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Systematics |
2Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18 D, SE-752 36, Uppsala, Sweden; 3The Bergius Foundations at the Royal Swedish Academy of Science, P. O. Box 50017, SE-104 05, Stockholm, Sweden
Received for publication October 11, 2001. Accepted for publication February 19, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Breoniinae • Cinchonoideae • chloroplast DNA • Corynantheinae • Naucleeae • nuclear DNA • Rubiaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Bremer [1996a
]; tribal: e.g., Bremer and Thulin [1998]
, Andersson and Rova [1999]
, Andreasen and Bremer [2000]
; generic: e.g., Nepokroeff, Bremer, and Sytsma [1999]
, Andersson [2001]
, Lantz, Andreasen, and Bremer [2002]
; species: e.g., McDowell and Bremer [1998]
; Persson [2000]
). One of the groups of Rubiaceae that has recently drawn our attention is the tribe Naucleeae of the subfamily Cinchonoideae because of the conflicting views about its circumscriptions and generic limits (Haviland, 1897
; Verdcourt, 1958
; Bremekamp, 1966
; Ridsdale, 1975
, 1978a
; Robbrecht, 1988
, 1994
; Bremer, Andreasen, and Olsson, 1995
). The results of the phylogenetic studies by Razafimandimbison and Bremer (2002)
based on molecular (internal transcribed spacer [ITS] and rbcL) and morphological data strongly support a broader circumscription for Naucleeae, which includes all members of Naucleeae sensu Ridsdale (1978a)
, Cephalanthus, Hallea, Mitragyna, Uncaria (as shown by Bremer, Andreasen, and Olsson, 1995
), Corynanthe, and Pausinystalia. Naucleeae sensu Razafimandimbison and Bremer (hereafter referred to as Naucleeae sensu lato [s.l.]) as presently circumscribed consists of 26 genera and 179 species of trees, shrubs, and woody climbers. Most representatives occur in tropical Asia, mostly in Southeast Asia, with 134 species and 13 genera, followed by Madagascar with 24 species and 4 genera, Africa with 22 species and 8 genera, and Central, North, and South America together with only 5 species and 2 genera. Neonauclea, Uncaria, and Breonia are the most speciose genera, with 65 (Ridsdale, 1989
), 34 (Ridsdale, 1978a
), and 20 (Razafimandimbison, 2002
) species, respectively. Nine genera (Adinauclea, Breonadia, Burttdavya, Diyaminauclea, Haldina, Janotia, Khasiaclunea, Metadina, and Sinoadina) are monotypic; the remaining genera contain two to four species (Tables 1 and 2). Naucleeae are a well-defined monophyletic group that can easily be recognized by numerous flowers arranged in globose inflorescences and epigynous floral nectaries deeply embedded in hypanthia (Bremer, Andreasen, and Olsson, 1995
; Razafimandimbison, 2002
; Razafimandimbison and Bremer, 2002
).
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recognized four subtribes: Anthocephalidae (containing Neolamarckia and Nauclea); Cephalanthidae (containing Adina, Breonia, Cephalanthus, and Neonauclea); Mitragynae (forming Mitragyna); and Uncariae (containing Uncaria). Haviland (1897)
tentatively placed the Malagasy genus Paracephaelis in Naucleeae, but its position within the tribe Pavetteae (subfamily Ixoroideae) has now widely been accepted (e.g., Bridson and Robbrecht, 1985
; Robbrecht, 1988
, 1994
). Ridsdale (1978a)
divided Anthocephalidae sensu Haviland into two subtribes: Anthocephalinae, currently known as Neolamarckiinae (Robbrecht, 1988
) (comprising one genus Neolamarckia), and Naucleinae (including Burttdavya, Nauclea sensu stricto (s.s.) (Ochreinauclea and Sarcocephalus). Ridsdale further recognized a third subtribe Adininae (comprising Adina, Breonia, and Neonauclea all sensu Haviland).
Generic limits in Naucleeae have also long been controversial and unsettled (see Table 2). Haviland's (1897)
first worldwide revision of Naucleeae consisted of nine genera: Adina, Breonia, Cephalanthus, Mitragyna, Nauclea, Neolamarckia, Neonauclea, Paracephaelis, and Uncaria. While the generic limits of Cephalanthus, Neolamarckia, and Uncaria have never been disputed, the delimitations of Adina, Breonia, Mitragyna, Nauclea, and Neonauclea have been a source of disagreement (Bremekamp, 1966
; Leroy, 1975a
; Ridsdale, 1975
, 1978a
, b
, 1989
; Razafimandimbison, 2002
). All genera described after Haviland's revision, except Burttdavya and Neobreonia, included only species previously described in Adina, Nauclea, and Neonauclea. These three genera were considered by Ridsdale (1975
, 1978a
) to be heterogeneous. He adopted new generic circumscriptions, leading to recognition of several small genera (Table 2).
Razafimandimbison and Bremer's (2002)
studies on Naucleeae s.l. show that the combined morphological and molecular data sets yield the best resolution at different areas of the cladograms. Reviews of arguments for combining different data sets are discussed thoroughly by several authors (e.g., de Queiroz, Donoghue, and Kim, 1995
; Bremer, 1996b
; Nixon and Carpenter, 1997
) and will not be repeated here.
The present study was intended to include a much larger taxon sampling than Razafimandimbison and Bremer (2002)
and one additional data set from trnT-F. It strengthened our previous conclusions on the circumscriptions of Naucleeae and also allowed us to elucidate its highly controversial intratribal classifications. The main objective of this study is to produce, with combined molecular and morphological data, robust phylogenies for Naucleeae s.l. The results of this phylogenetic study will subsequently be used to (1) substantiate new intratribal classification; (2) test the monophyly of previous subtribal circumscriptions; (3) and evaluate the status of all formally described genera currently placed in Naucleeae s.l.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and 11 were added to this study (http://ajbsupp.botany.org/v89/). The genus Luculia, which has been shown to be basal in Rubiaceae (e.g., Bremer et al., 1999
), was used to root the trees. Cinchona pubescens and Exostema lineatum, used as ougroup taxa in Razafimandimbison and Bremer's (2002)
study, were also included here in order to test the monophyly of Naucleeae s.l.
