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Volume 94 Annals Number 1 of the 2 () ¥issourt BOTANICAL Missouri MAY 1 1 2007 Botanical GARDEN LIBRARY Garden
A PHYLOGENETIC ANALYSIS OF Mary E. Endress, Raymond W. J. M. van der ALYXIEAE (APOCYNAC EAE) : a „ b 1 10 BASED ON RBCL, MATK, TRNL peur onn Powell David bo idi INTRON, TRNL-F SPACER Ylva-Maria Zimmerman," and Victor A. Albert“ SEQUENCES, AND
MORPHOLOGICAL CHARACTERS!
ABSTRACT
Within Rauvolfioideae (Apocynaceae). genera have long been assigned to tribes based mainly on only one or two superficial fruit and seed characters. Taxa with drupaceous fruits were included in Alyxieae. To elucidate relationships within Alyxieae, we analyzed phylogenetically a data set of sequences from four plastid DNA regions (rbcL, M, tral. intron, and trnL-F intergenic d and a morphological data set for 33 genera of Apocynaceae, inc luding. representatives of all genera previously included in Alyxieae and two non-Apocynaceae species. Results of parsimony analysis indicate that Alyxieae as previously delimited are polyphyletic, with most genera falling into two main clades. The Alyxia clade includes seven genera:
lyxta Banks ex R. Br., Lepinta Decne., Lepintopsis Valeton, Pteralyxia K. Schum., and CONI; Desf. together with Plectaneta Thouars. (earlier included in Plumerieae) and Chilocarpus Blume (earlier included in Chilocarpeae). The Vinca clade includes eight genera: Cabucala Pichon, Petchia Livera, Rauvolfia L. ia G. Don. Vinca L., Neisosperma Raf.,
Ochrosia Juss.. and Kopsia Blume. Vallesia Ruiz & Pav. and Anechites Griseb. are not related to either clade and come out as sister to Aspidosperma Mart. & Zucc. (Aspidospermeae) and Thevetia L. (Plumerieae), respectively. The fruit and seed
! We wish to thank the following persons who provided pleni material or DNA samples: A. Assi, Paul Berry, F. Billiet, pia Bremer, V. Ferreira, P. Garnock-Jones, P. Kessler. I. Koch. A. Leeuwe eia rg, S. Liede, ^ Ne ill.] R. Omlor, I q lignat,
„S. Tucker, and S. Zona. For technics da ssistance and photographie help with s pollen
>
révost, G. Ro mero, A. Spech contribution sincere thanks are die to Elisabeth Grafström and AS Hellbom, Palynological Laboratory, Swedish Museum
of Natural History. Stockholm, and Bertie Joan van Heuven and Wim Star, Nationaal Herbarium Nederland, Leiden. This study was supported by a grant from the Helge Ax:son Johnson d and the Swedish Foundation for International Cooperation Research (STE `) to B. Sennblad.
? Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland. mendress@systbot. unizh.ch.
zh. '*Naltionaal Herbarium Nederland, P.O. Box 9514, 2300 RA Leiden, The Netherlands. ‘Laboratoire, Dynamique de la Biodiversité, UMR 5172, Université Paul Sabatier, 31062 Toulouse, Cedex 9, France. ^ Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond Surrey, TW9 3DS, United Kingdom.
Stockholm Bioinformatics Center, Stockholm University, AlbaNova Research C enter, 8E-10691 Stockholm, Sweden. College of Veterinary Medicine, University of Illinois, Urbana, Illinois 60801. U.S.A. " National Tope ba Pone al Garde n, 3530 Papalina Road, Kalaheo Kauai, Hawaii 96741, U.S.A. "Institut fiir vie und Morphologie. Universität Witten/Herdecke, la Str. 10 D-58448. Witten. Germa
e Natural History Museum, University of Oslo, Box 1172 Blindern, NO-0318 Oslo, Norway T Siwert Nilsson passed away unexpectedly before the manuscript was completed. We los! boil an excellent collaborator
and a dear friend. We dedicate this paper to him.
ANN. Missourt Bor. GARD. 94: 1-35. PUBLISHED ON 26 APRIL 2007.
Annals of the Missouri Botanical Garden
characters previously used to demarcate Alyxieae are homoplasious, as are other morphological characters such as style head
structure and syncarpy versus apocarpy. Conversely, pollen morphology. which has not previously played much of a role in
tribal delimitation, was shown to be the most useful morphological character for delimiting Alyxieae from other tribes of
Rauvolfioideae. Key words:
Alyxieae, Apocynaceae, classification, matk, morphology, phylogeny, pollen, rbcL, systematics, tral. trnb-F.
Tribal
referred to as Plumerioideae in the older literature)
delimitation in Rauvolfioideae (usually
has previously been based on fruit and seed characters (Schumann, 1895; Pichon, 1949a, 1949c; Ly, 1986; Leeuwenberg, 1994a). One reason that fruit and seed characters have been so frequently used in classifications and keys is that they are readily observed, permitting easy recognilion of many genera. The j
classifications is that the flowers of many Rauvolfioi-
other main reason for the fruit-based tribal
deae tend to be superficially similar. Many are
relatively small with a whitish, salverform corolla,
and there are few obvious distinguishing floral characters useful for differentiating tribes in this subfamily. The most detailed studies of Rauvolfioi- deae were those of Pichon (1948a, 1948b, 1949a, 1950b), who published extensively on the family. His classificalion was a greal improvement over ils predecessors. lts main weakness was that tribal delimitation was based mainly on a single fruit character. He split the rauvolfioid tribes into two main groups, depending on whether the deeper layers of the mesocarp were fleshy or dry. The group with a fleshy inner mesocarp was divided into five tribes: Carisseae, Ambelanieae, and Macoubeeae comprised those taxa with indehiscent berries and Chilocarpeae and Tabernaemontaneae included those taxa with fruit consisting of dehiscent follicles with arillate seeds. In the group with a dry mesocarp, he recognized three tribes. Two of them are characterized by dehiscent fruits: Alstonieae (Plumerieae sensu Leeuwenberg, 1994a), in which the fruit consists of a pair of follicles. and the monotypic Allamandeae, in which the fruit is a spiny unilocular capsule. The last tribe, Rauvolfieae (Alyxieae sensu Leeuwenberg, 1994a), contained all taxa in which the fruit is an indehiscent drupe with a stony endocarp. It is the relationships within this group that are the focus of this paper.
Pichon (1949a) recognized five subtribes within his Rauvolfieae: Rauvolfinae (including Cabucala Pi- chon, Petchia Livera, Rauvolfia L., and Podochrosta Baill.), Alyxiinae (including Alyxia Banks ex R. Br.. Lepinia Deene., and Lepiniopsis Valeton), Ochrosiinae (including only Ochrosia Juss.), Vallesiinae (including Ruiz & Blume
Condylocarpinae (including Rhipidia Marker. and
Vallesia . and
Pav. and KO
Cond ylocarpon Desf.).
Pichon. included two genera
as incertae sedis: Anechites Griseb. and Notonerium
Benth. Notonerium has since been shown to belong to Boraginaceae (Crisp, 1983).
In his classification from 19% a, Leeuwenberg maintained Pichon’s (104% a) circumscription of Rau- volfieae as well as the five subtribes included there but gave no insight into the delimitation of. the subtribes, stating only that the relatively slight its subtribes are not easily
differences between
described in a concise way. The only differences between Leeuwenberg's (1994a) and Pichon’s (19494) tribal eireumscriptions are that Leeuwenberg changed the name of the tribe to Alyxieae, put Podochrosta into
synonymy under Rauvolfía and Rhipidia in Condylo-
carpon (following Fallen, 1983b), and included Anechites in Condylocarpinae, although Fallen
(1983a) had suggested earlier that a position closer to taxa previously included in Cerbereae (Cameraria L.. Cerbera L., Cerberiopsis Vieill. ex Pancher & Sebert, Thevetia L.. and Skytanthus Meyen) was more appropriate. More recently, Leeuwenberg (1997) pul Cabucala into synonymy under Petchia.
In addition to the taxa mentioned above, there are four other genera characterized by drupaceous fruits: Cerbera, Thevetia, Cerberiopsis, and Cameraria. These, together with Skytanthus, with follicular fruits, were split out of Rauvolfioideae and treated by Pichon (1948b) as a separate subfamily, Cerberoideae. The characters he used for delimitation of this subfamily are ambiguous. Leeuwenberg (1994a) recognized the
group as defined by Pichon, but at the tribal level as
—
Cerbereae. Morphological studies by Fallen (1985 suggested a close relationship between Cerbereae and Allamanda l., the sole genus placed in Allamandeae by Pichon (1949a) and Leeuwenberg (1994a). Studies based on molecular or combined morphological and molecular data (Endress et al., 1996; Sennblad & Bremer, 1996, 2000, 2002) indicated that the genera previously included in Cerbereae do form a natural group (see Potgieter & Albert, 2001, for a different opinion) and that they are only a part of a larger group that includes not only Allamanda, but also Plumeria L. (usually included in the Plumerieae: Rauvolfioi- deae). An analysis of Cerbereae is not the aim of this study, although some representatives from that tribe are included in our analyses.
Using fruit characters to delimit tribes in Rauvol- fioideae is appealing because it allows taxa to be
easily categorized and keys to be constructed.
Volume 94, Number 1 2007
Endress et al. 3 Phylogenetic Analysis of Alyxieae
other characters of these do not
indicate the same patterns of relationships. Phyloge-
However, taxa netic analyses of mainly molecular data have shown that these fruit- and seed-based classifications are considerably
more artificial than previously sus-
pected. An rbcL analysis by Sennblad and Bremer (1996) indicated that Catharanthus G. Don (with dry
dehiscent follicles and included in Plumerieae
—
Was more closely related to taxa previously placed i
~
al Alyxieae sensu Leeuwenberg (1994a) than to other Plumerieae. In larger studies (Sennblad. ; Sennblad & Bremer, 2000, 2002). Catharanthus and Vinca L. formed a well-supported clade together with Rauvolfia, Ochrosia, and Kopsia, which have fleshy
e S
í
drupes. In the same study, Chilocarpus Blume, which has always been thought to be most closely related to Carisseae, formed a strongly supported clade with Alyxia and Lepinia (Alyxieae), confirming results of a strongly supported Chilocarpus—Alyxieae clade reported previously by Civeyrel (1996) and van der Ham et al. (2001). Pichon (1949a) already realized that Geissospermum Allemao (with indehiscent fruits and seeds embedded in pulp) is probably the nearest relative of Aspidosperma Mart. Zucc. (with dry dehiscent follicles and wind-dispersed seeds with a diaphanous wing), a position supported by Potgieter Albert (2001) and Simões et al. (2007). In and Albert (2001) found that Vallesia (with drupaceous fruits and seeds embedded in juicy pulp) is closely related to Haplophyton A. DC.
(with dry dehiscent follicles and wind-dispersed Such that fruit characters in Apocynaceae are evolutionarily plastic
and
addition, Potgieter
comose seeds). results indicate in response to selective pressures for adaptations associated with wind or animal dispersal.
The most recent classifications of Apocynaceae s.l. (Endress & Bruyns, 2000; Sennblad & Bremer, 2002) attempted. to rectify some of these anomalies. In the classification of Endress and Bruyns (2000), Anechites was moved to a newly defined Plumerieae (including Cerbereae sensu Leeuwenberg (1994a) as well as Allamanda), a position suggested by Fallen (19832), and Vallesia was included with Aspidosperma, Geis- sospermum, and Haplophyton in a newly defined Alstonieae. The remainder of Alyxieae (sensu Leeu- wenberg, 1994a) was split into two tribes, Alyxieae and Vinceae, based on molecular results as well as additional. morphological characters. Alyxieae sensu
Endress and Bruyns
—
2000) included seven genera: Alyxia, Pteralyxia K. Schum., Lepinia, Lepiniopsis, Plectaneia Thouars, Condylocarpon, and Chilocarpus.
inceae Walter, Catharanthus, Vinca, Rauvolfia, Petchia, Kopsia, Neisosperma Raf., and Ochrosia. Rhazya Decne. was considered to be
synonymous with Amsonia, and Cabucala with Petchia
included Amsonia
(following Leeuwenberg, 1997). Simões et al. (2007)
treated Amsonia incertae sedis and
transferred Laxoplumeria Markgr.,
as a genus Tonduzia Pittier, and Kamettia Kostel. to Vinceae, bringing the total number of genera in the tribe up to 10.
The classification of Sennblad and Bremer (2002) proposed a new system that is compatible with traditional Linnaean nomenclature but uses a variant of the definitions used in phylogenetic nomenclature Although
they do not provide lists of included genera, their
Y classifications.
AO
to improve the stability
definitions of the tribes containing traditional Alyx- ieae genera are completely congruent with those of Endress and Bruyns (2000). The aim of this study is to cladistically evaluate and Vinceae as circumscribed by Endress and Bruyns (2000) in comparison
the
Alyxieae
with previous classifications, to re-
examine usefulness of fruit and seed characters for tribal delimitation within Rauvolfioideae, and to discover new morphological characters that have hitherto received little attention in classification of
this subfamily but show phylogenetic potential.
MATERIALS AND METHODS TAXON SAMPLING
The ingroup taxa were chosen to include represen- tatives of all genera of Alyxieae (sensu Leeuwenberg, 1994a), as well as other putatively related genera. The oulgroup taxa are one genus each of Loganiaceae and Gelsemiaceae, which several studies (Bremer & Struwe, 1992; Chase et al., 2000; Soltis et al., 2000) have demonstrated to be closely related to Apocynaceae (Appendices 1, 2). Other more ron focused studies on Gentianales (Struwe et al., 1996; Backlund et
al., 2000) ra uu indicated that Loganiaceae and
1993; Savolainen et al.,
Endress et al.,
Gelsemiaceae are the closest families to Apocyna- ceae. For the morphological analyses, we omitted the outgroups altogether because, in preliminary analyses, one or the other of these genera was embedded
different portions. of the ingroup due to obvious parallelisms of certain characters; we arranged the morphological tree with the same group sister to the
rest as in the molecular results.
FLORAL STRUCTURE
Fixed flowers at or near anthesis (only buds were avallable for Lepintopsis) were dehydrated in an alcohol-xylene series, embedded in paraplast, cut with a rotary microtome at 10 um, and stained with safranin and astra blue. For SEM studies, material was critical-point dried and then sputter-coated with
gold.
