major clades have only weak bootstrap support, that some ambiguity still exists in the placement of certain genera, and that several critical large genera have now been shown to be polyphyletic. Nonetheless, based on the results of our analyses that do have strong support, we propose a realignment of the genera of the Zingiberaceae into four subfamilies (Tables 2–4): the Siphonochiloideae (the genus Siphonochilus only), the Tamijioideae (the single genus Tamijia), the Alpinioideae (most of the former Alpinieae), and the Zingiberoideae (including the former tribes Hedychieae, Zingibereae, and Globbeae).

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Table 2. A key to the subfamilies and tribes of a new Linnaean classification of the Zingiberaceae (see Figs. 10 and 11 and Table 4). Authorities follow Reveal (2002)

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Fig. 11. One of 980 equally parsimonious trees of the combined analysis of the Zingiberaceae in which monophyletic genera have been collapsed into a single branch for clarity. Branch lengths (above the line) and bootstrap values (below the line if ≥50%) are shown for clades encompassing two or more genera. Asterisks indicate nodes that collapse in the strict consensus tree. Subfamilies and tribes of the new classification of the family are indicated (see Fig. 10 and Tables 2–4)

Within the Alpinioideae, we recognize the two main clades as tribes Alpinieae and Riedelieae; the placement of Siliquamomum is equivocal and requires further study (Fig. 11). The polyphyletic nature of three of the major genera in the Alpinieae (Alpinia, Amomum, and Etlingera) prevents us from formally recognizing the Renealmia clade and the Etlingera clade at this time, even though each clade has strong bootstrap support. Within the Zingiberoideae we recognize two tribes to distinguish most of the old Hedychieae (here called the Zingibereae) from the Globbeae (Fig. 11). Although strong molecular support is lacking for either tribe within the Zingiberoideae, we believe that it is better to recognize the Zingibereae and to retain the Globbeae until more evidence is accumulated. We also refrain from formally recognizing either the Kaempferia clade or the Curcuma clade at this time. We have not been able to place the genus Caulokaempferia into either tribe with any degree of certainty. A forthcoming investigation of the Globbeae and related taxa will address some of these issues in more depth (K. J. Williams, W. J. Kress, and P. S. Manos, unpublished data). The relationships among the genera are discussed below within the framework of this revised classification.

Basal lineages

The strong support from the coding region of matK for the position of Siphonochilus as the basal lineage in the family was at first surprising. The subsequent discovery of a 12-base insertion at the 3′ end of matK that this genus shares with all other Zingiberales, but not the rest of the Zingiberaceae, provided unequivocal support for this basal placement. Our morphological interpretation of this topology is that the partial to almost complete fusion of the lateral staminodes to the large labellum in Siphonochilus is a plesiomorphic character of the basal Zingiberaceae shared with the sister family Costaceae (Figs. 1–2, 7). The recent discovery of the monotypic genus Tamijia, a narrow endemic from northern Borneo (Sakai and Nagamasu, 2000)⇓, is equally interesting. Although the inflorescence structure and vegetative morphology are unlike Siphonochilus, Tamijia shares the same fusion of the lateral staminodes and labellum, and some relationship between the two genera was suggested by Sakai and Nagamasu (2000)⇓. The independent results of our molecular analysis place Tamijiaas sister to the remaining Zingiberaceae, excluding the basal Siphonochilus, and confirm that this floral character is a feature shared with the sister taxa of the Costaceae. Because of the distinctness of these two genera in the family and their basal positions, we have chosen to recognize each genus as a subfamily of the Zingiberaceae, the Siphonochiloideae and the Tamijioideae, to indicate their phylogenetic position in the Zingiberaceae (Tables 2–4).