Molecular data
Freshly collected, dried silica-gel leaves (Chase and Hills, 1991
) and/or herbarium specimen leaves were used for extracting DNA following the mini-prep procedure of Doyle and Doyle (1987)
. Three molecular data sets, ITS (regions of nrDNA), rbcL (a protein coding gene of cpDNA), and trnT-F (mainly noncoding regions of cpDNA), in combination with morphological data, were used in this study. Both amplification and sequencing of ITS and rbcL were done following the protocols described in Razafimandimbison and Bremer (2002)
.
The trnT-F consists of four regions: an intergenic spacer between the trnT (UGU) and trnL (UAA) 5' exon, the trnL 5' exon, a trnL (UAA) intron, and another intergenic spacer between the trnL (UAA) 3' exon and trnF (GAA) (Taberlet et al., 1991
). The primers used to amplify and sequence these four regions are given in Table 3. The primer pair trnT-F_aF or trnT-F_a1F/trnT-F_dR amplifies the region between the trnT and trnL exon 3'; the primer pair trnT-F_cF/trnT-F_fR amplifies the region between the trnL intron and trnF. The internal primers, trnT-F_bR, trnT-F_eF, trnT-F_jF, trnT-F_iR, and trnT-F_rF, were also used to produce complete sequences of the entire regions of the trnT-F, with at least partial sequence overlap. The four regions of the trnT-F were amplified separately for the taxa with degraded DNA. The positions of the primers used in Nicotiana tabacum and their directions are shown in Table 3. Polymerase chain reactions (PCR) for the trnT-F were 50-µL reactions including 28.25 µL sterilized H2O, 5 µL reaction buffer, 5 µL MgCL2, 5 µL TMACL (Chevet LeMaître, and Katinka, 1995
), 4 µL, 0.25 µL Taq (5U/µL), 0.5 µL 5' primer, 0.5 µL 3' primer, 0.5 µL BSA 1%, and 1 µL of DNA templates. The PCR amplifications, performed in a Eppendorf Mastercycle gradient (Bergman & Beving Instrument, Stockholm, Sweden), began with initial melting for 1 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min 30 s at 55°C, and 1 min 30 s at 72°C, and ended with a final extension phase of 7 min at 72°C.
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), 53 rbcL sequences (of which 45 are newly published here, while eight were previously published by the second author alone or with her co-authors [Bremer, Andreasen, and Olsson, 1995
; Bremer, 1996a
, b
; Bremer and Thulin, 1998
; Bremer et al., 1999
]), and 55 trnT-F sequences, all newly published here.
Morphological data
All 49 morphological characters investigated in this study were from Razafimandimbison and Bremer (2002)
. They represented variation in gross morphology, karyology, and phytochemical data.
Data analyses
Molecular analysis
Sequence data sets of each marker were aligned using CLUSTAL X (Thompson et al., 1997
) to produce an initial alignment; this was followed by manual alignment using Se-Al (Rambaut, 1995
). The sequence data were subsequently analyzed using the Sequencher (Gene Codes Corporation, Stockholm, Sweden) software package. All new sequences were submitted to EMBL. All accession numbers may be found at the American Journal of Botany's supplemental data site (http://ajbsupp.botany.org/v89/). The sequenced species were used as terminals.
Morphological analysis
Using species as terminals in morphological analyses provides a potential test for monophyly of highly variable genera (e.g., Wiens, 2000
). However, both our previous (Razafimandimbison and Bremer, 2002
) and present molecular analyses demonstrate that most Naucleeae genera with at least two species are monophyletic. In our morphological analysis, genera and species (sequenced species) were used as terminals for the monophyletic and paraphyletic (Cephalanthus, Neonauclea, and Pertusadina) genera, respectively.
Combined analysis
The morphological character states of the monophyletic genera were used to represent that of their sequenced species in the combined molecular-morphological analysis.
Search strategies
Parsimony analyses of the ITS, rbcL, and trnT-F matrices (excluding uninformative characters) were performed with PAUP* version 4.0b6 (Swofford, 2000
) using heuristic searches, with the MULTREES option off, nearest neighbor interchanges (NNI) branch swapping, and 10 000 random additions. The characters were unordered (i.e., using Fitch parsimony) and equally weighted. The shortest tree was saved for each replicate regardless of its length. All trees retained were submitted to a second round of tree bisection-reconnection (TBR) branch swapping with MULTREES on. Parsimony analyses of the combined molecular (ITS-rbcL-trnT-F) and molecular-morphological data sets were subsequently carried out using heuristic search, unordered and unweighted characters, MULTREES option on, TBR branch swapping, and 10 000 random addition sequences to search for multiple islands of most-parsimonious trees (Maddison, 1991
). In all analyses indels were treated as missing data and all potential phylogenetically informative indels were rescored as binary characters (0 and 1). The consistency index (CI; Kluge and Farris, 1969
) and retention index (RI; Farris, 1989
) were calculated to estimate homoplasy. Bootstrap (BS; Felsenstein, 1985
) and jackknife (JK; Farris et al., 1996
) values using heuristic searches, with the MULTREES option off, NNI branch swapping, five random additions, and 10 000 replicates were performed to assess relative support for the identified clades.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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studies. Accordingly, we present only the tree from our new trnT-F analysis as well as the strict consensus trees from the combined molecular and molecular-morphological analyses. All formally published generic names currently placed in Naucleeae s.l. (except Diyaminauclea and Khasiaclunea) are used in the trees (Figs. 1–3), although no single author accepts all these genera.
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) using the following settings: a deletion frequency of 37%, emulate "jac" resampling option on, 10 000 replicates, the MULTREES option off, NNI branch swapping, and five random addition sequences. All investigated taxa of Naucleeae s.l. were resolved with high support (JK = 100) as a monophyletic group (Fig. 1). The following clades, also revealed by the morphological, ITS and rbcL trees (results not presented), were retained: Clade A (consisting of Cephalanthus glabratus, C. occidentalis, and C. salicifolius; JK = 100); Clade B (consisting of Adina sensu Ridsdale, Adinauclea, Haldina, Metadina, and Pertusadina; JK = 56); Clade C (formed by Myrmeconauclea, Neonauclea, and Ochreinauclea; JK = 72); Clade D (including Breonadia, Breonia, Gyrostipula, and Janotia; JK = 96); Clade E (consisting of Hallea and Mitragyna, JK = 71); Clade F (containing Corynanthe and Pausinystalia; JK = 99); Clade G (formed by Uncaria africana and Uncaria sp.; JK = 67); Clade H (including Uncaria guianensis and U. rhynchophylla, JK = 82); Clade I (consisting Nauclea diderrichii and N. xanthoxylon; JK = 71); and Clade J (formed by Nauclea orientalis and N. subdita; JK = 89).