Annals of the Missouri Botanical Garden
POLLEN MORPHOLOGY
Pollen material was sampled from the 1 BISH, BR, COL, G, L, P, PTBG, QCA,
UB, WAG, and Z. Pollen studies were carried out in
herbaria: Leiden and Stockholm. For light microscopy (LM), pollen material was acetolyzed (except for Condylo- carpon and Vinca), mounted in glycerine jelly, and 10 pollen grains were
sealed with paraffin. Generally,
measured for polar axis (P) and equatorial diameter
(E). For SEM, pollen was sputter-coated with gold and examined with a JSM 5300 or JSM 6300 scanning
electron microscope (JEOL, Tokyo). Frozen sections
were made using an Ames Tissue-TEK Cryostat. For
transmission electron microscopy (TEM), unaceto-
lyzed material (whole anthers) was embedded in Spurr
resin or 3/7 Epon, sectioned with a LKB Ultrotome II or V, poststained with uranylacetate and lead citrate, and examined with a Zeiss 10, a JEOL 100-5, or a JEM 1010. Terminology is according to Punt et al. (1994).
OTHER MORPHOLOGICAL AND CHEMICAL CHARACTERS
Information on fruit and seeds were taken from observations of herbarium specimens as available. Several fruit and seed characters were taken from the literature, as were data on the presence of laticifers and intraxylary phloem (Solereder, 1892; Schumann, 1895; Valeton, 1895; Degener, 1946; Pichon, 1947a, 1947b, 1948a, 1948b, 1948c, 1948d, 1949a, 19400. 1949¢, 1950a, 1950b, 1950c, 1952; Gensel, 1969; Markgraf, 1971, 1976, 1979; Markgraf & Huber, 1975; Corner, 1976; 1980; Leeuwenberg & 1980; Rogers, 1986; Rudjiman, 1986; 1987; Metcalf & Chalk, 1989; Rosatti, 1989; Wagner et al., 1990; Sévenet et al., 1994; Forster & Williams, 1996; Omino, 1996; Leeuwenberg, 1997; Sidiyasa, 1998; Lin & Bernardello, 1999). Chemical data were taken from the literature (Johns et al., 1968; 1970, 1989; Coppen & Cobb, 1983; Kisakürek et al., 1983; Homberger & Hesse, 1984;
1987; Endress et al., 1990; Wagner et al., ; Zhu et al., 1990; Attaurrahman et al., 1989, Arambewela & Ranatunge, 1991; Jensen, 1992; 1994; Zeches et al., 1995; Kam et al.,
Conn, Leenhouts,
Pagen,
Hegnauer,
1991: Sévenet et al..
1997).
MOLECULAR METHODS
16 of matK, and 11 of
intron and trnl-F intergenic
Nine new sequences of rbcL, the n
produced for this study;
spacer were the other sequences were published previously (Appendix 2). Total DNA was extracted from fresh leaves, silica gel-dried material (Chase & Hills, 1991), or herbarium material using the methods of Saghai-Maroof et al. (1984) or modified
Doyle and Doyle (1987). For the latter. DNA samples
were purified by ultracentrifugat
in CsCl-ethidium bromide gradients (1.55 m Additional 1 (Qiagen,
cases with
using the QlAquick PCR purification Valencia, California) was performed in problematic polymerase chain reaction (PCR) ampli- Double- stranded DNA was amplified with PCR primers for rbcL from Fay et al. (1998); the trnL intron and trn L-F
fication using the manufacturer's. protocol.
intergenic spacer (hereafter, trnL-F) were amplified using the c and f primers of Taberlet et al. (1991): matk primers were those of Endress et al. (1996) and Johnson and Soltis (1994). Direct sequencing of PCR products was performed using the PCR primers plus internal sequencing primers. For rbcL, the internal primers were those of Fay et al. (1998); for trnL-F, we Taberlet et al. (1991); and for matK, we designed two new internal primers: 734F, | 5'-ATGTATGTGACTACGAATCA-3' 829R, 5'-ACTTTCTATTTTTCCATAGA-3'
ber of cases, we also used the internal sequencing
used the d and e primers of
and
In a num-
primers as PCR primers to amplify shorter products.
For sequencing, we used either the Dye Deoxy Terminator Cycle Sequencing or Big Dye kits of Applied Biosystems (ABI; Warrington, Cheshire,
United Kingdom). Sequencing reactions were carried out directly on the cleaned PCR products and run on an ABI 277a automated sequencer at Kew following the manufacturer's protocols.
CLADISTIC ANALYSES
The data matrix comprised four submatrices: each
the three
^ © —
DNA regions plus morphology. The morphological submatrix (Appendix 3) comprises 54 characters from floral, fruit, vegetative, and pollen morphology and phytochemistry (Appendix 4). All
PAUP* 4.0b10
(Swofford, 2002). Heuristic searches were performed
analyses were performed using with all characters given unit weight (Fitch parsimony; Fitch, 1971), separately before their joint combined analysis. Each
1000
random taxon entry, the subtree pruning
and each submatrix Was analyzed
of the searches used the following settings: replicates of re-grafting (SPR) branch swapping algorithm, and MULTREES on (saving multiple equally parsimonious
All the
shortest trees were then collected and used as starting
trees) but holding only 10 trees per replicate. trees for a search with a 25,000-tree limit. If the tree limit was reached, then swapping was allowed to continue until all 25,000 trees were swapped to completion.
BP) (Felsenstein, 1985)
were calculated with 500 replicates on each of the
Bootstrap percentages
—
submatrices as well as on the two combined matrices
Volume 94, Number 1 2007
Endress et al. 5 Phylogenetic Analysis of Alyxieae
(molecular combined and molecular/morphological We
branch swapping
used the following settings: SPR and MULTREES on, holding only All other settings were those of the PAUP* 4.0. This
produces results statistically indistinguishable from
combined).
10 trees per step. standard defaults of strategy other, more thorough bootstrap protocols (Salamin et al., 2003). We apply the following arbitrary scale in our discussion: 5096—7496, weakly supported; 7596— 84%, 85% - 100%, strongly supported. Alignment for rbcL and matK was a simple the had only a few easily characterized. insertions/ deletions (indels). irnL-F, alignment of Potgieter and Albert (2001) and added
moderately supported;
matter: former had no length variation, and the
atter For we started with the
the additional taxa needed for this analysis, which required adding a few more insertions; we did adjust their alignment in places following Kelchner (2000). We analyzed the trnL intron and trnL-F. intergenic spacer in a single analysis (which can be considered
“non-coding” because there is only about a 30 bp
region of the trnL exon included); this region, termed Irnl-F, number of variable sites is the lowest and, even when
is composed of two unrelated parts, but the combined, these produced a highly unresolved strict consensus tree. We do report statistics for these two regions separately (Appendix 2) but consider results only for the two combined.
Incongruence of different regions of plastid DNA
me
would be unexpected because recombination is
unknown in the generally uniparentally inherited plastid genome. Several tests for combinability have been developed, but we have not used any of them in this paper. Such tests have proven to be unreliable indicators of incongruence (Reeves et al., 2001), so we altach no particular significance to results of these tests but instead prefer to look for cases of strongly supported and incongruent patterns of relationships. Differences in relationships are to be expected with different matrix components simply due to sampling effects where there are too few variable characters to obtain clear patterns. If, however, there are only a few characters in a matrix, such as is the case here with the morphological characters, differentiating between sampling error and incongruent patterns is extremely difficult. estimates of relationships deviate from those based on DNA data, particularly for the Vinceae, but the small
We note that the morphologically based
number of morphological characters (only 54) does not permit us to say whether the differences between
molecular and morphological patterns are evidence of
true incongruence. Character state distributions. of selected morpho- logical characters were individually mapped onto the
total evidence tree (Figs. 10, 11) using MacClade 4.0
(Maddison & Maddison, 2000) to illustrate character evolution and compare the usefulness of characters that have previously been used in delimitation of
Alyxieae.
RESULTS MORPHOLOGY
Analysis of the morphological matrix produced 31 trees in three islands (18, 4, and 9 trees) of equally parsimonious trees, each of 246 steps with a consis- teney index (CI) of 0.37 and a retention index (RI) of 0.62. A strict (Fig. 1; numbers below the branches are BPs) shows that these morphological data, and the three islands place
consensus tree of all three islands
he position of several genera is unclear with
them in different relative positions to the clades that The glabra
are consistently resolved in all three islands. positions of Vallesia antillana Woodson, V.
Cav.) Link,
cathartica L., Anechites nerium (Aubl.) Urb.,
and the Plumeria clade (Allamanda
—
Plumeria rubra L., and Thevetia peruviana (Pers.) K. Schum.)
consistent in all three islands, as is a clade
are composed of all remaining taxa. Within the last, Acokanthera oblongifolia (Hochst.) Codd, A. oppositi-
folia (Lam.) Codd, er laxum (Benth.) Pichon, Durand & H. Pleiocarpa mutica iu Tabernaemontana divar- icata (L.) R. Br. & Schult., and T. pandacaqui Lam. occupy different positions in each the consistently resolved clades: (1) L.) R. Br. to Rhazya stricta Decne. and (2) Alyxia oblongata Domin and A. ruscifolia R. Br. to Nerium (Fig. 1). The other conspicuously differ- ently placed group is that composed of Amsonia ciliata Walter, and Catharanthus roseus (L.) G. Don + Vinca major L. and V. minor L., which in two of the islands (18 and 9
Picralima nitida (Stapf) Durand,
ex Roem.
three islands with respect to
^ © —
Alstonia scholaris
oleander L.
—
A. tabernaemontana Walter, Rhazya stricta,
tree islands) are a clade but in the other island form a grade. Clades that receive moderate to strong BPs are
the following: Neisosperma nakatana (Koidz.)
Fosberg & Sachet + Ochrosia coccinea (Teijsm. &
Binn.) Miq. (BP 83), Alyxia oblongata and A. ruscifolia + Lepinia marquisensis Lorence & W. L. Wagner, L. solomonensis Hemsl. and L. taitensis Decne. + Lepiniopsis ternatensis Valeton and L. trilocularis Markgr. + Pteralyxia kauaiensis Caum
and P. macrocarpa (Hillebr.) K. Schum. (BP 97; the last two genera with BP 96; Kibatalia gitingensis (Elmer) Woodson + Mascaren- . + Nerium oleander (BP 98;
2tcralima. nitida. +
the Alyxia clade), hasia arborescens the first two genera adi BP 94), Pleiocarpa mutica (BP 84), and Allamanda cathartica
6 Annals of the Missouri Botanical Garden
r- Acokanthera oblongifolia
L- Acokanthera oppositifolia Alstonia scholaris
7 Amsonia ciliata | Amsonia tabernaemontana —— Cabucala polysperma
— Kopsia fruticosa Neisosperma nakaiana T — ——— Petchia ceylanica Rauvolfia vomitoria Catharanthus roseus — Vinca minor fe Vinca major Craspidospermum verticillatum Rhazya Stricta Alyxia oblongata L Alyxia ruscifolia Lepinia marquisensis f 97 Lepinia solomonensis
Lepinia taitensis 96 Lepiniopsis ternatensis 70 95 Lepiniopsis trilocularis Pteralyxia kauaiensis Pteralyxia macrocarpa 58 r- Condylocarpon guyanense L- Condylocarpon isthmicum r- Chilocarpus denudatus I Chilocarpus suaveolens r- Plectaneia stenophylla — E Plectaneia thouarsii Kibatalia gitingensis 94 Mascarenhasia arborescens Nerium oleander Molongum laxum r—- Picralima nitida 84 1— Pleiocarpa mutica r— Tabernaemontana divaricata L— Tabernaemontana pandacaqui Allamanda cathartica oa Anechites nerium Bt Thevetia peruviana Plumeria rubra Vallesia antillana L— Vallesia glabra Aspidosperma parvifolium
98
Figure 1. Strict consensus tree of the three islands found in the morphological analysis. Numbers below the branches indicate bootstrap percentages greater than 50%.
Volume 94, Number 1 2007
Endress et al. 7 Phylogenetic Analysis of Alyxieae
+ Anechites nertum Catharanthus roseus + Vinca major and V.
(BP 72).
—
nearly reached the moderate level
ANALYSIS OF RBCL
Of the 1398 included positions, 244. (18%) were variable and 146 (10%) were potentially parsimony informative. Analysis produced two islands of equally parsimonious trees, one of 39 trees and the other of 16 they had 479 steps with CI (including un- informative positions) of 0.59 and RI of 0.63. The two
islands differ in the relative positions of Neisosperma
ume]
lrees:
oppositifolia (Lam.) Fosberg & Sachet and Rauvolfia mannii Stapf and Chilocarpus suaveolens Blume +
Condylocarpon guyanense Desf. In island one, the
—
atter form a clade with the Kibatalia gitingensis + Mascarenhasia arborescens + Nerium oleander clade, and Neisosperma oppositifolia and Rauvolfia mannii unresolved members of the
are including
Cabucala polysperma (Scott-Elliott) Pichon + Petchia
clade
ceylanica (Wight) Livera + Ochrosia coccinea. In
island two, „ oppositifolia + Rauvolfia
mannii are sister to Catharanthus roseus + Vinca
minor, and 1 1 suaveolens + Condylocarpon
guyanense are unresolved. The strict consensus of
oth islands (Fig. 2) therefore shows these taxa to be unresolved. Supported clades that are also found
the morphological analysis include the following: Allamanda cathartica + Plumeria inodora Jacq. (BP 100) and Anechites nerium + Thevetia peruviana (BP BP 025
ruscifolia + Lepinia taitensis + Lepiniopsis trilocularis
whole Plumeria clade, Alyxia
+ Pteralyxia kauaiensis (the Alyxia clade; BP 99
Picralima nitida sister to Pleiocarpa mutica (BP 100): Catharanthus roseus + Vinca minor (BP 68): and Kibatalia gitingensis + Mascarenhasia arborescens + BP 93). Well- supported clades that are not strongly in conflict with the
Nerium oleander (the Nerium clade;
morphological results include: Aspidosperma
triternatum Rojas Acosta as sister to Vallesia antillana (BP 100); Plectaneia stenophylla Jum. as sister to the Alyxia clade (BP 98), within which the topology is the same as in the morphological results; Amsonia tabernaemontana as sister to Rhaz zya stricta (BP 100); Cabucala polysperma as sister to Petchia ceylanica (BP 100); and Molongum laxum as sister to Tabernaemontana divaricata (B 5). Chilocarpus suaveolens as sister to Condylocarpon guyanense (BP 85) is contradicted in the morphological results by a weak BP 70 for the latter to be sister to Alyxia clade. Weakly supported results not contradicting relation- ships produced by morphology are the Plumeria clade (BP 62: Allamanda cathartica,
Plumeria inodora, and Thevetia peruviana); Picralima
Anechites nerium,
Thevetia peruviana (BP 84).
minor
nitida + Pleiocarpa mutica as sister to the Alyxia clade, plus Plectaneia stenophylla (BP 70); and the Vinca clade (BP 65; Cabucala polysperma, Petchia ceylanica, Ochrosia coccinea, Catharanthus roseus, Vinca minor, Neisosperma oppositifolia, and Rauvolfia
mannii).