Alpinioideae

The former tribe Alpinieae and new subfamily Alpinioideae are defined by a number of features (Tables 1, 2, 4). The most conspicuous floral feature is the great reduction or absence of the two lateral staminodes (Figs. 4, 7). These staminodes represent two modified stamens of the outer staminal whorl of the Zingiberaceae floral plan; the third member of this whorl is absent. The “labellum,” which is nearly ubiquitous in the family, is a petaloid structure formed by the fusion of the two lateral sterile stamens of the inner staminal whorl; the median member of this whorl is the single tetrasporangiate fertile stamen. The lateral staminodes are also absent in the genera Rhynchanthusand Pommereschea, usually placed in the traditional Alpinieae. However, in these two genera the labellum is also reduced or absent and our molecular results place them within the new Zingiberoideae. These two genera are clear examples of independent reductions in lateral staminodes in the family.

The second apomorphy of the Alpinioideae is the perpendicular orientation of the plane of distichy of the shoots with respect to the direction of growth of the rhizome (Burtt, 1972⇓). This character is present in all of the genera formerly placed in the Alpinieae, except for Rhynchanthus and Pommereschea in which the orientation is parallel. In accordance with the suggestions of others (e.g., K. Larsen [AAU], T. Wood [FLAS], and M. Newman [E], personal communication; Smith, 1980⇓), these two genera should be included in the Zingiberoideae. Sakai and Nagamasu (2000)⇓assigned their new genus Tamijia to the Alpinieae based on the perpendicular shoot orientation, but recognized that their genus was somewhat anomalous in this tribe. Indeed, according to our molecular results, Tamijia deserves recognition as a separate subfamily altogether.

Within the subfamily Alpinioideae are two major groups of genera, here recognized as tribes Alpinieae and Riedelieae (Tables 2–4). The first tribe contains the majority of genera in the subfamily and includes taxa distributed in Asia, Africa, and the Americas. The taxa in the Alpinieae fall into two main subclades, the Renealmia clade and the Etlingera clade, each of which is supported by strong bootstrap values. However, three of the largest genera in this tribe, Etlingera, Amomum, and Alpinia, are polyphyletic, and the latter two have species in both the Renealmia clade and the Etlingera clade. Because our sampling of Etlingera was modest (three species) we suggest that taxon sampling be increased before any firm conclusions be drawn about the status of this genus. However, it is clear from this investigation as well as others (Harris et al., 2000⇓; Rangsiruji, Newman, and Cronk, 2000b)⇓ that major additional taxonomic studies and realignments are necessary in both Alpinia and Amomum. In Renealmia, the one genus in the Alpinioideae that is distributed across continents, our limited sampling of six species provided good support for a distinct African clade and a tropical American clade (Figs. 8–10).

For Alpinia the results of our analysis identified four separate groups (Alpinia I–IV) in the 12 species we sampled. Each of these four groups corresponds to one of the clades recognized in the molecular analysis of Rangsiruji, Newman, and Cronk (2000b)⇓ and, as in their analysis, our results do not support the classification of Alpinia proposed by Smith (1990a)⇓. Alpinia galanga and A. conchigera (Alpinia I; part of the A. galanga clade of Rangsiruji, Newman, and Cronk) are placed in two separate sections of subgenus Alpinia by Smith, but here form a strongly supported clade basal to the other members of the Renealmia clade (Aframomum, Renealmia, Amomum I, Elettariopsis, and Paramomum; including African, Asian, and American species). The rest of Alpinia is scattered in the Etlingera clade. Alpinia II (A. elegans, A. luteocarpa, and A. vittata; part of the A. eubractea clade of Rangsiruji, Newman, and Cronk) includes the genus Vanoverberghia (from the Philippines and Taiwan) and is allied with the polyphyletic Etlingera, Hornstedtia, and Amomum II (all Asian). Alpinia III (six species included in four of Rangsiruji, Newman, and Cronk's clades) is united with the genus Plagiostachys whereas Alpinia IV (single species A. carolinensisin Rangsiruji, Newman, and Cronk's A. carolinensis clade) is unresolved with Alpinia II and III. A more extensive analysis of Alpinia is in progress (A.-Z. Liu and W. J. Kress, unpublished data).