Combined analyses
The partition of homogeneity tests performed between the ITS, rbcL, and morphological data sets of Naucleeae s.l. in Razafimandimbison and Bremer (2002)
showed that these three data sets were combinable. The visual inspection shows that the jackknife consensus trnT-F tree (Fig. 1) yields almost identical topologies as the strict consensus ITS, rbcL, and morphological trees from this study (results not shown). Therefore, we feel justified in merging all data sets in a large matrix for conducting combined analyses. We were not able to obtain ITS and trnT-L (corresponding to A–E regions, approximately 1200 bp) sequences from Sinoadina racemosa and Ludekia borneensis, respectively, mainly due to difficulties with amplification of their DNA templates from herbarium material. These two taxa were included in the combined analyses by inserting question marks (?) for their missing nucleotides. Fusion of the three separate molecular data sets of 53 taxa in one matrix yielded 4044 positions and 381 parsimony-informative characters (Table 4). A parsimony analysis resulted in three islands containing 15 most equally parsimonious trees each 1181 steps long, with CI of 0.466 (excluding uninformative characters) and RI of 0.651. The addition of 49 morphological characters to the combined molecular matrix, which contained 4093 characters, of which 426 were parsimony-informative (Table 4), resulted in 38 most parsimonious trees in four islands each 1429 steps long, with CI of 0.467 (excluding uninformative characters), and RI of 0.685. Both combined trees (Figs. 2 and 3) supported the monophyly of Naucleeae s.l. and diagnosed the same monophyletic groups, which we recognize here as subtribes: Cephalanthinae, Mitragyninae, Uncarinae, Corynantheinae, Naucleinae, Breoniinae (all highly supported), and "Adininae" (poorly supported). Within the "Adininae," the Neonauclea clade received moderate and high support in the combined molecular (JK = 79, BS = 73) and combined molecular-morphological trees (JK = 94, BS = 89), respectively. The relationships among these seven subtribes were largely unresolved.
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, represented by A. pilulifera and A. rubella here, was resolved with weak (JK = 58, BS < 50%) and strong support (JK = 94, BS = 88) as monophyletic in the combined molecular and combined molecular-morphological trees, respectively. Similarly, Cephalanthinae (including C. natalensis) had no support in the combined molecular tree, but received high support (JK = 95, BS = 93) in the combined molecular-morphological tree. Also, some differences were perceived within Corynantheinae: in the combined molecular tree (Fig. 2), Corynanthe paniculata was resolved with high support as sister to Pausinystalia macroceras and nested between this latter and P. johimbe; P. lane-poolei subsp. ituriense was resolved as sister to the rest of the members of Corynantheinae. However, in the combined molecular-morphological tree (Fig. 3), the three species of Pausinystalia formed a strongly monophyletic group and C. paniculata was resolved as sister to the remaining members of Corynantheinae.
Classification
We propose here a new subtribal classification of Naucleeae s.l. based on the combined molecular-morphological tree (Fig. 3) and supplemented by comprehensive information from literature. We provisionally recognize the subtribe Adininae to accommodate Adina, Adinauclea, Haldina, Metadina, Pertusadina, Sinoadina, and the members of Neonauclea clade (Lukedia, Myrmeconauclea, and Neonauclea).
Subtribe Cephalanthinae DC., Prodromus 4: 538 (1830) (as Cephalantheae)
—Cephalantheae H. B. K. [Nov. Gen. Sp. 3: 379 (1818), nom. Prov. (as Sectio) ex Kunth, Synop. Pl. Aequinoct. 4: 37 (1824) (as Sectio); Cham. & Schlecht. Linnea 4: 147 (1829) (as Sectio); Lindl., Intr. Nat. Syst. Bot., p. 204 (1830) (as Cephalantheae); Endl., Gen. Pl.: 530 (1838); Ench. Bot.: 271 (1841) (as Cephalantheae).
—Cephalanthidae Haviland. J. Linn. Soc. Bot. 33: 21 (1897)
Useful recent study: Ridsdale (1976)
Erect shrubs or trees. Stipules small, entire. Young inflorescences not surrounded by calyptra-like bracts. Inflorescences terminal. Flowers homostylous. Corolla lobes imbricate in bud, with large colleters in sinuses between corolla lobes (absent in Cephalanthus natalensis). Ovary bicarpellate, with one ovule in each locule, pendulous. Infructescences constituted by free, indehiscent fruits. Seeds unwinged, arillate (except C. natalensis).
Genus included: Cephalanthus
Subtribe Mitragyninae Haviland, J. Linn. Soc. Bot. 33: 21 (1897) (as Mitragyneae)
Useful recent studies: Leroy (1975a)
; Ridsdale (1978b)
Trees or shrubs. Stipules large, entire. Young inflorescences not surrounded by calyptra-like bracts. Inflorescences terminal. Flowers homostylous. Corolla lobe valvate in bud. Stigmas mitriform; ovary bicarpellate, with numerous ovules in each locule, basally attached, and ascendingly imbricate. Pollens 3-zonocolporate with H-shaped endoapertures. Infructescences formed by free, capsular fruits. Seeds winged.
Genus included: Mitragyna s.l.
Subtribe Uncarinae Haviland, J. Linn. Soc. Bot. 33: 21 (1897)
Useful recent study: Ridsdale (1978b)
Woody lianas. Stipules large, entire or shallowly to deeply bifid. Young inflorescences not surrounded by calyptra-like bracts. Paired fang hooks (modified inflorescence peduncles) always present. Inflorescences terminal or axillary. Flowers homostylous. Corolla lobe valvate in bud. Ovary bicarpellate, with numerous ovules in each locule, basally attached, and ascendingly imbricate. Infructescences constituted by free, capsular fruits. Seeds winged. Genus included: Uncaria
Subtribe Corynantheinae Razafimandimbison and B. Bremer, subtrib. nov.