ANALYSIS OF MATK
The aligned matK matrix contained 1647 bp, of which 561 (349) were variable and 250 (15%) were potentially parsimony informative. We were unable to mat k: Lepinia, Lepiniopsis, Ochrosia, and Plectaneta.
Anechites, Anal- ysis produced a single, most parsimonious tree of 970 steps with a CI of 0.73 and an RI of 0.59. Patterns of
relationships are nearly identical to those found with
amplify the following taxa for
rbcL, but in general, BPs are higher than with rbcL or irnL-F (Fig. 3). Patterns that were not observed with rbcL include: Chilocarpus suaveolens + Condylocarpon guyanense (BP 100) as sister (BP 99) to the Alyxia clade (BP. 100);
in an isolated position as sister (BP 93) to all but
Alstonia scholaris strongly supported
Aspidosperma triternatum + Vallesia antillana; and Aspidosperma triternatum + Vallesia antillana (BP 82) moderately supported as sister to the the ingroup (BP 97).
rest of
ANALYSIS OF TRNL-F
The aligned trnL-F matrix consisted of 1206 bp (761 bp from the trn L intron and 445 bp from the trnL- F spacer). We were unable to amplify the following irnL-F: Kibatalia G. Don and Lepinia. Analysis produced over 25,000 trees of 460 steps with a CI of 0.78 and an RI of 0.70 (trnL i steps with a CI of 0.76 and an RI of 0.69; trnL-F intergenic spacer: 168 steps with a CI of 0.82 and an RI of
largely
taxa for
0.72) (Fig. 4). Relationships from these two.
non-coding regions are similar to those
estimated from rbcL and matK (Figs. 2, 3). The major noteworthy result (also observed in the matK results,
but which received BP < 50) is a strongly supported
clade (BP 97) composed of Nerium oleander and Mascarenhasia arborescens of the Nerium clade, observed with morphology. Thel, and matK, with
Acokanthera oppositifolia weakly supported as sister (BP 60) also observed but without support with matK. and Allamanda indet. + Plumeria alba Kunth (BP 95), ) A. DC. (the
last two unresolved with respect to the Nerium clade)
Anechites nerium, and Thevetia ahouai (L.
COMBINED MOLECULAR ANALYSES
The combined data set produced 46 equally
parsimonious trees of 1925 steps with a CL of 0.70
Annals of the Missouri Botanical Garden
[—— Geniostoma
|» UL Gelsemium Acokanthera Allamanda 100 Plumeria 62 Ly Anechites 97 Thevetia | Alstonia Aspidosperma 100 pr IBN Vallesia Alyxia Lepinia 99 93 Lepiniopsis 98 79 Pteralyxia Plectanela e r—— Picralima 100 - ——— Pleiocarpa r—— Amsonia DOS 100 - ——— Rhazya Cabucala 100 Petchia 53 Ochrosia
r—— Catharanthus
68 ——— Vinca Neisosperma Rauvolfia
r—— Chilocarpus 85 |. ——— Condylocarpon Craspidospermum
Kibatalia e Mascarenhasia 93 OL. Nerium Kopsia
r—— Molongum 95 L——— Tabernaemontana
Strict consensus tree of the two islands found with the rbel, data. Numbers below the branches indicate
65
Figure 2. bootstrap percentages greater than 50%.
Volume 94, Number 1 2007
Endress et al. 9 Phylogenetic Analysis of Alyxieae
Geniostoma Gelsemium Acokanthera Kibatalia Mascarenhasia Nerium 14 [22 Allamanda 100.17. Plumeria 58 40 Thevetia 4 26 —— Amsonia B 98 US Rhazya a P Craspidospermum à 142 2 Picralima 25/90 100L2_ Pleiocarpa
35 LL Alyxia Pteralyxia
Chilocarpus Condylocarpon Cabucala Petchia Catharanthus Rauvolfia Vinca Neisosperma
Kopsia
Molongum
Tabernaemontana Alstonia
29
19. Aspidosperma
82
Figure 3.
Vallesia
‘he single most parsimonious tree found with the matK data. Numbers above the branches indicate estimated
substitutions, ACCTRAN optimization. Numbers below the branches indicate bootstrap percentages greater than 50%.
and an RI of 0.62. One of the shortest individual trees is shown in Figure 5, with estimated substitutions (ACCTRAN
branches and BPs below: groups not present in al
optimization) indicated above the
m
shortest trees are marked with an arrowhead. We show a single tree to illustrate. relative levels of genetic divergence. The contribution of each region to this tree was: rbcL, 488 steps (vs. 479 for the rbcL trees); matK, 974 steps (vs. 970 for the matK tree); trnL
intron, 293 steps (vs. 292 on the trnL-F trees); and
irnL-F intergenic spacer, 170 steps (vs. 168 on the irnL-F trees). Patterns of relationships are much like those in the previous analyses, and BPs are generally
higher than in any of the individual analyses.
ANALYSIS OF ALL DATA COMBINED
The combined data produced a single, most
parsimonious tree of 2226 steps with a CI of 0.65 and an RI of 0.60 (Fig. 6). The DNA optimized onto
Annals of the Missouri Botanical Garden
— Geniostoma
95
| | L- Gelsemium
60 | 100
Acokanthera Mascarenhasia Nerium Allamanda Plumeria Anechites
Thevetia
Alyxia Lepiniopsis Pteralyxia
Plectaneia p Chilocarpus
— — Condylocarpon Amsonia
E
L__ Rhazya Cabucala Petchia Catharanthus Neisosperma
Ochrosia
56
Rauvolfia
Vinca
~ 5 L
Craspidospermum Kopsia
Molongum Tabernaemontana
— Picralima
91
... 1L ——. Pleiocarpa
Alstonia
— Aspidosperma
99
Fig 4.
the bra ane che ss indicate bootstrap percentages greater than 5
this tree is 1926 steps, combined DNA tree: Kopsia fruticosa (Ker.
one step longer than the this step is caused by shifting Gawl.) A. DC.
laxum + Tabernaemontana (two spp.,
and Molongum Appendix 2) from an unresolved position with respect to the major clades into positions as a grade with respect to the Vinca clade. Otherwise, relationships are exactly as The data optimized onto the combined trees (ACCTRAN a CI of 0.33 and an RI of 0.55, versus 266 steps with a CI of 0.38 and an
with the combined molecular data. morphological
16 steps with
optimization) was 2
RI of 0.61 in the morphological analysis.
LL Vallesia
Strict consensus tree of the 25,000 equally pus t pe wsimonious trees found with the trnb- data. Numbers below 5066
DISCUSSION
EVALUATION OF TRADITIONAL CIRCUMSCRIPTIONS OF ALYNIEAE
Both the morphological and the molecular analysis indicate that Alyxieae as previously eireumscribed are polyphyletic. Of the individual data sets analyzed, the tree best
based on matK provided the support,
followed by that of trnL-F. BPs in the tree based on
rbcl 4
much of the tree a polytomy,
and the morphological data set were low, with and the positions of
several genera were equivocal. Even then, however,
Endress et al. 11
Volume 94, Number 1 Phylogenetic Analysis of Alyxieae
84 Geniostoma
L83 Gelsemium outgroups 1 Acokanthera Carisseae 19 Kibatalia — 28 j l Mascarenhasia Apocynoideae 13 Nerium 45 Allamanda Plumerieae Vinceae Alyxieae 61/100 70 p 19% 39 ra Chilocarpus 100 L65. Condylocarpon 5 " pss Craspidospermum _ Melodineae Y 57 Picralima united m Pleiocarpa E ullis 7 7 Is Vinceae > ¿ES SF} 39 24 Molongum 15 100 " Tabernaemontana | Tabernaemontaneae 98 Cabucala da rosia 21 Eas l 97 27 Vinca Vinceae 99 Rauvolfia — Neisosperma 37 nia DT 49 f Aspidosperma Alstonieae 100 L4Z Vallesia =
Figure 5. One of the most parsimonious trees found with the combined molecular data. Numbers above the branches indicate estimated substitutions, ACCTRAN optimization. Numbers below the branches indicate bootstrap percentages greater
than 50%. Groups not present in all 46 shortest trees are noted with an arrowhead.
clusters of genera are present. The combined data tree is similar to that produced by the combined molecular data and provides much better support for patterns of relationship already seen in each of the individual trees. Therefore, the remainder of the discussion will be based on the total combined tree (Fig. 6). This tree is not intended to represent relationships within or among tribes other than Alyxieae and Vinceae.
All genera traditionally included in Alyxieae are preceded by a dot in Figure 6. Vallesia and Anechites,
both included in Alyxieae by Pichon (1949a, 1950b,
as Rauvolfieae) and maintained there by Leeuwenberg
1994), are not closely affiliated with any of the other
—
Vallesia is sister to Aspidosperma, a that previous phylogenetic studies (Sennblad & Bremer, 2000, 2002; Potgieter & Albert, 2001; 2007). It is unlikely that a close relationship between Vallesia and Aspidosperma would have been predicted
based on their floral or fruit structures; they are too
members of the ingroup.
position supports results of
Simoes et al.,
siomorphic to be of much help, and the small
indehiscent drupaceous fruits of the former look very
Annals of the Missouri Botanical Garden
E Geniostoma L89. Gelsemium outgroups 47 Acokanthera Carisseae 32 Kibatalia o] 0 28 Mascarenhasia Apocynoideae Plumerieae 58 Amsonia ; 100 Er Rhazya B 10 Alyxia Lepinia Lepiniop Pteralyxia Alyxieae 68/100 10 Plectaneia (The Alyxia clade) A 42 22. Chilocarpus 100 m non mii Craspidospermum Melodineae 13 Laso” Picralima 100L9.. Pleiocarpa . |Funterieae 16 2 bucala EJ Petchia Rauvolfia 75 ooo Vinceae 26 E ^ ico (The Vinca clade) 94 1158 Ochrosia opsia — ogee _ |Tabernaemontaneae 100 Tabernaemontana | 3 Alstonia 54 39 F Alstonieae 100 — 46 Valles
Figure 6. data.
indicate bootstre ap percentages great
Numbers above the branches indic er iini 50% dier
. The Al
and brackets. « Alyxieae (Rauvolfieae in es
te estimated substitutions, ACCT yxta clade and the Vinca clade are classific: ations). A =
The single most ane caine tree found in the total combined analysis of the morphological and molecular
—
the branches
indicated with thicker branches
AN optimization. Numbers below
taxa not recognized at generic rank by Leeuwenberg
(19944). Tribal n. names on right follow the classification of Endress and Bruyns (2000).
different from the dehiscent follicular latter. A close relationship between the two genera is . In. both genera the pollen has five or six apertures (as opposed the
however, supported by pollen morphology which are The
Endress
Lo usual 3-aperturate condition),
surrounded by distinctive prominent. ridges.
inclusion of Vallesia in Alstonieae sensu and Bruyns (2000) is supported by previous rbcL data (Sennblad € Bremer, 2000, 2002), but not by trnL-F
data (Potgieter & Albert, 2001). which included more
fruits of the
genera near the base of Apocynaceae. In our analysis, Anechites is sister to Thevetia, grouping with members of Cerbereae of traditional classifications (Cerberoi- deae of Pichon, 1948b). which confirms results based on morphology (Fallen, 1983a; Alvarado-Cárdenas & 2007) 2000,
Bruyns’ (2000) inclusion of Anechites in Plumerieae.
Ochoterena, and earlier rbcL data (Sennblad &
Bremer, 2002) and supports Endress and
The remainder of the genera of Alyxieae fall into two
main clades.
Volume 94, Number 1 2007
Endress et al. 13 Phylogenetic Analysis of Alyxieae
RELATIONSHIPS WITHIN VINCEAE AND ALYXIEAE
The first main cluster of Alyxieae in the combined
analysis (Fig. 6) is the Alyxia clade and includes Alyxia, Lepinia, Lepiniopsis, and Condylocarpon. In
addition, the placement of Chilocarpus, Plectaneia,
and Pteralyxia in this clade is well supported. This
corroborates the positions of Alyxia, Lepinia, and
Chilocarpus in the study by Sennblad and Bremer
(2000, 2002) based on rbcL data. Except for Condylocarpon, the members of this group have
irregular pollen grains with relatively large porate The Lepinia and Lepiniopsis have three or four apertures 2001). Within the Alyxia clade,
Alyxia, Lepinia, Lepiniopsis, Pteralyxia, and Plecta-
apertures. aperture number is usually two:
(van der Ham et al., The first three. genera
(19492)
Alyxiinae. Pteralyxia was considered to be
neia form a clade (Fig. 6).
correspond to Pichon’s and Leeuwenberg’s
(1994a
a synonym of Alyxia by Pichon (1949a) and a synonym
—
of either Ochrosia by Leeuwenberg (see
2001: 169,
has previously been
Alyxia or Gunn et al.. 1992 and van der Ham et al., 187).
included in
Plectaneia, in contrast,
Plumerieae and has usually been considered to be related to genera such as Gonioma E. Mey., Stephanostegia Baill., and Craspidospermum
Bojer ex A. DC. 1895;
Plectaneiinae of Pichon, 1949a: Craspidosperminae of
(Alstoniinae of Schumann, Leeuwenberg, 1994a), all of which are included
Melodineae in Endress and Bruyns (2000). In terms of Alyxia, Lepinia, Lepiniopsis, and tight-knit
(Fig. 7). The porate apertures can be large (maximum
pollen morphology,
Pteralyxia form a monophyletic group
less
SEA
21-33 Um), their margins are clearly thickened clearly also in Chilocarpus), the inner exine surface is
granular (also in subfamily Apocynoideae), the inner
PEN
exine layer (nexine) is completely endexinous, the
—
infratectum is hardly recognizable (being reduced to + sparse gaps in the inner ectexine), and a relatively thick tectum is present (also in several other taxa).
Lepinia and Lepiniopsis share the presence of an
rt
ornamentation consisting of anastomosed verrucae
(van der Ham et al., 2001). In the large analysis of trnL-F by Potgieter and Albert (2001), Alyxia, Lepiniopsis, Condylocarpon, and Plectaneia were
whereas Pteralyxia affined unlikely
Pteralyxia considering its distinctive pollen,
supported in Alyxieae, with Plumerieae. This is an position. for which is a synapomorphy of Alyxieae and found nowhere else
2001). As the
same vouchered specimen was used as the source of
1 Apocynaceae (van der Ham et al.,
DNA for both studies, the reason for this discrepancy between our trn L-F results and those of Potgieter and Albert (2001) is most likely due to a mix-up in the
—
aboratory. It is noteworthy that of the seven genera in
the Alyxieae, four, Lepinia, Lepiniopsis, Pteralyxia, and Plectaneia, are island endemics (Leeuwenberg, 1997; Lorence & Wagner, 1997), and Alyxia has its ereatest species diversity in the Pacific (Middleton, 2000, 2002).