Taxonomic matters are made worse by the polyphyletic nature of the genus Amomum. Although we only sampled four species in this genus, these four species form two separate lineages: one within the Renealmia clade as sister to the genus Elettariopsis, which has a striking similarity in floral structure and is often difficult to distinguish from Amomum (Holttum, 1950⇓; Kiew, 1982⇓), and the other in the Etlingera clade, which is strongly supported to include a species of Etlingera. The polyphyly of Amomum was also suggested by Harris et al. (2000)⇓ in their investigation of the molecular systematics of the genus Aframomum. In a separate analysis of over 30 species of Amomum using ITS and matK sequence data, we have identified four separate lineages of Amomum scattered among the other genera of the Alpinieae (Y.-M. Xia, W. J. Kress, and L. M. Prince, unpublished data). Needless to say, the generic boundaries and definitions of all the taxa in this tribe require more extensive and careful molecular and morphological study before a new classification can be proposed. For this reason we have chosen not to give formal taxonomic status to either the Renealmia clade or the Etlingera clade, even though strong molecular support exists for both lineages.

The Riedelieae (Tables 2–4) include four genera united by both molecular and morphological characters: Burbidgea (Borneo), Pleuranthodium (primarily New Guinea and Bismarck Archipelago), Riedelia (New Guinea), and Siamanthus (Thailand). The affinities of these four genera are indicated by their common possession of long, slender silique-like capsules that open by longitudinal slits to the base (Smith, 1972⇓, 1990b⇓; Larsen and Mood, 1998⇓). Larsen and Mood (1998)⇓ also pointed out that the close relationship of at least three of these genera (Burbidgea, Riedelia, and Siamanthus) is suggested by unique “nectary pits” on the dorsal midrib of the leaf blade. We have now confirmed the presence of these extrafloral nectaries in Pleuranthodium as well (W. J. Kress and M. Bordelon, unpublished data). These vegetative and reproductive characters taken together make the Riedelieae a readily recognizable tribe in the Alpinioideae.

The long silique-like fruits of Siliquamomum (Southern China and Vietnam) are similar to the fruits found in the Riedelieae, but the presence of a well-developed labellum and lateral staminodes as well as the absence of the extrafloral nectaries on the leaf midribs separate this genus from other members of that tribe. The placement of Siliquamomum in either the Riedelieae or the Alpinieae is unresolved by our molecular analysis. For these reasons we prefer to place this genus as incertae sedis until further evidence confirms its position in the Alpinioideae.

Within the Alpinioideae, we included in our molecular analysis four of the six monospecific genera. Cyphostigma pulchellum (Thw.) Benth., restricted in distribution to Sri Lanka, is poorly known (Dassanayake, 1983⇓), and no tissue was available for our investigation. Leptosolena haenkei Presl, endemic to the lower slopes of Mt. Pinatubo in Northern Luzon, Philippines, has not been collected since it was described in the early 1900s and is presumed extinct (Madulid, 1996⇓). Based on morphological features we have tentatively placed these small genera (along with the African Aulotandra and Asian Geocharis and Geostachys) in the Alpinieae. Vanoverberghia, also from the Philippines, was only known from a single species, V. sepulchrei Merr., until a second species, V. sasakiana H. Funak. & H. Ohashi, was described from the Lanyu Islands southeast of Taiwan (Funakoshi and Ohashi, 2000)⇓. Merrill (1912)⇓, in his original description of the genus, compared it to Riedelia, but also recognized its affinity to Alpinia as did Smith (1990a)⇓. Our results show that this genus is a member of the Alpinia II clade that includes other species from the Philippines (e.g., A. elegansand A. luteocarpa). As noted above, a phylogenetic and taxonomic revision of Alpiniaand its closely related genera, including Vanoverberghia and Plagiostachys, is needed.