Type genus: Corynanthe
Useful recent studies: Stoffelen, Robbrecht, and Smets (1996)
Subtribus a subtribubus aliis differt appendicibus glabris prominentibus e corollae lobis prolongatis.
Trees. Stipules entire. Young inflorescences not surrounded by calyptra-like bracts. Inflorescences terminal or axillary or both. Flowers homostylous. Corolla lobes valvate in bud, prolonged by glabrous, well-developed appendages. Ovary bicarpellate, with numerous ovules, basally attached, and ascendingly imbricate. Infructescences constituted by free, capsular fruits. Seeds winged.
Genera included: Corynanthe, Pausinystalia, Pseudocinchona.
Subtribe Naucleinae DC., Prodromus Systematis Naturalis 4: 343 (1830) (as Cinchoneae subtribe Naucleeae)
—Subtribe Sarcocephalinae DC., Prodromus Systematis Naturalis 4: 367 (1830) (as Gardeniaceae subtribe Sarcocephalinae)
—Anthocephalinae Ridsdale, Blumea 24: 320 (1978a)
—Neolamarckiinae Robbrecht, Opera Botanica Belgica 1: 179 (1988)
Shrubs or trees. Stipules entire or shallowly bifid. Young inflorescences not surrounded by calyptra-like bracts. Inflorescences terminal. Flowers homostylous. Corolla lobe imbricate in bud. Stigmas fusiform (spindle-shaped), with receptive areas restricted to the base; ovary bicarpellate, with numerous ovules in each locule, mainly pendulous. Infructescences multiple fruits or formed by free fruits, indehiscent. Seeds unwinged.
Genera included: Burttdavya, Nauclea, Neolamarckia, Ochreinauclea, Sarcocephalus
Subtribe Breoniinae Razafimandimbison and B. Bremer, subtrib. nov.
Type genus: Breonia
Useful recent treatments: Homolle (1938)
, Leroy (1975a)
, Ridsdale (1975)
, Razafimandimbison (2002)
Subtribes distinctus bracteis calyptriformibus inflorescentiam juvenem cingentibus et discis ad infructescentiae maturitatem e floris nectariis accrescentibus.
Trees or shrubs. Stipules entire. Young inflorescences completely surrounded by calyptra-like bracts. Inflorescences axillary. Flowers homostylous. Corolla lobe imbricate in bud. Stigmas clavate to globose; ovary bicarpellate, with 1 to 15 ovules in each locule, pendulous. Infructescences multiple fruits, indehiscent, with accrescent nectary disks derived from the post-growth of floral nectaries during infructescence development. Seeds unwinged or winged.
Genera included: Breonadia, Breonia, Gyrostipula, Janotia.
Subtribe Adininae Ridsdale, Blumea 24: 319 (1978a).
Useful recent study: Ridsdale (1978a)
Trees or shrubs. Stipules entire or shallowly bifid. Young inflorescences surrounded or not (in Ludekia) by calyptra-like bracts. Inflorescences terminal or axillary. Flowers homostylous. Corolla lobes valvate but subimbricate at the apex or imbricate. Stigmas clavate to globose, ovary bicarpellate, with ovary 1 to 14 ovules in each locule. Infructescences formed by free, capsular fruits. Seeds winged.
Genera included: Adina, Adinauclea, Haldina, Ludekia, Metadina, Myrmeconauclea, Neonauclea, Pertusadina, Sinoadina.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Person, 2000
; Lantz, Andreasen, and Bremer, 2002
).
Several studies (e.g., Gielly and Taberlet, 1994
; McDade and Moody, 1999
) have shown that the spacer portion of the trnT-F region is evolving more rapidly than the intron. This is also corroborated by our results, with both trnL-F and trnT-L spacers providing the same number of variable sites (71) and more than that of the trnL intron (31). Our studies also showed that the trnT-L spacer had more phylogenetically informative characters (all variable sites) than the trnL-F spacer (only 31 of 71 variable sites), suggesting that the combination of these three adjacent regions evolving at different rates is useful for inferring phylogenetic relationships in Rubiaceae.
Subtribal limits in Naucleeae s.l
Both strict consensus trees (Figs. 2 and 3) from the combined analyses identify the same lineages and also receive much higher support than any of the strict consensus trees from the separate analyses. We chose the strict consensus tree from the combined molecular-morphological analysis (Fig. 3) over the strict consensus tree from the combined molecular analysis (Fig. 2) for making conclusions on intratribal classifications, the generic limits, and relationships among the subtribes of Naucleeae s.l. because it is the best supported hypothesis, maximizing congruence among all of the characters sampled (e.g., Nixon and Carpenter, 1997
).
Haviland (1897)
and Ridsdale's (1978a)
subtribal limits are partly supported by our results. The combined molecular-morphological tree (Fig. 3) highly supports (JK = 100, BS = 100) the monophyly of the subtribes Anthocephalidae sensu Haviland (Naucleinae sensu Razafimandimbison and Bremer), Mitragynae sensu Haviland (Mitragyninae), and Uncariae sensu Haviland (Uncarinae). We perceive no support for the subtribe Cephalanthidae sensu Haviland because its members came out in five separate and distinct clades (Cephalanthinae, Breoniinae, Adininae). The subtribe Naucleinae sensu Ridsdale (same as Naucleinae sensu Razafimandimbison and Bremer excluding Neolamarckia) constitutes a moderately monophyletic group (JK = 84, BS = 76). Naucleinae sensu Ridsdale and Neolamarckiinae sensu Robbrecht (containing the single genus Neolamarckia) are resolved together as a monophyletic group (JK = 100, BS = 99), supporting the inclusion of Neolamarckia in Naucleinae sensu Ridsdale. Recognition of Neolamackiinae (Haviland, 1897
; Ridsdale, 1978a
) as a separate subtribe is mainly based on one autapomorphic character state: ovaries with upper parts divided into two or four cavities due to false septa. The subtribe Adininae sensu Ridsdale, containing Breoniinae, and Adininae sensu Razafimandimbison and Bremer, is not resolved as a monophyletic group.