Chilocarpus and Condylocarpon are sister genera
that are the sister group to the remainder of Alyxieae
—
Fig. 6). Because of its syncarpous ovary, Chilocarpus was included in Carisseae (Willughbeieae sensu Endress € Bruyns, 2000) by Schumann (1895,
Arduineae) and the invalid Chilocarpinae by Pichon (1948a). Leeuwenberg (1994a) provided a Latin di-
agnosis, raised Pichon's subtribe to tribal level, and in
—
his recent revision of Chilocarpus (Leeuwenberg,
2002) considers Chilocarpeae to fall somewhere between Carisseae and Ambelanieae. New World
exhibits a set of deviating pollen features (tetrads,
Condylocarpon is the only member of Alyxieae and inaperturate, and reduced exine; Fig. 7) that strongly indicate the pollen to be paedomorphic (i.e... un- derdeveloped regarding pollen wall features, vet viable). The basically decussate tetrad configuration indicates Condylocarpon pollen to be derived from a 2- aperturate rather than from a 3-aperturate ancestor, providing additional support for its present position in Alyxieae (van der Ham et al., 2001). Despite its aberrant, inaperturate, nearly exineless pollen, Con- dylocarpon resembles Chilocarpus in a number of other morphological aspects, especially the distinctive globose head of the flower buds. The fruits exhibit some superficial similarities; in most species of both genera, they are moniliform and woody, although the gynoecium is syncarpous and dehiscent in Chilocar- pus and apocarpous and indehiscent in Condylocarpon
(Fallen, 1983b
The second cluster of
main Alyxieae in the combined analysis (Fig. 6) is the Vinca clade and includes Cabucala, Petchia, Rauvolfia, Ochrosia,
Vinca. All
taxa in this clade are characterized by a differentiated
Neisosperma, Kopsia, Catharanthus, and style head with a distinct annulus at the base, but this is a plesiomorphic feature and is also found in other
Alstonia R. Br.,
Plumerieae). Similarly,
tribes (e.g., Alstonieae; Allamanda, the pollen morphology of this clade is unspecialized (Fig. 8). The occurrence of well-developed colpal and mesocolpial plates due to the presence of distinct supplementary endocolpi
(absent in Vinca, weak Kopsia and several taxa outside the Vinceae: Acokanthera G. Don, Allamanda, Aspidosperma, and Plumeria) is their most discrimi- nating feature. Of the
Rauvolfia,
Schumann’s (1895) classification. He included Rau-
taxa in the Vinca clade,
Ochrosia, and Kopsia were treated in
volfia in Rauvolfinae, together with nine other genera,
none of which shows a close relationship with
14 Annals of the Missouri Botanical Garden
Figure 7. Apocynaceae, Alyxieae. SEM and TEM images of pollen grains. A-C. Alyxia ruscifolia. A. Barrel-shaped 2- orate sula n grain d aed unknown) with differently sized pores. —B. Detail of A, showing psilate, perforate ornamentation. —C. TEM section of exine and intine, showing lectum (above), infratectum (gaps), and endexinous inner layer with dark ine fever be dark granules on the inner surface (arrow). D, E. Pleralyxia kauaiensis. —D. Barrel-shaped 2-porate pollen grain (orientation 1 with differently sized pores. —E. Detail of undulate ornamentation with perforations in the depressions. —F. Pleralyxia macrocarpa. TEM section d exine and intine, showing thick tectum (above). infratectum e line and several small gaps), and ende inous inner layer with small dark inclusions and larger dark surface granules (arrow). G, H. Lepinia solomonensis. —G. Tetraporate pollen grain (orientation unknown) with unequal sides and differently sized pores. —H. Detail of verrucate ornamentation. —1. Lepinia taitensis. TEM section of exine and intine. showing thick tectum (above), infratectum (commissural line and gap). and endexinous inner layer with small dark inclusions
D ge B Pa Z — * 2 Q m n S 2
„ T nsis. —]. ¿ 3-porale polle n grain (orie nlalion unknown) with unequal sides and differently sized pores. —K. Detail of J, showing + verrucate ornamentation. psilate annulus, and inner surface granules (arrow) inside pore. —L. TEM section P exine and intine, showing thick tectum (above ). infratectum (commissural line and sparse gaps), and endexinous inner laver with small dark inclusions anc i: large dark surface granules (arrow). M. O. Chilocarpus denudatus. —M. Psilate to finely fossulate s pollen grain (orientation unknown). —
— -
0. TEM section of exine and intine, showing tectum (above), granular infratectum, and ectexinous inner verrucate la iver. N Plectaneia thouarsii. —N. Psilate, perforate 2-porate polle n grain (orientation a —R. TE Mo section of exine and
Volume 94, Number 1 2007
Endress et al. 15 Phylogenetic Analysis of Alyxieae
Rauvolfía im this study or other recent analyses (Sennblad & Bremer, 2000, 2002; Potgieter & Albert, 2001; 2007), Kopsia were included in a different subtribe, Cerber- Endress & 2000). circumscribed by (1949a)
The Rauvolfinae comprised
Simoes et al., whereas Ochrosia and
eae (Plumerieae of Bruyns,
Rauvolfieae as Pichon included four subtribes. Cabucala, Petchia, and Rauvolfia, which are sup- ported as monophyletic in the Vinca clade. Ochrosi- nae contained only Ochrosia (including Neisosperma), and Vallesinae included Vallesia and Kopsia, which His
Alyxinae and Condylocarpinae, belong to
are only distantly related here. other two subtribes, elements of Alyxia clade in our study. Pichon (1949a) included Anechites as a genus incertae sedis, noting that he did not have sufficient material to place it, but he thought that it most probably belonged with Condylocarpon or in a tribe of its own.
The classification of Leeuwenberg (1994a) followed Pichon’s, with the same tribal circumscription of Alyxieae and the same subtribal circumscriptions. The only differences were that Anechites was included as a member of Condylocarpinae and the name of the
More
Leeuwenberg (1997) placed Cabucala in synonymy
tribe was changed to Alyxieae. recently,
under Petchia. Although there are some differences in pollen morphology (differing colpus length, tectum
thickness and ornamentation, and the presence/ absence of deviating mesocolpium centers) in the species studied, the floral structure of the two genera is nearly identical, and our results do not contradict the synonymy of Cabucala. Neisosperma, which was placed into synonymy under Ochrosia by Pichon (1949a) there by Leeuwenberg (1994a) and Hendrian (2004), is often considered to
be a distinct genus, especially by specialists dealing
and maintained
with species of the Pacific Basin, where both genera
have their greatest species density (Fosberg et al.,
1977; Markgraf, 1979; Boiteau, 1981; Smith, 1988; Wagner et al., 1990; Forster € Williams, 1996).
Macromorphologically, plants of Neisosperma and Ochrosia have a clear resemblance (e.g., trees with corolla lobe aestivation dextrorsely Their
fers in several aspects (size,
whorled leaves,
contort, fruits large, fleshy colorful drupes).
—
pollen, however, di
ectoaperture shape and margin, endoaperture margin,
and ornamentation). In our analyses, inclusion of Neisosperma in Ochrosia is only moderately supported. A detailed study including more species of both genera is needed in order to elucidate their relation- ship.
Also in the Vinca clade (Fig. 6) are two genera included in Plumerieae: Catharanthus 1895 as
Leeuwenberg,
conventionally and Vinea (Schumann, Alstoniinae; Pichon,
1949a as 1994). The
pollen of Vinca is peculiar and unique by its indistinct
Alstonieae; ectoapertures, relatively large endoapertures, and thin exine, which makes it difficult to compare with the pollen of other taxa. Vinca also shows some derived floral characteristies, such as the enlarged spathulate, apical anther appendage, which most likely plays in inhibiting desiccation of the secondarily 1908), possibly related to
the temperate habitat of this genus.
a role
presented pollen (Church,
The genera included here in the Vinca clade are the same as those included in Vinceae of Endress and Bruyns (2000), with the exception of Amsonia and Rhazya. These
considered to be closely related to Catharanthus and
two genera have traditionally been and thus conventionally included in Plumer- 1895 as
Catharanthus,
Vinca,
ieae (Schumann, Alstoniinae). Amsonia,
Rhazya,
nerinae of Pichon (1949a) and Catharanthaninae of
and Vinca constituted Loch-
Leeuwenberg (1994a). Flowers, fruits, seeds, and pollen of Rhazya scarcely differ from those of Amsonia (Pichon, 1949a; Nilsson, 1986), which was therefore treated as a synonym of Amsonia by Endress and
Bruyns (2000) and not contradicted by our study
(Fig. 6). However, Amsonia and Rhazya together group here with neither Alyxieae nor Vinceae.
Instead, they are placed as sister to the Plumerieae and Carisseae + Apocynoideae, although bootstrap support for this is less than 50% (Fig. 6). In the study by Potgieter and Albert (2001). Amsonia was in a clade together with Thevetia peruviana, which was included in a large polytomy. Floral structure of Amsonia and Rhazya, including details of the style head, is similar to that of Catharanthus and Vinca and does not agree with the more derived position for the former two genera indicated by the molecular analysis. Similarly, Amsonia and Rhazya occupy an unexpected position tree considering their secondary
in the molecular
— intine, ee tectum (above), undulate granular infratectum, loose?). P, Q, S. Condylocarpon isthmicum. —P. Tetr:
members in decussate configuration. —Q. TE
constituent members, loc T tion through exte rnal wall, showing the thin tectu = 10 um in A, D, G, J
eclexinous inner layer. Scale bar
ad almost filling anther locule,
'ally fused joa walls with sparse pores (arrow
and ectexinous inner layer with surface verrucae (partly showing the four constituent, psilate
`M section through tetrad almost filling anther locule, showing three of the four
). and thin, locally thickened, external walls. —S.
m, the sp defined infratectum (arrow). a locally ES kene x M
> Q; scale bar = 1 um in B, C. K. L,
16 Annals of the Missouri Botanical Garden
r” zu Lon v
Figure 8. bec d in Vinceae (AP). Melodineae (Q—5). SEM and TEM images of pollen grains. —A. Petchia ceylanica. Psik:
AE colporate polle n grain in oblique view: colpi short with relative ly l: arge ¢ ae B, C. Cabucala caudata. —B. Inside of dta polle n grain showing Iwo colpal plates, « sach ene losing ane ndopore surrounded by a distinel
endoannulus: in the center a mesocolpial plate delimited from the colpal plates by supplementary endocolpi (arrows). —C. TEM section through supplementary endocolpus (arrow). colpal plate (left); and mesocolpial plate (right). —D. Kopsta Jruticosa, SEM section, showing psilate to scabrate exine stratified into two equally thick strata (tectum above) separated by a thin infratectum. —E. Ochrosia coccinea. Verrucate 3-colporate pollen grain in polar view. F-H. Neisosperma nakaiana. — F. Psilate/finely rere tricolporate pollen grain in polar view. —G. TEM section through mesocolpial exine and intine, showing tectum (above), irregular infratectum and foot layer, and spongy endexine (arrow). —H. TEM section through apertural exine, showing tectum (above), regula ar ane ctum, foot layer, which is much thie ke ned under the colpus (arrow), and spongy endexine. I-K. Rhazya stricta, . Psilate, perforate pollen grain in equatorial view; colpi short and wide with relatively large e ron: —J. Inside of pollen grain showing lalongate e i ed delimited polewards D horizontally oriented colpal 2d o the left and to the right, psilate Ve cuis plates delimited by wide verrucate zon s (supple mentary
endocolpi). —K. TEM section through exine and intine, showing tectum (above), slightly thinner, zat ilate infratectum, and inner layer of the same x no ss as tectum. I. M. Vinca minor. (eritical- pou dric —L. Psilate 4-
aperturate pollen grain in polar view. silate d-aperturate pollen grain in equatorial view, HCM the indistinel ecloaperture (porous area) in center of depresse ‘ oblong zone that is 1 by 185 endoapertur: np costae. N-P. Vinca major. -N. TEM section through thin me 1 exine and intine, showing tectum (above) with indistinct perforations. distinct
Volume 94, Number 1 2007
Endress et al. 17 Phylogenetic Analysis of Alyxieae
chemistry. Both genera contain numerous complex indole alkaloids of the plumerane type (Ganzinger & 1976: Kisakürek et al., 1983), whereas in all
genera above them in the uppermost clades of the tree
Hesse,
and in Alyxieae, indole alkaloids have been lost. In
the comparative study by Nilsson (1986), however. pollen of Amsonia and Rhazya was found to be nearly identical but showed no close relationships with Cabucala (Vinceae).
pollen of Catharanthus or
Because the focus of our study was Alyxieae. this was the only group densely sampled. To better assess the phylogenetic position of Amsonia and Rhazya. additional representatives of other tribes, especially previously unstudied genera of Melodineae sensu
Endress and Bruyns (2000)
The unexpected positioning of Amsonia and Rhazya
should be included.
in the combined molecular tree (Fig. 5) away from the case of the
Amsonta and Rhazya did not
rest of Vinceae could be regarded as a
incongruence with morphology. However, in morphological analysis, always appear together with Catharanthus + Vinca, and their position was relatively unclear (e.g., no BP > 50). the DNA and
morphological better
resolution and higher support in the all-data combined
Furthermore, combining
data resulted in generally tree relative to the combined DNA data tree, which would not be expected if there were highly in- congruent basic patterns in each of them. The exact position of Amsonia/Rhazya is also not clear with the DNA data, except that their exclusion from the clade with the rest of Vinceae is not strongly supported (Figs. 5, 6), which concurs with the results of Simões et al. (2007).
SIGNIFICANCE OF MORPHOLOGICAL AND CHEMICAL CHARACTERS
been demonstrated here and elsewhere
1983a; 2000, 2002;
Potgieter & Albert, 2001), superficial resemblance of
has
(Fallen, Sennblad & Bremer,
characters such as fruits and seeds of Apocynaceae that are correlated with dispersal mode are extremely labile, and there is a tendency for the repeated independent evolution of certain fruit and seed types. Similar findings for other angiosperm families have and Eriksson (1992),
been reported by Bremer
Armbruster (1996), Endress (1996), Hufford (1997). and Clausing et al. (2000). Fruit and seed characters are thus particularly unreliable when used alone for determining relationships among genera in Apocyna- fruit
dehiscence, mesocarp consistency, and seed appen-
ceae. Examples of these characters include: dages. It was the use of such simple, single character- that led to the artificial tribal classifications of Schumann (1895), Pichon (1949a).