The monotypic Paramomum, endemic to Yunnan, China, was recently moved by Wu (1997)⇓ into the genus Amomum. In the original description of Paramomum, Tong (1985)⇓ compared it to Costus because of its supposed spiral phyllotaxy. Our molecular results place this genus as sister to Elettariopsis in a clade with species of Amomum I, thus supporting its separation from Amomum and clear distance from Costus. The monotypic and distinct nature of the remaining two genera in the Alpinioideae, Siliquamomum and Siamanthus, is also supported here by the long branch lengths and isolated positions of these taxa in or near the Riedelieae.

Zingiberoideae

Our subfamily Zingiberoideae contains the remaining genera in the family, which were formerly placed within the three tribes Zingibereae, Hedychieae, and Globbeae (Table 3). The most recognizable floral features of this subfamily are the conspicuous and often well-developed lateral staminodes (Figs. 3, 5, 6) that are generally absent in the Alpinioideae (Tables 2, 4). These lateral staminodes are fused to the corolla tube and free from the labellum in most of the former Hedychieae, whereas they are fused to the labellum in the genus Zingiber (former Zingibereae; Fig. 5) and connate to the filament in Globba and Mantisia (part of the former Globbeae; Fig. 6). In a few genera the lateral staminodes and/or the labellum are reduced or absent (Rhynchanthus, Pommereschea, Nanochilus, Stadiochilus, Laosanthus, Hedychium horsfieldii, and H. bordelonianum; Smith, 1980⇓; Newman, 1990⇓; Larsen and Jenjittikul, 2001⇓; Williams, Kress, and Thet Tun, 2002⇓). Hence, the presence of well-developed lateral staminodes is a feature not shared by all members of the Zingiberoideae as recognized here.

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Table 3. Placement of genera in the new classification of the family Zingiberaceae. (Asterisks indicate taxa not sampled in the molecular phylogenetic analyses; these genera are tentatively placed based on morphological features)

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Table 4. Characteristics of the subfamilies and tribes of the new classification of the Zingiberaceae presented here (Burtt, 1972; Burtt and Smith, 1972; Larsen et al., 1998; Sakai and Nagamasu, 2000; W. J. Kress, personal observation). Some nondiagnostic features are included as a reference to the previous classification (see Table 1)

The most prominent vegetative feature that is universal in this subfamily is the parallel orientation of the plane of distichy of the leafy shoots with respect to the rhizome (perpendicular orientation in all other Zingiberaceae). As pointed out earlier, this character was the basis for some authors to suggest that genera such as Rhynchanthus and Pommereschea with parallel orientation were not properly placed in the Alpinieae (Burtt, 1972⇓; Smith, 1980⇓; Wood, Whitten, and Williams, 2000⇓). A second vegetative trait that characterizes the Zingiberoideae (and Siphonochilus), briefly discussed by Wood, Whitten, and Williams (2000)⇓, is the capacity for members of this subfamily to go into dormancy during the dry season in monsoonal climates. Unlike the Alpinioideae and Tamijioideae, in which individuals are evergreen throughout the year, the Zingiberoideae, including taxa of the former Hedychieae, Globbeae, and Zingibereae, have a forced dormancy period during which all aboveground parts are shed and the plant overwinters as a thick, often fleshy underground rhizome (sometimes with starch-filled roots or tubers). Either just prior to or at the earliest sign of the wet season, individuals will break dormancy with either vegetative shoots or reproductive shoots (e.g., Hemiorchis, Gagnepainia, Mantisia, some kaempferias and many curcumas). However, some species in the Zingiberoideae can also be evergreen in the wet forest habitats where they occur (e.g., some globbas, zingibers, and hedychiums), but these same species can be forced into dormancy under stress or in greenhouse environments (M. Bordelon [US] and T. Wood [FLAS], personal communication). These latter taxa may be descendents of ancestors from monsoonal climates that have secondarily colonized evergreen habitats. As far as we know, members of Alpinioideae cannot be forced into dormancy, possess tough fibrous rather than fleshy rhizomes, and are always evergreen in their natural habitats.