The present study shows that a new intratribal classification of Naucleeae s.l. is needed. The combined molecular-morphological tree (Fig. 3) depicts that the tribe can be subdivided into seven subtribes: Cephalanthinae, Mitragyninae, Uncarinae, Corynantheinae, Naucleinae, Breoniinae (all strongly supported), and "Adininae" (poorly supported). Both Naucleinae (as Anthocephalidae sensu Haviland) and Neolamarckiinae (as Anthocephalinae sensu Ridsdale) have been used in earlier subtribal classifications (Haviland, 1897
; Ridsdale, 1978a
). Naucleinae (Candolle, 1830
) has priority over Neolamarckiinae (Robbrecht, 1988
). "Adininae" receives poor support (JK and BS < 50) in both combined trees (Figs. 2 and 3). However, we have chosen a conservative approach to provisionally maintain Adininae as a separate subtribe to accommodate Adina, Adinauclea, Haldina, Metadina, Ludekia, Myrmeconauclea, Neonauclea, Pertusadina, and Sinoadina.
If compared with the previous subtribal classifications of Naucleeae (Haviland, 1897
; Ridsdale, 1978a
) it appears that Haviland's scheme is best supported by our data. Of the seven subtribes recognized here, two (Breoniinae and Corynantheinae) are newly described, two (Adininae and Cephalanthinae) have received very different circumscriptions (Table 2), and two (Mitragyninae and Uncarinae) have identical circumscriptions as Haviland's (1897)
. The circumscription of Anthocephalidae sensu Haviland differs from our Naucleinae only because of the inclusion of Burttdavya, which was described after his worldwide revision for Naucleeae (Haviland, 1897
).
Relationships among subtribes
Our results support the basal position of Cephalanthinae in Naucleeae s.l. Cephalanthinae is resolved with high support (JK = 100, BS = 100) as sister to the rest of Naucleeae placed in a large clade. However, even the combination of four data sets (three molecular and one morphological) did not provide enough informative characters to resolve the relationships among the six subtribes within this large clade. The poor resolution can be explained by the inadequate number of synapomorphic characteristics shared among these lineages, which are mostly united by homoplastic characters. In other words, this pattern may also reflect rapid early diversification of these subtribes, such that few or no mutations apparently became fixed in their common ancestors. Although Ridsdale's subtribal concepts (1978a)
are only partly supported by our results, his conclusions about the relationships between his subtribes are corroborated by our studies: he says "the subtribes here recognized are relatively homogeneous but these subtribes have only a rather low level of relationship with each other" (Ridsdale, 1978a
, p. 309). Most of the synapomorphic characteristics support the nodes that define both the subtribes and their internal nodes; this suggests that the diversification of and within the subtribes at both molecular and morphological levels must have occurred rather slowly after their early rapid radiation, such that there has been enough time for both morphological and molecular changes to accumulate along branches.
Relationships within subtribes and generic limits
Our intention is to establish new generic limits that are as consistent as possible with the previous classifications of Naucleeae and to minimize nomenclatural changes. In this study, we used two criteria for evaluating the status of the genera (with two or more species) currently placed in Naucleeae s.1. as well as generic recognition (Backlund and Bremer, 1998
): (1) genera must be monophyletic and diagnosed by inclusive morphological synapomorphies, allowing recognition of their members; and (2) they must be easy to identify and separate from each other. Assessment and recognition of the monotypic genera are done based on a combination of the following three criteria: (a) if they are not nested within well-defined genera; (b) if they have at least two autapomorphic characters, allowing them to be recognized very easily; (c) and if their relationships with other genera are not strongly supported.
Cephalanthinae
Unlike its taxonomic position, the recognition Cephalanthus at generic level has been widely accepted. We maintain Cephalanthus sensu Ridsdale (1976)
, represented by three species here, as a separate genus because it is strongly supported as a monophyletic group, although we have been unable to find any unique morphological synapomorphy that unites C. natalensis and the American and Asian species.
Mitragyninae
Leroy (1975a)
segregated three of the four African species (Mitragyna ciliata, M. rubrostipulata, and M. stipulosa) from Mitragyna s.1. and placed them in the new genus Hallea based on the following characters: monopodial growth, relatively large leaves, axillary inflorescences on lateral twigs, the abaxial side of corolla lobes densely pubescent and prolonged into short appendages, and stigmas with the receptive areas covering the entire surfaces of the stigmatic lobes. Mitragyna s.s. are characterized by having small leaves, terminal inflorescences, sympodial growth, the outside of corolla lobes glabrous and without appendages, and stigmas with the receptive areas restricted towards the distal and proximal ends of the stigmatic lobes. These architectural and floral structure differences correlate with wood and leaf anatomy (see Leroy, 1975a
) and some differences in the spectrum of alkaloids. Also, they are supported by chorology: Hallea are exclusively restricted to rheophytic habitats such as swampy forest in East and Central Africa, whereas Mitragyna are mostly Asian, with only one African representative (M. inermis), which is restricted to the Sudanian regions. Hallea has been recognized as a separate genus by several authors (e.g., Huysman, Robbrecht, and Smets, 1994
). However, Ridsdale (1978a)
retained Hallea in Mitragyna.
Our strict consensus combined molecular-morphological tree (Fig. 3) strongly shows with high support (JK = 100, BS = 100) that Hallea and Mitragyna s.s. are more closely related to each other than they are to the rest of Naucleeae s.1.; this is consistent with morphological evidence. Mitragyna s.1. (including Hallea), an Afro-Asian genus with nine species (four African and five Asian), is diagnosed by mitriform stigmatic lobes and three-zonocolporate pollen grains with endoapertures that are always H-shaped (Huysmans, Robbrecht, and Smets, 1994
). Also, the combined molecular-morphological tree supports the monophyly of Mitragyna s.s., represented by three species here, but neither reject nor support the monophyly of Hallea. However, the paraphyly of Hallea is highly supported by the combined molecular tree (Fig. 2); therefore, we merge Hallea with Mitragyna, as suggested by Ridsdale (1978b)
.
Uncarinae
The generic status of Uncaria, represented in our analysis by four species, has never been disputed due to its unique climbing habit and paired fang hooks that make this genus very distinct from the other members of Naucleeae s.1. Our study strongly supports the monophyly of Uncaria, supporting its generic status.