The most reliable char-
based categories
and Leeuwenberg (1994a).
acters are likely to be more subtle, and one must be
lama]
willing to invest some effort to determine what they
are. It is also unrealistic to believe that any single character is going to provide a non-homoplasious synapomorphy for any large genus or tribe, but rather it is more reasonable to expect that these groups can be circumscribed by a specific combination of characters.
Alyxieae pollen is characterized by large porate apertures (Fig. 7), which are distinct from the usual small aperturate porate grains found in Apocynoideae. completely different from the colporate grains found in Vinceae, and characteristic for all other Rauvol- fioideae (Figs. 8, 9). Details of the pollen ectoaper- tures proved to be the most important morphological characters for defining Alyxieae because the unusual and distinct pollen type is synapomorphic for the tribe. Aperture type is less informative in the other tribes of Rauvolfioideae because, with a few excep- 8). all Rauvolfioi-
deae have colporate pollen grains. Within Alyxieae,
tions (e.g., Craspidospermum; Fig.
aberrant, inaperturate, nearly exineless pollen that remains in tetrads is an autapomorphy for Condylo- carpon. Inaperturate pollen is otherwise known in the family only in Secamonoideae and Asclepiadoideae as well as some genera of Periplocoideae that have pollinia (e.g., Finlaysonia Wall., Hemidesmus R. Br.: Schill & Jäkel, 1978; Verhoeven & Venter, 2001). Condylocarpon is also of interest biogeographically, being the only Neotropical member of Alyxieae: all other genera are found in southeastern Asia and the Pacific. Aperture type thus provides a clear distinc- tion between the Alyxieae and Vinceae.
Other morphological characteristics that distin-
guish Alyxieae from Vinceae (but not necessarily
«—
infratectum, and inner laver consisting of f oot layer and spongy endexine (arrow). — P.
costa (arrow) in ectoapertural area. — EM s continuous tecti
ains, showing small ectopores and h
ction through endoapertural costa outside
eavily costate DE e (arrow). —S. TEM
O. TEM section through endoapertural
'cloapertural area, showing
im (arrow), discontinuous inner exine, and Shae mae intine. Q—5. Cae verticillatum. —Q. Rhomboidal tetrad, showing several pores (in adjacent positions) near
ures. —R. TEM section through two adjacent pores of section through two adjacent
vollen grains, aa ae exine stratigraphy with joint tecta, separate infratecta, and inner exine layers; E 3 E rn I F, L, M, Q: s
(arrow). Scale bar = 10 um in A, E, F, L, scale bar
= 5 um in
1 I-K; scale bar = 1 um in B-D,
Annals of the Missouri Botanical Garden
Figure 9. Apocynaceae. Aspidospermeae (A, B, D- | Alstonieae (C), Hunterieae (G-I ). Plumerieae (J). Carisseae (K, L) Malouetieae (Apocynoideae) (M-O). SEM and TEM images of 1 n grains. A, B. arma ahaha tum. —A, Psilate/ fossulate 6-colporate pollen grain in slightly oblique polar view, showing prominent arcus-like ri surrounding the colpi. —
B. SEM section of ridge, showing a thin, psilate, pe ie tectum, thick granular infratectum, M thin i inner exine layer. —C. Alstonia scholaris. TEM section through exine and inline, showing die silate, n ie tectum, granular infratectum, dimerous foot layer, and indistinct spongy e mdexine (arrow). a. allesia yn —D. Psilate pentacolporate polle n grain in polar view, showing prominent ridges surrounding colpi. —E. Part of fractured pollen grain showing three “empty? ridges and psilate to scabrate inside with elongated, unevenly distributed granular marks (arrow). —F. va llesia antillana. VEM section ua. lectum subtended by granular infratectum (arrow) and cavity, and a + dimerous inner layer. G, H
Scabrate inside of ai pollen
through ridge, . Picralima nitida. TEM section
Pleiocarpa mutica grain showing two apertures through exine and intine; exine consists subtended by a thin foot layer and thin indis Acokanthera . jys of endocracks (supplementary endocolpi:
tion dh supplementary e C» 'olpus. (arrow) subtended by thicke —M. Psilate triporate pollen grain, showing
. Psilate, perforate tricolporate pollen grain in polar view. — eft); endo} vores have pol: ar costae (arrow). undulate d tectum, slightly thinner granular-reticulate infratectum
J. An finely perforate tric olporate
(right and extreme of relatively ‘ick. tinct endexine (arrow). —J. Anechites nerium. Psilate, "reris of tric olporate polle n grain showing colpal pl ates with arrows) delimiting psilate ned intine
polle n grain in pol: ir view endopores (left and right) and
—L. TE! d
lalongate
mesocolpial and polar plates.
(left) and mesocolpial exine m N
. Mascarenhasta arborescens pores with weak annulus (arrow). —N. Exi P nt showing part of psilate, sparse n perforate tec 1155 inner side beset with diverseh
Volume 94, Number 1 2007
Endress et al. 19 Phylogenetic Analysis of Alyxieae
from other tribes of Rauvolfioideae) include the simple style head, which is uniformly secretory and receptive and lacks a pollen-trapping annulus at the base (vs. the style head differentiated. into distinct morphological and functional zones, with the re- ceptive region located beneath an annulus at the base). Except for Plectaneia, Alyxieae seeds have a tough and usually conspicuously ruminate endo- sperm (vs. a smooth and soft endosperm in Vinceae). within Rauvolfioideae were so
1895:
it was
Because tribes artificial in earlier classifications (Schumann,
Pichon, 1949a, 1950b: 19944).
impossible to understand the evolution of secondary
Leeuwenberg,
chemistry in this subfamily. As tribal cireumscriptions become more natural, it is now possible to gain a better understanding of the phylogenetic. pattern of indole alka-
for Rauvol-
alkaloids and cardenolides. Generally, indole loids are considered to be characteristic fioideae. However, indole alkaloids characterize only five of the tribes included in this study (Alstonieae. Tabernaemontaneae, Vinceae, Hunterieae, and Melo- dineae). whereas indole alkaloids have not been reported in the other three tribes (Fig. 6). Acokanthera and Carissa L. contain cardenolides. Based on their
syncarpous gynoecium, they were conventionally included with indole alkaloid-containing genera in 1994a). Here and in other phylogenetic studies (Endress et al.,
1996; Potgieter € Albert, 2001; 2007),
the Carisseae sensu Endress and Bruyns (2000) are
a polyphyletic Carisseae (Leeuwenberg,
—
Simóes et al.,
placed with other taxa in which indole alkaloids have been lost or replaced by other secondary compounds (Johns et al., 1968; Hegnauer, 1970; Coppen & Cobb, 1983; Kisakürek et al., 1983; Jensen, 1992).
All genera of Vinceae contain various complex indole alkaloids (Hegnauer, 1970, 1989; Ganzinger & 1976; Kisakürek et al., 1983). In contrast, all Alyxieae studied for secondary chemistry
Hesse, genera of lack indole alkaloids. Alyxia and Lepiniopsis contain 1970, 1989),
and although Lepinia has never been analyzed for
coumarins (Johns et al., 1968; Hegnauer, secondary compounds, the crushed leaves are known to emit a strong coumarin scent (D. Lorence, pers. comm., 1999). Coumarins are absent from Plectaneia,
and no reliable data are available on the secondary
CHARACTER EVOLUTION
Gynoecium, fruit, and seed morphology are com- plex and homoplasious in Rauvolfioideae. In this analysis, the plesiomorphic state of the gynoecium is apocarpous, in concurrence with Potgieter and Albert (2001: but see Sennblad & Bremer, 2000, 2002, for an alternative view), and the majority of the genera are apocarpous. Even in the small sample here, almost every clade includes at least one syncarpous genus; similar findings are reported by Simóes et al. (2007).
The style head is a useful character for distinguish- n Apocynaceae (see Fig. 10), but it is
me
ing genera structurally complex, making it difficult to break down into meaningful character states for coding morpho- logical characters. The two taxa sister to the rest in this analysis, Vallesia and Aspidosperma, have a simple style head that is vertically undifferentiated. A simple Alyxieae and for the Hunterieae and Carisseae (Fallen, 1986; 1996; 2000). A
similar type of style head is found in some (but not all)
style head 1s also characteristic for all
Endress et al., Endress & Bruyns,
genera of Alstonieae and Plumerieae. Tabernaemon-
~ ~ =
va s.l., as currently circumscribed by Leeuwenberg (1991, differentiated style head, such as the species included
1994b), includes species with a simple, un-
in this study, as well as ones with a complex,
vertically differentiated style head with distinct functional regions and a broad pollen-trapping flange at the base (e.g.. all the New World species). These results indicate that style head specialization has probably evolved in parallel in various clades of Rauvolfioideae, as was suggested by Potgieter and
Albert (2001), and that this probably has proceeded in
=
both directions (Fig.
Within Alyxieae, Lepinia and Lepiniopsis have a 3- to 5-carpellate ovary, which is partially to d A 3- to 5-carpellate ovary is otherwise b in duae:
All other
syncarpous, respectively (Endress et al.,
ynaceae only in Pleiocarpa (Hunterieae). genera have two carpels.
Fruit optimized: Vallesia has naked seeds in a small juicy
type and seed margin are equivocally
drupe, whereas Aspidosperma has thick woody follicles and seeds with a diaphanous wing. This is in ge the be of
Carisseae (a conglomeration of Willughbeieae, Melo-
contrast to the traditional view of
dineae, Hunterieae, and Carisseae, sensu Endress &
chemistry of. Pteralyxia, Chilocarpus, and Condylo- carpon.
Pes
sized granules, and endoannulus (arrow). —O
ecto- and endoannulus; innermost exine layer consists of granu the intine contains numerous radially oriented dark inclusions (arrow). O.
granular infrate C tum;
scale bar = 5 um in K, M; scale bar = 1 Um in B, C, E,
TEM section through exine and intine near pore; exine is thickened into an
les or irregular elements that are separated from tectum by
Scale bar = 10 um in A, D, G-
STYLE HEAD BODY C] undifferentiated MH Differentiated E Equivocal
Figure 10. Evolution of style head body differentiation and pollen aperture type mapped onto the single most parsimonious tree from the combined analysis of morphological and molecular : ) ) | | YI PI 2 I j I E
data using ACCTRAN optimization.
Micron
CIL epiniopsis OPteralyxia OPlectaneia Chilocarpus Condylocarpon
„
OPicralima
POLLEN
— Colporate
Annulate porate — Non annulate porate EN inaperturate
DT abernaemontana
ill Alstonia DAspidosperma
100 OVallesia
po qODoOoDoOoqoqQ 2 2
USPIBE) jeoiuejog unossi|A
ay} Jo sjeuuy
FRUIT TYPE
ehiscen follicle or capsule Ma Fleshy dehiscent follicle or capsule
E Equivocal
Figure 11. Evolution of fruit type and seed margin mapped onto the single most
ACCTRAN optimization.
Geniostoma
Ej OOOO
O Acokanthera
Im Kibatalia
Po II Mascarenhasia
100 du
100
E Condylocarpon
il Craspidospermum O Picralima
O Pleiocarpa
(3 Cabucala
EJ Petchia
O Molongum
E Tab
ll Alstonia Bl Aspidosperma B Vallesia
SEED MARGIN C] Naked
EN Fimbriate
parsimonious tree from the combined analysis of morphological and molecular data using
20082
| JequinN FE euinjoA
eealXÁ|V jo sisÁjeuy oneusBojAyd
"Je Ja sselpuy
Le
Annals of the Missouri Botanical Garden
Bruyns, 2000) as the most unspecialized fruit type in the family. Vinceae are homogeneous with regard to seed margin, with the seeds being unwinged (see Simóes et al., 2007, regarding Kamettia and Tonduzia. not included here). analysis, the seeds are enclosed an indehiscent
Only
having delicate, papery dehiscent follicles (Fig. 11). 8 paper Pig
drupe. Catharanthus and Vinca deviate, in
Catharanthus and Vinca also have a derived herba- ceous habit, whereas the other genera in Vinceae are
trees or shrubs.
The seed Alyxieae (Fig. 11).
specialized genera
margin is evolutionarily plastic ii
The four morphologically most
Alyxia, "epu Lepiniopsis, and ll have
distinctive. cylindrical. seeds that are qu
Pteralyxia, as well as C a
rolled with a deep hilar groove. Plectaneia. in contrast, has flat seeds with a wing at each end. In
Chilocarpus the seeds have a small pink corky aril on
the funiculus and are presented in an unusual type of
leathery dehiscent fruit that splits apart along one or
Arils are rare ir
two valves (Leeuwenberg, 2002). Apocynaceae, otherwise known only in Tabernaemon- taneae. Similar types of fruits are known as display 1983: Smith, 2000) al.. 2000) or as & Green,
fruits in Gesneriaceae (Wiehler.
and Melast (Clausing e dehiscent berries in Oleaceae (Lawrence 1993: Li et al.. 2002). Seed
Plumerieae (Pichon,
margins also 1940b,
this
vary 1950b
diversity ir
considerably in
contrast to
as Cerberoideae). In
Rauvolfioideae, in Apocynoideae and all other
subfamilies, the fruit and seed type is uniform: the fruit is a pair of follicles (rarely postgenitally united) that dehisce to release small, comose seeds. Palynologically, starting from the basic regular 3- colporate condition typical in the Rauvolfioideae (Figs. 8. 9; Nilsson, 1986), an entire suite of changes (Fig. 10):
pollen grain sha ye irre ular, aperture number mostly D J
characterizes the derivation of Alyxieae
two (sometimes three), and ectoapertures porate with thickened margins (Fig. 7). Within the clade, several other characters change: maximum pore diameter is 9 um in Chilocarpus, 12 um in Plectaneta, and 21— 33 um in the subclade including Alyxia, Pteralyxta, Lepinia, and Lepiniopsis. Possibly due to paedomor- phosis, Condylocarpon pollen is inaperturate (van der Ham et al., 2001). Together with the larger maximum also shows much
pore diameter, the Alyxia clade
larger. barrel-shaped (two pores) or depressed (three
—
or four pores) pollen grains, an endexinous inner exine
layer with a granular surface, and an indistinct (reduced) infratectum. Within the Alyxia clade, there is a change toward a more heavily sculptured tectum in the subelade including Alyxia, Pteralyxta. Lepinia.
and Lepiniopsis, from psilate in Alyxia, via undulate in
In the genera included in our
Pteralyxia, toward + verrucale in Lepinia and
Lepiniopsis. Pollen of the last two genera mostly has more than two apertures: three (less often two)
Lepiniopsis and three or four in Lepinia (van der Ham 2001).
aperture conditions, as the apertures are still irregular
et al., This is not simply a reversal lo basic
or diverse within a single grain regarding size, configuration, and orientation.