Within the Zingiberoideae, we have chosen, for historical consistency, to recognize two tribes even though our molecular data do not provide strong support for these clades (Tables 2–4). The Zingibereae include most of the former Hedychieae as well as the genus Zingiber. All members of this tribe possess a trilocular ovary, which in some genera may be incompletely closed towards the apex (e.g., Scaphochlamys and Paracautleya), and axial or rarely basal placentation. Within the Zingibereae several groups of genera are supported by varying bootstrap values. A Kaempferia clade, including Haniffia, Zingiber, Kaempferia, Distichochlamys, Scaphochlamys, Boesenbergia, Curcumorpha, and Cornukaempferia (and probably Haplochorema and Parakaempferia not included in our analysis) is only weakly supported here, but has also been recognized by others (Newman, 1995⇓; Searle and Hedderson, 2000⇓; Wood, Whitten, and Williams, 2000⇓). These taxa lack the distinctive pseudostem (except for Zingiber and Haniffia) found in other members of the subfamily. Although it has been suggested that the genus Cornukaempferia is allied with either Kaempferia(Mood and Larsen, 1997⇓) or Zingiber (Searle and Hedderson, 2000⇓; Wood, Whitten, and Williams, 2000)⇓, our results do not support either of these hypotheses and resolution among the genera of the Kaempferia clade is generally low.

The close relationship between Distichochlamys and Scaphochlamys as well as their generic boundaries have been recognized (Newman, 1995⇓). However, the taxonomic position of Curcumorpha and Boesenbergia requires some discussion. Boesenbergia is a genus of about 60 species found throughout tropical Asia, including India, Myanmar, Thailand, Malaysia, and Borneo (Sirirugsa, 1992⇓), and is distinguished by its two-ranked inflorescence bracts, saccate labellum, and phenological pattern of flowers opening from the apex toward the base (Smith, 1987⇓). Curcumorpha longiflora was described as a monotypic genus from northeastern India (now known to also occur in Myanmar and Thailand) distinctive in its radical inflorescences with spirally arranged bracts and the presence of a staminodial cup (Rao and Verma, 1971⇓). Larsen (1997)⇓ placed Curcumorpha into Boesenbergia stating that the characters cited by the original authors did not sufficiently distinguish it from that genus. Our results suggest that Boesenbergia itself may be polyphyletic with one group of species (here represented by B. pulcherrima) allied to Curcumorpha. This taxonomic complex is yet another example of the problems that exist in defining clear generic boundaries in the Zingiberaceae.

The Curcuma clade has strong bootstrap support and includes Curcuma, Stahlianthus, Hitchenia, and Smithatris, which share cone-like inflorescences of few-flowered, congested bracts (although much reduced in Stahlianthus). Curcuma, with about 50 species, has been circumscribed by its “pouched” inflorescence bracts and versatile, usually spurred anthers. However, these features are neither unique nor universal in the genus, and our results along with those of others (Wood, Whitten, and Williams, 2000)⇓ strongly suggest that Curcuma is paraphyletic with Hitchenia and Stahlianthus, and maybe Smithatris. Expanded taxon sampling within these genera is necessary to realign species and distinguish boundaries among the taxa (T. Rehse, W. J. Kress, and P. S. Manos, unpublished data). Rather than accepting an enlarged concept of Curcuma to include these associated genera, the strong support of several clades within the Curcuma complex may necessitate the splitting up of Curcuma into several smaller genera.

Among the remaining genera in the Zingibereae two primarily Himalayan clades have strong support: the Roscoea + Cautleya clade in the western end of the range from Kashmir to southwestern China and the Pommereschea + Rhynchanthus clade in the eastern end straddling the borders of Yunnan, Myanmar, and Thailand. Both of these clades have moderate to strong support and at least in the ITS analysis form a weakly supported Himalayan alliance of four genera. The placement of Hedychium (a large, strongly supported monophyletic genus), the monotypic Pyrgophyllum endemic to southwestern China, and the few-species Camptandra from the lowland wet forests of Thailand and Malaysia is still unresolved, although the latter two genera are allied with the Curcuma clade in the ITS analysis.