Corynantheinae
The African genus Corynanthe was originally described by Welwitsch (1869)
based on a single species (C. paniculata) from Angola. Later, more species (including the African "yohimbe tree" [C. johimbe]) were recognized. Because of the presence of johimbine (or yohimbe), a substance with medicinal properties, Corynanthe attracted early systematic attention. The anatomical and pharmacological studies by Dupouy and Beille (1905)
, which confirmed J.-B. Pierre's conclusions (unpublished data, Laboratoire Phanérogamie, Paris) based on morphological observations, revealed some evidence to support the distinctions between Corynanthe paniculata (with an infundibular corolla tube, exserted style and anthers, spherical and undivided stigmas, and mainly loculicidal capsules) and C. johimbe (with corolla tubes differentiated in a basal narrowly and shortly cylindrical part bearing an apical bladder, included style and anthers, bilobed stigmas, and mainly septicidal capsules). Consequently, they placed C. johimbe in the separate genus, Pausinystalia. These morphological distinctions between Corynanthe and Pausinystalia defined by Dupouy and Beille (1905)
have been followed by several authors (e.g., Brandt, 1922
; De Wildeman, 1922
; Good, 1926
; Hallé, 1966
; Stoffelen, Robbrecht, and Smets, 1996
). Chevalier (1909)
questioned the homogeneity of Corynanthe sensu Dupouy and Beille (containing C. pachyceras and C. paniculata) and adopted a new circumcription by restricting it to the type species with five-merous flowers, exserted style and anthers, and loculicidal capsules and placing the species with four-merous flowers, exserted style and anthers, and largely septicidal capsules (C. pachyceras) in his new genus Pseudocinchona (P. africana). Chevalier (1926)
subsequently made the new combination Pseudocinchona pachyceras (K. Schum.) A. Chev. because the epithet "pachyceras" (Schumann, 1901
) has priority over that of "africana" (Chevalier, 1909
). Pseudocinchona is additionally characterized by the occurrence of two alkaloids, corynanthine and corynantheine, which are absent in Pausinystalia and Corynanthe s.s. (C. paniculata). Raymond-Hamet's (1941)
study confirmed that johimbine is indeed absent in Pseudocinchona but present in Pausinystalia johimbe and Corynanthe paniculata. Also, he found out that johimbine is absent in Pausinystalia mayumbensis (Good, 1926
) and therefore transferred this latter to Pseudocinchona. Hallé (1966)
treated the two species of pseudocinchona (P. mayumbensis and P. pachyceras) in Corynanthe and instead recognized them at subgeneric level. However, Stoffelen, Robbrecht, and Smets (1996)
also included Pseudocinchona in Corynanthe, but without recognizing them at the infrageneric level.
The combined molecular-morphological tree (Fig. 3) strongly supports Pausinystalia sensu Stoffelen, Robbrecht, and Smets (1996)
(represented by three species here) as a separate monophyletic group. However, Corynanthe sensu Stoffelen, Robbrecht, and Smets (1996)
, represented here by all three species, is resolved with high support as paraphyletic, with its type species C. paniculata placed as sister to a clade containing Pausinystalia sensu Stoffelen, Robbrecht, and Smets, Corynanthe mayumbensis, and C. pachyceras. The combined molecular tree (Fig. 2) places C. paniculata with high support nested between Pausinystalia johimbe and P. macroceras, making Pausinystalia sensu Stoffelen, Robbrecht, and Smets (1996)
paraphyletic; this result is also consistent with phytochemical data (C. paniculata and Pausinystalia have johimbine, a substance not found in Pseudocinchona [Chevalier, 1909
; Raymond-Hamet, 1941
]). In both combined trees, Pseudocinchona (represented by P. mayumbensis and P. pachyceras) is shown with strong support to be monophyletic. Based on all evidence presented above, we argue that the generic status of Corynanthe sensu Stoffelen, Robbrecht, and Smets (1996)
is untenable. Three alternative solutions are possible. One is to restrict Corynanthe to C. paniculata, resurrect Pseudocinchona sensu Chevalier (1909
, 1926
), and maintain the current circumscription of Pausinystalia (Stoffelen, Robbrecht, and Smets, 1996
). The second is to sink Pausinystalia in Corynanthe (the older name) and retain all the described taxa within the Corynanthe lineage without infrageneric subdivision; this would maximize the nomenclature stability, but would make Corynanthe rather heterogenous morphologically. Third is to include Pausinystalia in Corynanthe sensu Stoffelen, Robbrecht, and Smets (1996)
and recognize Corynanthe paniculata, Pseudocinchona, and Pausinystalia at the subgeneric level. We favor the first alternative because this does not require any nomenclatural change (maximizing stability) and also reflects the distinctness of these three genera.
Naucleinae sensu Razafimandimbison and Bremer
Nauclea sensu Haviland (1897)
is characterized by a combination of two characters: spindle-shaped stigmatic lobes with the receptive areas restricted only to the base and multiple fruits. Ridsdale's narrow generic concept of Nauclea includes only the species with Y-shaped placentae attached to the upper third of the septum and multiple fruits (Ridsdale, 1975
, 1978a
). Consequently, he reinstated the African genus Sarcocephalus and recognized the new genus Ochreinauclea to accommodate Nauclea, both maingayi and N. missionis, which Ridsdale thought to have pseudomultiple fruits.
Nauclea sensu Ridsdale (1975
, 1978a)
, represented by four species in our analysis, is resolved with high support (JK = 99, BS = 96) as monophyletic. The Y-shaped placenta appears to be the single morphological synapomorphy of Nauclea sensu Ridsdale. Therefore, we will maintain the present circumscription of Nauclea (Ridsdale, 1978a
).
The two species of Sarcocephalus sensu Ridsdale (1975)
analyzed here form a highly supported monophyletic group (JK = 100, BS = 99). Both Nauclea sensu Ridsdale and Sarcocephalus have multiple fruits, but this latter is distinct from the former by its small deltoid stipules with obtuse to slightly notched apices, calyx lobes prolonged by small appendages, discoidal placentae rather than large stipules and corolla lobes without appendages, and Y-shaped placentae as in Nauclea sensu Ridsdale (1975)
. We therefore maintain the generic status of Sarcocephalus.
The generic status of the East African monotypic genus Burttdavya is maintained based on its linear-oblong placentae and free indehiscent fruits without exocarps (Ridsdale, 1975
); Burttdavya is placed with weak support (JK = 50, JK < 50%) as sister to Sarcocephalus.