The functional significance of the remarkable shift in pollen morphology toward and within Alyxieae is not understood. The oldest fossils of the Alyxia pollen type date from the Paleocene of northwestern Borneo
(Muller.
age of the syndrome.
1981). which demonstrates the considerable
CLASSIFICATION
The topologies of this study support recognition of Vinceae and Endress and (2000). as Anechites from either tribe and their. placement in (2007) respectively. Our results do not support Rhazya in
si these two genera are not supported in any
Alyxieae sensu Bruyns
well as the exclusion of Vallesia and
Aspidospermeae sensu Simões et al. and
Plumerieae,
inclusion of Amsonia and Vinceae:
` the other included in this study. The
^
groups end of Amsonia was also not resolved in the study by Simoes et al. (2007).
Amsonia and Rhazya remains equivocal, these genera
Because the position. of are withdrawn from Vinceae and left unplaced for the time being. Although it is unsatisfying to leave them in limbo, a classification should reflect phylogeny, so it seems best to keep them as unplaced genera until
more data are available to place them more definitely.
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their indole alkaloid
tana (Apocynaceae) based on
content. PL Syst. Evol. 172: 13-3: APPENDIX .
Voucher specimens used for morphologic al character
assessment in the Alyxieae study. Herbarium acronyms are in parentheses. Specimens used to study floral structure are indicated with FS, those used for light microscopy, scanning e le ctron. miseroscopy, and transmission electron microscopy indicated by LM, SEM, and TEM, respec live ly, following the he rbarium ac ronym. APOCYNACEAE
ee d 9 st.) Codd
ningen, Aas et al. s.n. (Z) ES eri mc 0 am.) Code Bayliss BRI 544 (S) LM, SEM.
Allamanda cathartica L.
of pollen grains are
cult. Gard.
South Africa, TEM
w, 1983, Fallen s.n. (Z) FS;
cult. Royal Bot. Gard. 0 (WAG) LM, SEM, TEM
Gabon, Leeuwenberg | Alstonia scholaris (L.) R. S “ult. Fairchild un a (L)
Gillis ee i) FS: New unea, Schodde 2 , TEN
G LM, SEM Alyxia mala Domin
Australia, das 835 (L) LM IM ruscifolia R.
ult. | 1 b Give 1055 (Z) ES: Australia, Clark
et al. 1753 (L) SEM, TEM Amsonia ciliata Walter
U.S.A., Sasseen s.n. (W AG) LM, SEM, Amsonia a ig eae Walter
cult. Bot. Gard. Zürich, Endress s.n. (Z) ES Anechites nertum m ) Urb.
5 16471 (Z) FS; Dominican Republic,
TEM
Ec uador,
van 15239 (S) LM, i e parti A. Brazil, Ferreira s.n. a a Brazil, Heringer 10672 (UB) LM, SEM Cabucala buio Ma rkgr ladagascar, Capuron 23701-5F (P) LM, SEM, TEM
Pl pobipérmia (Scott -E 71 v En ; Civeyrel 1281 (
Giod roseus (U G. De
Madagascar,
cult. Bot. Gard. Zurich, Endres s.n. (J) FS: Liberia, Van Harten 29 (WAG) I SEM, TEM Chilocarpus denudatus Blume. cult. B Gard. Bogor. Burck s.n. (Z) FS: India, Ridsdale 757 (L) LM: Java, Blume s.n. (L) LM: Java. ion. S) SEM; S ale Richards 1463 (L) TEM
at Chilocarpus al "ns Blume
Java, Hochreutiner 2547 ~ Z) FS Cond yloc arpon guyanense Des French Guiana, Sastre x (P22) ES
Kibatalia gitingensis (E 1 Woo dson
Philippines, Liede . s (Z) FS; Philippines, Wenzel 652 (G) LM, SEM, TEM Kopsia fruticosa (Ker p ) is Java, Prévost (Z pn n ictoria, Trinidad,
Broadway 5965 LM. a M. TE
Lepinia marquisensis Lorence & ign Fatu Hiva, Marquesas da Perlman 10271 (BISH. Z) FS
Lepinia solomonensis Hen
isl. Solomon Islands, BSIP 13496 (L) LM,
TEM Lepinia laitensis ecne. Soci Islands. Moorea. Perlman et al. 15071 (PTBG, 7) qo Islands. Tahiti, Whistler 4932 (BISH) a ternatensis Valet Moluc . Mochtar 306 (D LM. SEM: PNH 17362 (L) TE M
Lepiniopsis trilocularis Markgr. Palau Islands, Lorence 8265 (PTBG, Z) FS Mascarenhasia arborescens A. DC cult. Fairchild Bot. Gardi 2 n. (Z) FS; Madagascar, Schlieben 8128 (Z) LM, SEM: 22808-5F (P) LM. SEM, TEM Molongum laxum (Benth.) Pichon Venezuela, 13 pus 110 Z) FS; Colombia. Dunt 30207 7 (C OL) I SEM, TEM Visser pm TUE ) 18 & Sachet
Neill 5291 (J
Madagascar, Capuron
E aimea 5 tum, Hawaiian Is. . LM. SEM, TEN Motín y 1 " cult. Bot. Gard. Zurich. Fallen s.n. (Z) ES Segal 252 (WAG) LM, TEM: cult. Perpignan, Leeuwen- berg 12206 (WAG) LM, SEM, TEM Ochrosia coccinea (Teijsm. & Binn.) cult. Bogor Bot. Gard., FS, LM, SEM, TEM Petchia ceylanica (Wight) Livera ult. Bot. Gard. Kaiserslautern, Omlor s.n. „S: Sri Lanka. Wambeck 2510 (S) LM. Pic 1 1 5 e (Stapf) T. Durand & H. 1 cult. Bot. Gard. Wageningen. a 10779 (2) FS: Zaire, Gille 100 (BR) LM, SEM,
Plectaneta 0 Jum.
France,
30/8/1982 (Z)
Mic Java, anon. s.n.
E ui S. Il.
FS
ult. Madagascar, Pe lignat s.n. (Z Plectaneia thouarsii Roem. & Schult. Madagascar, Bernardi 11820 (L) LM. SEM, Pleiocarpa mutica Benth. cult. Bot. Gard. Wageningen, van Setten 415 (WAG, Z) FS: Ivory Cont Leeuwenberg 12145 (WAG) LM, SEM Plumeria rubra L.
TEM
ps Bot. Gard. Zurich, Fallen s.n. (Z) FS: Ghana, eeuwenberg 11089 i AG) LM, SEM, TEM e kauaiensis Cau m (Z) FS: Hawaii, Flynn 269
aual, Ds (PTBG) SEM
Preralyxta Do cea (Hillebr.) K. Se = M, TE!
^
Hawaii, Swezey s.n. (L) LM. M vomitoria Afze Ivory Coast. JJ. 337 (WAG) LM, SEM, berg 12122 (WAG) LM, SE
Aké , isi s.n. (Z) F
TEN:
; Nigeria, Leeuwenberg
Ivory Coast, Leeuwen-
Volume 94, Number 1 2007
Endress et al. 2 Phylogenetic Analysis of Alyxieae
Rhazya stricta De Yemen, ONE A A FS: Saudi Arabia, Schimper 812 (L) LM, SEM, TEM
Tabernae a iar (L.) R.
Br. ex Roem. & Schult. cult. Bot. Gard. ) FS
calcutta, anon. s.n. ( Tabernaemontana 12 9 om Lam. LM, SEM, TEM Thevetia peruviana (Pers.) E Schum. cult. Bot. Gard. Zurich, Fallen s.n., Florida, Gillis 9227 (S) LM, SEM, TE Vallesia pu An Woodson ult. Fairchild Trop. Gard, Zona s.n. (Z) FS; Florida, , 43415 5 (S) TEM Vallesia 77 (Cav. Link A. & H. Andersen 1009 (QCA) LM, SEM
ustra la, Alkin s.n.
e FS; cult.
alapagos Is.,
Vinca minor
(Z) FS: cult. ed SEM: cult.
cult. Zurich, Fallen s.n. Ne therlands, De Kort s.n. Nilsson s.n. (S) SE
Schipluiden, Sollentuna. Sweden, 11 major L. cult. Pijnacker, Netherlands, Van der Ham s.n. (L) LM. TEM GELSEMIACEAE
) J. St.-Hil. 28771 /) ES; i. (S) LM, SEM, TEM
Gelsemium sempervirens (L.) U.S.A., Louisiana, Tucker South Carolina, Wall. s
LOGANIACEAE
Geniostoma rupe: (J. R. Forst. & G.
B. J. Conn
Zealand, Garnock-Jones s.n. (WELTU, Z)
e New Zealand, NZ 9 (S) LM, SEM,
TEM
Forst.) var. „ ium n ( W 1
Nilsson
Annals
of the Missouri Botanical Garden
Voucher
Appendix 2.
specimens used for molecular analyses and GenBank accession numbers,
Taxon GenBank Accession No. APOCYNACEAE Voucher/Literature Citation math rbcL irnl, trnL-F Acokanthera oblongifolia Endress et al., 1996 Z70182 (Hochst.) Codd Sennblad & Bremer, 1996 X91758 Acokanthera oppositifolia Potgieter & Albert, 2001 AF214302 AF214148 (Lam.) Codd Mlamanda cathartica |. Endress et al., Z70190 Sennblad & B mer, e X91759 Allamanda det. Potgieter & Albert, 2001 AF214304 AF214150 Alstonia boonei De Wild. Potgieter & Albert, 2001 AF102374 AF214151 Alstonia scholaris (L.) R. Endress et al., 1996 Z70189 Br. Sennblad € Bremer, 1996 X91700 Alyxia buxifolia R. Br Potgieter & Albert, 2001 AF214300 AF214152 Alyxia ruscifolia R. Br cult. Montpellier, Civeyrel 1055 1008375306 (TL) Sennblad € Bremer, 2002 AJ419731 Amsonia tabernaemontana Potgieter & Albert, 2001 AF214307 AF214153 Walter P Royal Bot. Gard. Kew, AM295066 — AM295078 Aveyrel 1057 (TL) Anechites nerium (Aubl) E & Bremer, 2002 \J419733 AM295087 A M295087 Urb. Aspidosperma quebracho- Potgieter & Albert, 2001 AF214319 AF214105 blanco Schltdl. Aspidosperma triternatum cult. Bot. Garden, Meise, Bremer AM295077 Rojas Acosta 3029 (UPS) Sennblad & Bremer, 2002 AJ419735 Cabucala polysperma Madagascar, Civeyrel 1281 (TL) AM295067 AM295079 AM295088 AM295088 (Scott-Elliot) Pichon Catharanthus roseus (L.) Potgieter & Albert, 2001 AF102392 AF214175 G. Don cult. Stockholm Univ., Bremer A M2950068 28 (UPS) Sennblad & Bremer, 1996 X91757 Chilocarpus suaveolens Endress et al., 1996 Z70184 X92445 Blume a m Gard. Bogor, Chase AM295089 AM295089 Condylocarpon ins & Albert, 2001 AF214337 AF214183 amazonicum (Markgr.) Ducke Condylocarpon guyanense French Guiana, M. F. Prevost s.n. 0837537 AM295080 esf. (CAY) Craspidospermum Madagascar, Civeyrel 1234 (TL) DQ837538 AM295090 295090 verticillatum Bojer ex Sennblad € Bremer. 2002 AJ419743 A. DC. Kibatalia gitingensis Philippines, Liede 3268 (Z) AM295069 (Elmer) Woodson Sennblad & Bremer, 2002 AJ419745 Kopsia fruticosa cult. Bot. Gard. Meise, Bremer AM295091 AM295091 (Ker Gawl.) A. DC. 3033 (UPS Endress et al., 1996 Z70178 Sennblad & Bremer, 1996 X91763
Lepinia taitensis Decne. Lepiniopsis ternatensis ‘aleton
Lepiniopsis trilocularis Markgr
Sennblad € Bremer, 2002 Potgieter € Albert, 2001
Palau Islands, Lorence 8265 (PTBG)
AJ419746 AF214374
AM295081
AF214220
Volume 94, Number 1 Endress et al. 29 2007 Phylogenetic Analysis of Alyxieae
Continued.
Appendix 2.
GenBank Accession No.
Taxon APOCYNACEAE Voucher/Literature Citation matK rbcL irnl, irnL-F Mascarenhasta arborescens Potgieter & Albert, 2001 AF214378 AF214224 : cult. bs ageningen Agric. Univ., 1M295070 )-16, Setten 625 (WAG) one et al., 1998 AJ002885 Molongum laxum (Benth.) Potgieter & Albert, 2001 AF214383 AF214229 Pichon Endress et al., 1996 Z70185 X91765 Neisosperma nakaiana Potgieter & Albert, 2001 AF214385 AF214231 (Koidz.) Fosberg & Sachet Neisosperma oppositifolia cult. Nat. Trop. Bot. Gard., AM295071 | AM295082 Lam.) Fosberg & Kauai, Lorence s.n., NTBG Sachet 970511 (PTBG) Nerium oleander L. Fotgie ater & Albert, 2001 AF214386 AF214232 rel et al., 1998 798173 Sennblad et a 190 AJ002886 Ochrosia coccinea Sennblad & Bremer, 2002 AJ419752 (Teijsm. & Binn.) cult. Wageningen Agric. Univ AM295092 AM295092 Miq. nr. 78PTHB 354, van der Laan 389 (WAG) Petchia ceylanica (Wight) cult. Univ. Kaiserslautern, AM295072 AM295083 AM295093 AM295093 avera Germany, Omlor s.n. (Z) Picralima nitida (Stapf) T. Potgieter & Albert, 2001 AF214404 AF214250 Durand € H. Durand Endress et al. 1996 Z70179 aha & Bremer, 1996 X91766 Plectaneia stenophylla Madagascar, Petignat s.n. (Z AM295084 Jum. Potgieter & Albert, 2001 AF214405 AF214251 Pleiocarpa mutica Benth. Potgieter & Albert, 2001 AF214407 AF214253 cult. Royal Bot. on Kew, DQ837539 Civeyrel 1086 (TI Sennblad & Pena E AJ419757 Plumeria alba Kunth Potgieter & Albe 01 AF214408 AF214254 Plumeria inodora Jacq. Sennblad & ipe 11 X91767 Plumeria rubra dress et al., 6 Z70191 Pteralyxia kauaiensis Kauai, Hawaii, Lorence 7768 AM 295073 | AM295085 AM295094 — AM295094. aum (PTBG, Z) Rauvolfia mannii Stapf Endress et al., 1996 Z10181 Sennblad & Bremer, 1996 X91769 Rauvolfia serpentina Potgieter & Albert, 2001 AF214415 AF214261 Benth. ex Kurz Rhazya stricta Decne. Agosti 29 (Z) AM295074 AM295086 AM295095 AM295095 Tabernaemontana Potgieter & Albert, 2001 AF214431 AF214277 citrifolia L. Tabernaemontana Endress et al., 1996 Z70187 divaricata (L.) R. Br Sennblad & Bremer, 1996 X91772 ex Roem. & Schult Thevetia ahouai (L.) A. Potgieter & Albert, 2001 AF214435 AF214281 Bs etia peruviana Pers.) Endress et al., 1996 Z70188 . Schum. Sennblad & Bremer, 1996 X91773 1 antillana cult. o 1 Gard., AM295075 Woodson Meagher 966 (FTG Sennblad & eid 2002 AJ419767 AF214447 AF214293
Potgieter & Albert, 2001
30 Annals of the Missouri Botanical Garden
Appendix 2.