The four genera in the traditionally recognized Globbeae (Tables 2–4; Fig. 6) are united by several morphological characters, such as the unilocular ovary with parietal placentation (convergent with Tamijia) and the long-arching filament to which the labellum and/or lateral staminodes are fused (in Globba and Mantisia only). Our molecular analysis strongly places the four genera of the Globbeae within the Zingiberoideae, but only weakly outside the taxa comprising the Zingibereae (see discussion below on Caulokaempferia). A number of vegetative and floral characters unite Globba with Mantisia and Hemiorchis with Gagnepainia, a result that is strongly supported by both the ITS and matK sequence data as well. Our analysis shows that the Indo-Burmese Mantisia is sister to an Indo-Burmese clade of Globba suggesting that the latter genus is paraphyletic; the relationships within this clade are currently under investigation (K. J. Williams, W. J. Kress, and P. S. Manos, unpublished data). Our ITS data provide only weak support to unite the four genera, and the combined analysis does not resolve this relationship. However, a more comprehensively sampled data set (K. J. Williams, W. J. Kress, and P. S. Manos, unpublished data) suggests that the tribe, as traditionally recognized, is in fact monophyletic. Because of this additional evidence and in the interest of maintaining stability with past classifications we advocate retaining the Globbeae as earlier recognized until forthcoming evidence proves otherwise.

Caulokaempferia, first included within the genus Kaempferia, was subsequently recognized as distinct by Larsen (1964)⇓. Its possible relationship to both Camptandra and Boesenbergia have been noted (Larsen and Smith, 1972⇓). Our results to not closely ally Caulokaempferia to any of the above three genera, and the combined analyses of the two molecular data sets leaves the placement of the genus within the Zingiberoideae unresolved with respect to the Zingibereae and the Globbeae. We therefore at present recognize Caulokaempferia as incertae sediswithin the Zingiberoideae in our revised classification.

We have been able to address the phylogenetic placement and distinctiveness of a number of the monotypic genera within the Zingiberoideae. Taxa such as Haniffia, Cornukaempferia, and Pyrgophyllum are characterized by relatively long branch lengths in the combined analyses (32–41 bp), while others, such as Curcumorpha, Hitchenia, Stahlianthus, and Smithatris, are less distinct (9–20 bp). As exploration of new habitats in Southeast Asia increases, additional species are being found in many genera that were previously only known from a single species (e.g., Curcumorpha, Cornukaempferia, Haniffia, Distichochlamys, and Smithatris). In addition, several monotypic genera such as Nanochilus, Paracautleya, Parakaempferia, Laosanthus, and Stadiochilus are yet to be sampled for inclusion in molecular investigations. We have tentatively placed these genera in the Zingibereae based on their morphology alone. A more thorough sampling and expanded phylogenetic analyses should provide additional answers to the status of the many small genera in the family.

Future directions

We recommend that more species of the large paraphyletic genera Curcuma, Alpinia, Amomum, and Etlingera be added to future molecular analyses to better delimit generic boundaries in these taxa and their allied genera. Taxa from important, but undersampled, genera such as Hornstedtia, Aulotandra, Elettaria, Geocharis, Geostachys, Riedelia, Burbidgea, and Scaphochlamys should be included in future investigations. We are especially interested in the placement and taxonomic status of the remaining monotypic genera (Stadiochilus, Nanochilus, Cyphostigma, Laosanthus, Paracautleya, Parakaempferia, and Leptosolena), some of which are exceedingly rare and may be extinct in the wild. We plan to continue our taxon sampling throughout the family with the hopes of recognizing appropriate monophyletic genera and eventually proposing a more detailed classification of the Zingiberaceae. We are also currently utilizing our phylogenetic results to investigate patterns of biogeographic history and character evolution in this interesting and diverse pantropical family.