The Asian genus Ochreinauclea was not included in our combined analyses because O. maingayi appeared in two different clades in the nuclear and chloroplast-based phylogenies (see Razafimandimbison and Bremer, 2002
). However, it was included in the ITS, rbcL, and trnT-F analyses. In the ITS tree, O. maingayi was placed within Naucleinae sensu Razafimandimbison and Bremer with high support as closely related to Neolamarckia cadamba. This relationship is corroborated by the occurrence of both of the two morphological synapomorphies (spindle-shaped stigmas and receptive areas restricted to the base of the stigmatic lobes) for Naucleinae in Ochreinauclea. However, the rbcL (Razafimandimbison and Bremer, 2002
) and trnT-F (Fig. 1) trees both placed Ochreinauclea in the Neonauclea clade. We have interpreted Ochreinauclea maingayi as a nothospecies, i.e., a hybrid between Neolamarckia and one taxon from the Neonauclea clade (Razafimandimbison and Bremer, 2002
). Ochreinauclea is characterized by multiple fruits, a feature also present in Nauclea and Sarcocephalus, and winged seeds, a feature commonly found in the Neonauclea clade, but absent in Naucleinae. Neolamarckia and Ochreinauclea both have conical terminal vegetative buds. The combination of indehiscent, multiple fruits and winged seeds together are unique for Ochreinauclea and rare in Rubiaceae. The Article H.5 in the International Code of Botanical Nomenclature (Saint Louis Code) 2000 (Greuter et al. 2000
: p. 107) states that "the appropriate rank of a nothotaxon is that of the postulated or known parent taxa." Therefore, like Neolamarckia, the generic status of Ochreinauclea can be maintained. Its unique features make Ochreinauclea easy to distinguish from the other Naucleeae genera.
Neolamarckia is an Asian genus, which was originally described by Richard (1830)
as Anthocephalus based on a nonexistent specimen of Sonnerat (see discussions in Razafimandimbison [2002]
and references therein). Neolamarckia is diagnosed by having branched placentae attached to the upper third of the septa and ovaries that in their upper parts are split into two or four locules, but their lower parts are divided into two locules by the false septa in the upper parts. The combined molecular-morphological tree (Fig. 3) placed Neolamarckia with high support (JK = 100, BS = 99) as sister to a clade containing Burttdavya, Nauclea sensu Ridsdale, and Sarcocephalus; this suggests that Neolamarckia is a distinct lineage and thus its generic status could be maintained.
Breoniinae
Breoniinae contains all the investigated members of the Malagasy Naucleeae (Breonia, Gyrostipula, and Janotia) and the Afro-Malagasy genus Breonadia.
Breonadia can easily be recognized by its verticillate leaves and intrapetiolar stipules. Our results placed Breonadia as basal and sister to a highly supported clade (JK = 92, BS = 86) containing Breonia, Gyrostipula, and Janotia. This shows that Breonadia is not closely related to Adina sensu Haviland, as previously suggested by Haviland (1897)
, but instead its position is consistent with Ridsdale's decision to separate Breonadia from Adina s.1. Its basal position and unique features suggest that its generic status could be maintained.
Breonia was originally described by Richard (1830)
as an endemic genus from Madagascar. Ridsdale (1975)
, in his revision of the African and Malagasy Naucleeae, adopted a narrow circumscription of Breonia, excluding Breonia decaryana and B. keliravina, both of which have flattened terminal vegetative buds, calyx tubes of adjacent flowers partly fused, and carpels containing a single ovule per locule. These two species were recognized as a separate genus Neobreonia (Ridsdale, 1975
). Recently, a revision of Breonia has been completed (Razafimandimbison, 2002
). It contains 20 species and Neobreonia decaryana has been reincluded; this decision was based on the results of our phylogenetic analyses of the ITS and rbcL and morphological data sets (Razafimandimbison and Bremer, 2002
) and is further supported by the present study.
Capuron (1972)
originally described two new species of Neonauclea from Madagascar, N. foveolata and N. macrostipula, mainly because of their dehiscent capsular fruits and bicornate seeds. A. Homolle (unpublished data, Laboratoire Phanérogamie, Paris) considered these two species of Neonauclea to be better treated in Adina. Leroy (1975b)
strongly argued that the characters observed in these two species (axillary inflorescence, absence of interfloral bracteoles and calyx appendages) did not fit either Adina (presence of interfloral bracteoles) or Neonauclea (inflorescence terminal and calyx lobes prolonged by long appendages). As a result, he classified N. foveolata and N. macrostipula in the separate genera Gyrostipula, with two species (G. comoriensis and G. foveolata), and Janotia, with one species (J. macrostipula).
In the strict consensus tree of the combined molecular-morphological data (Fig. 3), Gyrostipula appears monophyletic with high support (JK = 94, BS = 92). This is corroborated by a number of autapomorphies: convolute, red, long terminal vegetative buds, calyx tubes densely pubescent, and red placentae persistently attached to the septa after the fruits dehisce and release the mature seeds. Gyrostipula and Janotia are resolved as more closely related to each other than they are to the other members of Breoniinae, a relationship also corroborated by one morphological synapomorphy, ovules attached side by side to the base of the placentae. Janotia can easily be distinguished from Gyrostipula by its terminal vegetative buds with semi-persistent, complanate large foliaceous stipules and long filiform calyx lobes. We here retain both genera based on the principle of ease of identification (Backlund and Bremer, 1998
).