Continued.
Taxon
APOCYNACEAE
GenBank Accession No.
Voucher/Literature Citation math rbeL trol, trnL-F Vinca minor L. cult. Bot. Gard. Uppsala, Sennblad AM295070 230 (UPS) Sennblad & Bremer, 2002 AJ419708 Potgieter & Albert, 2001 AF214449 AF214295 GELSEMIACEAE Gelsemium sempervirens Endress et al., 1996 Z10195 (L.) J. St.-Hil. Olmstead et al., 1993 1,14397 cult. Royal Bot. Gard. Kew AM2950960 AM295090 Civeyrel 1069 (TL) LOGANIACEAE Geniostoma rupestre J. R. Endress et al., 1996 Z 10194. 708828 “orst. & G. Forst. Wellington, New Zealand, AM295097 AM295097
Garnock-Jones 2200 (WELTU)
NDIX 3. Matrix based on the morphological character coding. For some genera more than one species was used for character e 'oding. a = species used to code characters 1-37 (the non- PP | SI |
E E ters): b = species used to code characters 38-54 (the pollen characters). See Appendices 1 and 4.
i 2 3 4 5 123456789012345678901234567890123456789012345678901234
Acokanthera 4i i A. oppositifolia” Allamanda cathar
Alstonia scholaris
Alyxia oblongata”, A. ruscifolia"
Amsonia ciliata", A. tabernaemontana? Inechites nerium
Aspidosperma parvifolium
Cabucala caudata”, C. polysperma" Catharanthus roseus
Chilocarpus denudatus*”, C. suaveolens* Condylocarpon guyanense*. C. isthmicum®” Craspidospermum verticillatum
Kibatalia gitingensis
Kopsia fruticosa
Lepinia marquisensis", L. solomonensis”, L. taitensis*"
Lepiniopsis ternatensis”, L. trilocularis’ Mascarenhasia arborescens
Wolongum laxum
Veisosperma nakaiana
Verium oleander
Ochrosia coccinea
Petchia ceylanica
Picralima nitida
Plectaneia stenophylla, P. thouarsii” Pleiocarpa mutica
Plumeria rubra
Pteralyxia kauaiensis*", P. macrocarpa” Rauvolfia vomitoria
Rhazya stricta
Tabernaemontana divaricata’, T. pandacaqui" Thevetia peruviana
Vallesia antillana*”, V. glabra”
Vinca major". V. minor Gelsemium sempervirens
Geniostoma rupestre
010000000000000010000000000000201101000020100110011001 112000011001100121111012010200200010001020000100101200 012000100000000020110012000300202120000020002010001100
012010000000000010000010101002020100001112210010322000 211000010000000021100012001000202130000020000210011200 1100010110000011201110120100002021???0?0200000001?1240 0110001000000000100100120102002010300000303001000?1201 012000000000000021100010100000001120000020001200011101 210000000000000021102012001001202130001020002200001201 1100100000100000100100120130011000???00112201010211100 1120000000100000100000121010021201???1010????0?1011200 012000100000000020110002000100202130010133001000011100 010002010101110230001012012013101100100023101000311200 0100000001000000211020111000001--130001020002100011101 QOTIIOOOUODOIDOODOTI000011T1101002020100001122?10010222010 011000000010000010000111101002020100001122210010322010 010001010100110230001012012013101100100023101010311000 0100010000011101213100100030003011???200031201010211100 012000000100000021200011100100101120000020002200001001 0120011101000102312100120120131011010001231010?0311000 012000000100000021200010100100101120001021200200001021
0120000000000000211000101000000011??200021001200211000 010001100000000010000011003000300120000021002000101040 11000000000000001000001201020100012??00112010010311100
0100001000000000100001110030003001300000210020001?1100 011000010000000010010002000200202010000020000110101201
0110000000000000100000111010020201???01112210010322000 012000000000000021101010100000001120001020003200011100 211000000000000021100012000100212120000020000210011200
0100010000100000100100100030013211400000200020?0211200 0110010110011011201110011132002020010010200000?0101230 0110000000000000110000111030002--030000030300000011200 210000000000000121102012001001200120001024023001001200 100100010200002-0---0012000200201120000020000000000250 0001020102000000000000020130012011???00023101010101100
2005
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Annals of the Missouri Botanical Garden
APPENDIX. 4.
Characters and character states for the mee sde val
matrix used in the cladistic analyses. See \ppen ix 3. cler states used in 15 study are
re characters and cha
based on the exemplar me ‘Goal only the characteristics of the
species used in the analysis are considered in assigning codes. In cases of large genera with a range of states. this is indicated here.
|. Habit:
perennial herbs.
Q = trees or shrubs: | lianas or vines: 2
The species used to represent Alyxta here.
A. ruscifolia, is a shrub: the great majority of. Alyxia species, us ver, are lianas. . Non-articulated laticifers: 0 = absent; | = present
| Ta articulated laticifers are one of the key characters that
distinguish Apocynaceae s.l. from other Gentianales.
3. Phyllotaxis: 0 = leaves opposite; | = leaves alternate: 2
= E verticillate. Some taxa have leaves that are
predominantly verticillate but may have some
with only two leaves. These taxa are coded as verticillate here
|l. Stipules: 0 = absent: 1 = present. Apocynaceae are
here considered to be estipulate in the sense of Cronquist (1981) and Rosatti (1989). The colleters or interpetiolar ridges found in some taxa are not considered to b
homologous with true stipules, nor are the adaxial outgrowths at the base of the petiole in Alstonia scholaris (Sidiyasa. 1998
some
Small bract- or scale-like organs that are found in species of Rauvolfia have sometimes been called
stipules. In a recent revision of the Neotropical species.
however, Koch (2002) argued convincingly that these organs are : be tter interpreted as cata phy lls.
5. Supernumerary bracteoles: 0 = absent: | = present. Supernumerary bracteoles are e ost rs of 15 acteoles on the
These bracteoles often re-
pedicel subtending the calyx. semble the sepals.
Calveine colleters: 0 = absent; 1 = several. across the inner fac e of the se pal (these sometimes coalesced at the base); 2 = few, mostly in the sepal sinuses. Calycine colleters are a common feature in Gentianales, and their lack
r presence and arrangement is often used in ir ynaceae genera (e.g Stapf, 1902 338-330; Eze ‘urra et al.,
38: Middleton, 1999, fig. 1). le
as an aid in distinguishing e 1933: Rosatti, A QO: Omino, 1996: 87
‘ kei. gu ia ruscifolia, although col
1080:
of tthe sepals themselves, they are we I developed in the many the calyx. In
having
supernumerary bracteoles clustered below
Plumeria oded as
Endress et al. (1996). was a continuous row of calyeine colleters. Dele aile d examination, however, has shown that they are not homologous to typica calycine colleters in that the entire inner surface of the upper part of the sepal is glandular. Because no other taxon shares this condition in this study, it is non-informative and thus not coded here.
7. Fusion of corolla tube: O = fused just above the level of insertion; | =
stamen unfused (with gaps) just above the
evel of stamen insertion. In Apocynaceae, the lower corolla ube (composed of the united petal and stamen primordia) is congenitally fused; the upper part fuses postgenitally and basipetally, so that the last region to fuse is just above the insertion of the stamens. In some genera, flowers reach upper corolla is complete. 3 Nishino,
anthesis before hoan of ZW resulting in gaps in the corolla tube (Boke, 194€ 1982: Erbar, 1991).
\sprdosperma, Geissospermum. Haplophyton. and Jeune
These gaps are especially long in l g
tegia, resulting in consple uous slits that are easily isible
with a dissecting microscope (Woodson, 1951: Fallen. 1080
Leeuwenberg, 1997). Shorter gaps of some microns in length
(visible in microtome serial sections) are also found in
n tonta, C raspidospermum. and Hunterieae, whereas in other
genera epidermal remnants are still Wu 0 8 gh there 1 199
are no distinc gaps. In. Endress et al. ). epidermal
remnants and gaps were treated together as a 2 character state: corolla incompletely fused. Because. however, the tube may be fused yet still show epidermal remnants, here only
the presence of distinct gaps, visible with dissecting
microsc A or in serial sections, is coded as unfused.
8. rolla tube mouth: O = constricted: 1 = not cit |. Infrastaminal appendages: 0 = absen = present,
ene al appendages is a term used by P ic 2d 1948b)
for 5 of the lower, congenitally fused part of the
1 the staminal sectors (see Alvarado-Cárdenas
found mainly in tax l
Cerbera, Thevetia,
as ga
previously inch
Cerbertopsis). These genera long, thin style and
a disproportionate ‘ly large, broad style head. LO. Corolla lobe aestivation: 0 = sinistrorsely contort; | 2 = imbricate. Corolla lobe aestivation is
dextrorsely conte f the Apocynaceae. Vith a few
one most importan M. characters in
exceptions. the genus. Un Rauvolfioideae
constant within a
are almost always sinistrorsely contort, whereas in Apoc-
vnoideae. > normally dextrorsely contort or. rarely, valvate. n Aopsia, Ochrosia, and Neisosperma (all moldea. are 1 dextrorsely
2004: Middleton. 2004) and . Alstonia is one of the few genera in the
contort pen thus an
\ which both sinistrorsely as well dextrorsely contort sal occur, and this feature is constant only at the ES cles lev
. Pe e in bud: 0 = contorted petals in bud are spiraled upward
not inflexed: | = inflexed. In most
a
into a tip. I Petals that are inflexed in bud are. instead, folded downward and eer into the mouth of the corolla tube and | . Infle elatively uncommon andit in pom vnaceae.
only unfold at anthes xed petals is a
12. Gorolline corona below petal sinus; behind and/or just
above pom O = absent; | = a compact protruding lobe.
All outgrowths in the staminal sector and above the insertion
of the anther are inte 1 5 ted here as a corona. These include
the vertical ridges in Molongum Pichon, as well as the
ao rances termed casi appendages by Pichon ) ibr
(1948b) in Thevetia; the fin © lobes of Allamanda are also inte n ted as a corona (se e cde ss et al.. 1996). Ll thers: 0 =
atop filaments that arise from the corolla m — sessile upon 1 staminal ridges
rails: = absent: | = present.
14. Lignified guide Lignifie 4 ane rails are a spec n of the late al parts of
the anther and have a function in the complex pollination
mechanism in VH M ee ps are absent in most
Rauvolfioideae. but are characteristic for Apocvnoideae. It
is important to note that re guide rails are also
characteristic for the majority of Tabernaemontana species (including all of the Ne ixl “al vue although absent in the Ms
all of
Leeuwenberg,
wo 's used in study and in
'epresentalive spec section Ervatamia to e h they dd (sce 1994b: xv). TI or this cha Tabernaemontana would show more affinity morphologically i resentative 199
Thus. for racter, most species of
to olongum than ds parent from the repr
species 1 ised here oa with Endress et : » in whic ho a oo val species was used in the mol!
analys
Volume 94, Number 1 007
Endress et al. 33 Phylogenetic Analysis of Alyxieae
Anther dehiscence: 0 = introrse; | = latrorse; 2 = extrorse.
16. Anther/style situated above or below, but not closely synorganized with, the style head; | nthers at about cn same level as, connivent over and in dis the s st agglutinated to the style head via is pads and adhesive.
head synorganization: anthers and e head: 2 — anthers Synorganization of the anthers and the style head has always
been a key character in Apocynaceae. It is the mos! important traditional character that separates Apocynoideae (in which the anthers are postgenitally united with the style
in which the anthers are free from
head) from Rauvolfioideae the style heac
The lack of close synorganization of the anthers and style
—
head in Tabernaemontana divaricata is not typical of the whole genus (as defined by Leeuwenberg, 1991). In all Neotropical species of Tabernaemontana and in some
Paleotropical ones as well, the style head and anthers are more closely svnorganized and would be coded as character state l in this study.
0 =
un
H7. Style apex e cretory epithelium: 1 = style e style head with epithelium of the body uniformly
style apex without transformed into an 2 = style apex transformed into an
secretory and re live
enlarged style he ad with qe of the a vertically
differentiated with stigmatic region at base: : style apex transformed into an enlarge d style head, swith ente ‘lium of the body vertically differentiated, stigmatic zone at base. and
radially mechanically interrupted by the adnate 1 TES All
; h 1
e and manner of histological diffe -rentiation of the style head and the e pithelium is uiae. with a specific type often characteristic of a tribe (Schick, 1980; Fallen, 1986). Although the gynoecium apex in Geniostoma J. ; & C.
articular
Forst. is
irritable hairs with an abscissable lip are found scattered among the more numerous smaller, normal papillae (Endress et al., 1996). Specialized glue hairs like those found in Geniostoma are unknown in Be cae Style head upper hair wreath: 0 = absent; | = present. Some style heads have a wreath of longer hairs just below the unfused carpel tips. The main function of the wreath is for pollen deposition and secondary presentation. The flowers are protandrous; shortly before anthesis, the anthers ie and shed their pollen toward the center of the flower. If the style head has an upper wreath, the pollen is shed onto this ring of hairs, which plays a role in the complex pollination mechanism of Apocynaceae (Church, 1908: Schick, 1980: Fallen, 1986). 19. Style head pase, : = a distinct, thin collar; 2 = with a wreath of longer hairs; 3 = with thic e often equipped with a means of seraping off donor pollen from the
without collar or flange; | = with
flange. base of the style head is
proboscis of an insect visitor. The presence (or absence) and type of scraper is often „ of a particular tribe, and thus a useful character in 19 0 ily. When a scraper is present at the base of the located beneath it (Schick. in 20. Style head unfused apices: a = a less than 1/3 the conical and tapering to blunt and clavate, at least 1/3 the length of the total style 21. Free dise 5 0 = absent:
. the 1 7 ui zone is Fallen. 6)
inconspicuous, length of ihe ie style head; 1 = enlarged,
read.
entire, annular; M
|= = two se 17 lobes. In Apocynaceae, a free nectar dise is
often present. Sometimes the nectar dise is adnate to the base 50 ll R
of the ovary ne taxa (especially in Rauvolfioideae) are
tarless ar m appare um use deceit pollination (Haber. 1983 Lin & Bernardello, 1999). In some cases it is difficult to distinguish mx ther or not the base of the ovary is nectariferous. For this reason, only the presence versus
absence of a distinct nectary disc is coded here. : the large
genus Alstonia, this character varies from spe 0 species.