Adininae sensu Razafimandimbison and Bremer
Adina sensu Haviland, consisting of nine species (Adina cordifolia, A. microcephala, A. multifolia, A. oligocephala, A. pilulifera, A. polycephala, A. racemosa, A. rubella, and A. rubescens), is characterized by filiform to clavate interfloral bracteoles, valvate (though sometimes apically subimbricate) corolla lobes, and free, capsular fruits with the calyx remnants falling off together with the central axis. Ridsdale (1978a)
considered Haviland's Adina as a heterogeneous assemblage of taxa; he adopted a much narrower generic concept for Adina by restricting it to the three Asian species A. pilulifera, A. rubella, and A. dissimilis, which are diagnosed by having loosely defined terminal vegetative buds surrounded by spreading stipules. Ridsdale subsequently recognized all of the other species in separate genera: Adina fagifolia as Adinauclea, A. microcephala as Breonadia; A. cordifolia as Haldina; A. oligocephala as Metadina; A. polycephala as Khasiaclunea; A. multifolia and A. rubescens as Pertusadina multifolia and P. eurhyncha, respectively, and A. racemosa as Sinoadina. He stated that only few small characters separated these minor genera. Adinauclea has spathulate to spathulate-clavate interfloral bracts and valvate but subimbricate at the apex corolla lobes. Ridsdale (1978a)
argued that these features suggest that Adinauclea is more closely related to Adina than it is to Neonauclea. However, Adinauclea also has flattened terminal vegetative buds, a feature absent in Adina and Ridsdale's segregate genera but commonly found in Neonauclea and its satellite genera. Metadina can be easily diagnosed by having numerous (20–30) heads arranged in a compound thyrse. Pertusadina is characterized by a combination of conical terminal vegetative buds, axillary inflorescences, and capsular fruits. Finally, Sinoadina is characterized by terminal inflorescences with 7–11 heads arranged in a simple thyrse.
Our results are largely inconclusive for assessing the relationships and generic limits among Adinauclea, Metadina, and Pertusadina. For now, we maintain their generic status. Adina sensu Ridsdale (1978a)
is resolved with strong support (JK = 94, BS = 88) as a monophyletic group, supporting its generic status. Haldina and Sinoadina are left unresolved as separate lineages within a collapsed clade containing Naucleinae, Breoniinae, and Adininae; we also continue to maintain their generic status. Adding one more data set from a low-copy nuclear gene, such as phytochrome B, which has been shown to be useful for resolving relationships among and generic limits of many groups (e.g., Simmons et al., 2000
and Mathews, Tsai, and Kellogg, 2000
), would perhaps be helpful in resolving relationships among Adina sensu Haviland (1897)
and rigorously evaluate their generic status.
Neonauclea sensu Merrill (1915)
was a large Asian genus including about 70 species and characterized by terminal inflorescences and free capsular fruits. Merrill (1920)
recognized Neonauclea strigosa as the separate genus Myrmeconauclea because of its unique features (pseudomultiple fruits and seeds with very long ventral wings). Ridsdale accepted Myrmeconauclea, but still viewed Neonauclea sensu Merrill as an overly heterogeneous assemblage of taxa; he adopted a new circumscription for Neonauclea by restricting it to the species with corolla lobes prolonged by obtrigonal to spathulated, deciduous appendages. He recognized the following genera as separate genera: Neonauclea bernardoi as Ludekia, N. oligocephala as Khasiaclunea, and N. zeylanica as Diyaminauclea. Ludekia is distinct from the rest of Naucleeae genera by its globose stigmatic lobes with 7–9 prominently longitudinal ridges.
Ludekia borneensis, Myrmeconauclea strigosa, Neonauclea brassii, N. clemensii, N. forsteri, and N. longipedunculata constitute a strongly monophyletic group (JK = 94, BS = 89), corroborating their close relationships. However, the relationships between Ludekia and Neonauclea sensu Ridsdale are unresolved. Myrmeconauclea is nested within Neonauclea sensu Ridsdale, represented here by four species, indicating that Neonauclea sensu Ridsdale (1989)
is paraphyletic and needs to be recircumscribed. Two alternative solutions are possible. One would be to merge Ludekia and Myrmeconauclea in Neonauclea, making this latter as monophyletic; this would maximize taxonomic stability and minimize nomenclatural changes, but would also make Neonauclea rather heterogeneous morphologically. A second would be to retain the two well-defined genera (Ludekia and Myrmeconauclea) as separate genera; this would require then considerable splitting of the paraphyletic Neonauclea s.s. into several small genera, which might also cause a lot of nomenclatural changes. The first alternative is a more logical approach, but we have avoided recognizing morphologically heterogeneous genera. Also, we have investigated only four (of 65) species Neonauclea sensu Ridsdale (1989)
. A molecular phylogenetic study using fast-evolving markers and including several species of Neonauclea sensu Ridsdale is needed to specifically address the limits of the genera within Neonauclea clade. For now, we continue to maintain the generic status of Lukedia, Myrmeconauclea, and Neonauclea.
In conclusion, phylogenetic studies of Naucleeae s.l. based on the four data sets (three molecular, ITS, rbcL, and trnT-F, and one morphological) further confirm the monophyly of Naucleeae s.l., also including Cephalanthus, Corynanthe, Mitragyna, s.l. (including Hallea), Pausinystalia, and Uncaria. Anthocephalidae, Mitragynae, and Uncariae all sensu Haviland are highly supported as monophyletic; however, Cephalanthidae sensu Haviland is shown to be paraphyletic. Naucleinae sensu Ridsdale (1975)
is moderately supported as monophyletic. Naucleinae sensu Ridsdale and Neolamackiinae sensu Robbrecht (1994)
form a strongly supported monophyletic group. Our results neither support nor reject the monophyly of Adininae sensu Ridsdale (containing Adininae sensu Razafimandimbison and Bremer and Breoniinae). We disregard Haviland and Ridsdale's subtribal limits and recognize a new subtribal classification that contains seven morphologically distinct subtribes: Cephalanthinae, Mitragyninae, Uncarinae, Corynantheinae, Naucleinae, Breoniinae, and Adininae. Cephalanthinae is resolved as basal and sister to the rest of Naucleeae; however, the relationships among the other major lineages remain largely unresolved. Finally, we retain the generic status of the following 22 genera: Adina, Adinauclea, Breonadia, Breonia, Burttdavya, Cephalanthus, Gyrostipula, Haldina, Janotia, Ludekia, Metadina, Mitragyna, Myrmeconauclea, Nauclea, Neolamarckia, Neonauclea, Ochreinauclea, Pausinystalia, Pertusadina, Sarcocephalus, Sinoadina, and Uncaria. Corynanthe is restricted to its type species, C. paniculata. Hallea is reincluded in Mitragyna. The African genus Pseudocinchona is reestablished. We were unable to assess the relationships among Adinauclea, Adina, Metadina, and Pertusadina due to lack of resolution. Finally, all accepted genera and their synonymies are given in Table 5.
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4 Author for reprint requests (Fax: 46 18 471 6457; sylvain.razafimandimbison{at}ebc.uu.se)
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