The species included in this study, A. a. 1 a shallow
disc. In some other species of Alstonia, a slight
thickening can be discerned at the base of the ovary, and in vet others there is no nee ‘ation of a nectary al all. | = 3-5-carpellate.
22. Ovary: 0 = 2-carpellate;
brush Apocynaceae a the n is composed
of two carpels. The only exceptions are found in Lepinia and
i di ac. in the Alyxieae and in Pleiocarpa in Hunterieae )
en A s et al., 1997).
“Pl acentas: 0 = lignified or indurated in fruit; 1 = not iei e or indurated in fruit. . Mesocarp consistency: 0 = fleshy, without fibers; | =
a with fibers; dry or woody.
25. Endocarp: 0 = not forming a stone around the seed: |
E ied or sclerified and forming a stone around the seed. eeds: 0 = l= funi
2 due shape: 0 = broad, compressed, not folded, mostly
= cylindrical, as if longitudinally rolled:
sessile: culate.
circular to ovoid: |
2 = narrowly fusiform, flattened, with a longitudinal fold: 3 = irregularly shaped, globular or angular, not flattened. the other
1:0 = with neither flattened e s nor wing: 2
flattened on one side only, "side con
28. Seed marg | = with a narrow clara] edge, this sometimes dissected: = with a well-developed, usually membranous wing(s); 3 = fimbriate.
29. Seed coma: 0 = absent: | = present. A coma is a tufl of hairs 7 arising from a small restricted region at the end(s) of a seed. not considered to be homologous to the fimbria that are Tee around the margin of the seed in, for example,
Alstonic
30. Hilar depressi 0 = absent: 1 = an ipa depression, less than 50% | the length of the seed: 2 = a deep, broad furrow, traversing the entire seed length: 3 =
a deep, narrow fissure, traversing 75%-80% of 1 5 E of the seed.
31. Hilum e = linear, traversing the » TEE of the
seed: | = linea ae die than the seed: = md
circular (punc im; 3 = ovate, cove nup a pod ar
with shallow.
—
32. Endos "sperm: not ruminate:
irregular 1 or ruminations; 2 = with deep longitudinal ruminations. Ruminate ‘oop although relatively rare in Apocynaceae, is racteristic for Taber-
naemontana and is also found in 8 genera of Alyxieae. Chilocarpus is unusual in this aspect in that the genus can be divided into two groups: those with smooth and those with 1949¢; Markgraf, 1971). The
representative species used in the analysis here be 1 Lo
ruminale endosperm (Pichon,
smooth endosperm: IM had a JEC ie S from the
Chilocarpus would fit better with
the group with other group been selected, other Alyxieae as to this character.
33. Endospe rm: 0 = tough A corneous to subcartilagi- cate, soft or mealy.
nous; 1 = firm, fleshy or starchy; 2 = de
There is consi e thickness of the endosperm. For 7 the endosperm of Alyxia, Chilo- carpus, Condylocarpon, Lepinia, Lepiniopsis, and Pteralyxia is especially thick and tough (even difficult to cut with a razor blade). In Allamanda, Picralima, Plectaneia, and Pleiocarpa. endosperm is also tough but much thinner, but because no
34
Annals of the Missouri Botanical Garden
“thick” and “thin” could be
consistency of the sk rm is code
clear demarcation between
found, only th
nol aurict M
iculate at the base in IO
91). this was nol
7
Tabernaemontaneae (sensu encia l
alkaloit
iridoids present, indole alkaloids e nl
= Uae indole alkaloids prese nt. but only those 15 secologanin skeleton; 3 = dominant indole with rearranged secologa-
st nin skeleton of the eburnan and/or Place da type;
A = dominant indole alkaloids B nt, including those with rearranged secologanin pin of the ibogan type
36. Cardenolides: 0 = absent; | = present 37. Steroidal alkaloids 0 = absent: | esent.
= tetrad. Tetrads are rare in Rauvolfoideses ind of the taxa sampled here they oceur only in 1 arpon and Craspidospermum.
. Pollen grain: 0 small (S 51 um); 60 Average largest pollen grain size (either the length of the polar axis, P, or the diameter of the equatorial plane, E) varies between 25 and 90 um. It appears that a relatively 60 um and that only
cs large [zm
large gap exists between 51 and (56 Um) falls between. € coding polle n size either as
60 Um). with one ambiguous
Inechites small (= 5 case, gives two rather we e separated size classes
um) or as large (=
40. Pollen grain shape: O = regular; | = irregular. Pollen
grains with a regular quie have a * rlurale ape rlure f h i
apertures that are unequally spaced and sized and have an oblique orientation. Irregular pollen grains with three porate apertures have unequally spaced and sized apertures with oblique orientations.
= one or two; 2 = three
41. Aperture number: 0 = zero: |
or four; 3 — five or more. In Rauvolfioideae, only Condylocarpon has inaperturate pollen (aperture number = Two- e pollen (sometimes mixed with l-
1 Chilocarpus, Plectaneta, and eS a
within a single sample) is found in most other genera. A few
zero). l-aperturate pollen (often mixed genera NE, ive or more apertures (up to 10 apertures are found in Craspidospermum). In most samples studied, minor percentages of polle n grains with deviating aperture numbers are found, which is a common phenomenon in dicots. The coding given is for the dominant 1 numbers.
42. E cloape rtures: 0 = long colpi; ! = 5 E
r than ca.
large port; 3 = small po
ectoapertures are either longe 2/3 (long colpi) or shorter than ca. 1/3 (short ado) the length of the polar axis (P). Large least 6 Um: if 6 Um (Chilocarpus, Plectaneta), then they are always accompanied by larger pori (up to 9 and 12 um, respectively) in the same E n grain. size in
pori are al
Small pores are 2-5 Um and do not vary much i
a single grain. Due to its thin outer exine, Vinca 0 has
indistinct ecloapertures.
43. Ee cloaperture margin: 0 = P pid: thickened: akly ardly distinc ly
= outwardly thickened; 3 = ectoaperture margin is usually not thickened. In genera Dun
ickened:
viti: conspicuous aret ns ridge
S
arge pores, but also in the brac hycolpate Molongum. it i distinctly thickened into a well-de limite d, protruding margin
(aspidate pollen). In the genera with small pores, the I | 8 |
ecloaperture margin is nol or only weakly thic kened.
\spidosperma and Vallesia have conspicuous ridges (massive respectively) along the colpi joining
9 partly hollow,
toward the poles.
14. Endoapertures: 0 = distinct from and smaller than ecloapertures: = not distinct from ecloapertures; 2 = distinct from and larger tl ectoapertures. ! xia, Lepinia, Lepiniopsis, Plectaneta, and Pteralyxia (all with
porate polle n), the e ndoape rtures are nol delimited from the ectoapertures (e ndo- and e cloapertures congruent). ra. the endopore is distinct by being situated in in all other taxa
porate gent a differentiated inner exine laye the endo- and ectoapertures are incongruent. In colporate
er, and also 1
pollen grains, the endoapertures are E ways smaller than vo which the endoa- the
the ectoapertures, except in Vinca,
pertures (delimited by costae) are larger than V
. Endoaperture margin: : 0 = = nol inwardly bus kened:
ings at the 1 1 8 of circular to lalongate endoaper- lures.
16. ve mentary endocolpi: 0 = absent (no endoplates recognizable weak (zones P endocracks: endoplates indistinct): = Supplementary endocolpi are narrow (e.g.. Cabucula) to wide
distinct (endoplates well recognizable)
(e.g... Rhazya) zones along the colpi (one at each side) where
the inner exine layer is more or less missing. They may join interaperturally toward the poles. In some genera (e.g. Catharanthus). they seem to have taken over at least some of
the function of the ectocolpi, bordering on distinctly thicker X
intine parts (see El-Ghazaly. 1990, fig. 17). Usually.
supple mentary e pt have a granular inner surface and
smooth
fea T a
7. Intine A 0 = absent; present. Coding of argely based on data provided by Pichon
1950a. b)
ae charac ter is
Protruding intine bulges at o O
intricately channe led structure. In Alyxia, there s to be a kind of relatively rigid operculum topping the protrusion (Huang. 1986).
Exine: O = (thin). Condylocarpon and Vinca have a thin exine (0.1—0.6 um and ca. 0.1 Um. respectively), whereas in the other genera, exine thickness is at least 0.9 um, but is usually much
reduced reduced
nol
48
thicker. male;
¿0 = psilate; | = 2 or the inner
This c 1 ‘ter m
49. Inner exine surface
verrucale: 3 = granular. ornamentation of the exine (nexine surface) and was taken from scabrate surface has elements smaller than | um. Verrucate and granular elements are larger than 1 um, the former with
the inside of the mesocolpia (mesoporia) centers. A
a broad base, the latter with a constricted base.
50. Inner exine layer 1 O = inous (foot. layer/endexine); | = ect = endexinous (foot layer absent).
ectexinous/endex- xxinous (endex This character
xine absent): 2 codes for the composition of the inner exine layer (nexine).
Volume 94, Number 1 2007
Endress et al. 35 Phylogenetic Analysis of Alyxieae
a lamellate de
Endexinous parts are indicated by e lines; . Ectexinous parts
structure and/or a + spongy oe
homogeneous. Usually endexinous and ectexinous parts is in contrast. nfratectum: 0 = columellate: J = granular, reticulate or a gular: = not recognizable (commissural line). |
RP NUN s e is found only in Gelsemium. In most other genera, the infratectum is granular, and varying in thickness. In Alyxia, Lepinia,
Pieralyxia, an indis-
reticulate
or irregular, infratectum is
Lepiniopsis, and i the contact be-
tinct (sparse gaps in inner exine layer), tween ectexine and endexine being largely a commissural
line. 52. Tectum: O = thicker than infratectum + inner exine layer; 1 = equal to infratectum + inner exine layer; 2 =
thinner than infratectum + inner exine layer. This character codes for the thickness of the tectum ene with the rest of the exine (infratectum + inner exine layer). It is also an position of the
When
approximate measure for the relative infratectum. Usually the tectum is well delimited.
the boundary is irregular (e.g., in Plectaneia), the average
tectum thickness was measured. Outer exine surface: 0 = psilate (even to undulate `); with angular
srrucale, anastomosing verrucae; 4 =
verrucate, with + circular isolated verrucae: 3 = micro- reticulate; 4 = seabrate; 5 = _ striate-reticulate. This character codes for the outer ornamentation of the exine (tectum surface). Most genera have psilate pollen (no
protube rances), with an even to undulate, often perforate surface. Lepinia and Lepiniopsis have verrucate pollen with anastomosing verrucae. The other states, except for scabrate exine, occur in single genera.
. Mesoc pump ium centers surrour adie exi
0 = outer bus e = outer
hardly or not deviating from ne surface cle arly de >V ed from surrounding exine. cn about 1/ 4 of the sampled genera with colporate polle n, the outer n es of the mesocolpium centers have a different ornamentation compared with the 3 areas. Usually microfossulate to
the mesocolpium centers have a rugulate,
verrucate, or a less distinctly perforate ornamentation.
PHYLOGENETIC RELATIONSHIPS Livia Wanntorp?? and Paul I. Forster! BETWEEN HOYA AND THE
MONOTYPIC GENERA MADANGIA,
ABSOLMSIA, AND MICHOLITZIA
(APOCYNACEAE,
MARSDENIEAE): INSIGHTS FROM
FLOWER MORPHOLOGY'
ABSTRACT
The e genera Absolmsia Kuntze, Madangia P. I. Forst, Liddle & I. Addle, and V 5 zia N. E. Br. (Apocynaceae, 9 85 nieae) are, according to molecular phylogenetic results, nested a in the genus Hoya R. Br. a with Dischidia R. Br., these genera are considered to e omprise a well-supported clade within the tribe Va mieae. Flower
dpi of a Madangia, and Micholitzia is examined by SEM and light microscopy and compared to that of the related H. caudata Wook. f., H. curtisii King € Gamble, H. lc Schltr., H. lacunosa Blume, H. ao Schltr., H. retusa Dalzell, and H. venusta Schltr. We discuss the Morpholngic val evidence that supports the reduction of these genera into Hoya
Among characters supporting such a position is the presence of : Sh id margin on the pollinia of Absolmsta, Madangia., Micholitzia, and Hoya that is absent on the pollinia of Dischidia. 9 and Madangia vem a staminal corona with outer er lobes, similar to that of Hoya. While the outer corona processes of Absolmsta nd Micholitzia are free and revolute,
as In most species of Hoya, those of Madangia are laterally fused forming a continuous skirt resembling the corona of certain species of Hoya. The flowers of Micholitzia are superfic ial similar to those of Dischidia in its urceolate corolla, but the
sli E corona with revolute outer processes dec idedly links Micholitzia to Hoya. The new combination M. inflata (P. J. N.
Forst, Liddle & I. M. Liddle) L. Wanntorp & P. I. Forst. is proposed. For Micholitzia obcordata E. Br., the name A. yuennanensis Hand.-Mazz. has nomenclatural bs and should therefore P ised when including M. obcordata in Hoya. ey words: Absolmála, Apocynaceae, corolla, corona, Dischidia, Hoya, Madangia, Marsdenieae, Micholitzia, morpho-
logy, pellucid margin, phylogeny, pollinaria, pollinia.
The tribe Marsdenieae Bentham (1868) (Apocyna- these studies, leaving several relationships within the ceae, Asclepiadoideae) is thought to include 29 Marsdenieae unresolved. genera and over 570 species distributed in the New One of the genera in the Marsdenieae whose and the Old Worlds (Omlor, 1998). The taxonomic taxonomic delimitations remain unclear is Hoya, the positions of the genera Cionura Griseb., Dregea E. wax plants ofo ornamental horticulture, a large tropical
Mey., Dischidia R. Br., Gymnema R. Br., Hoya R. Br., genus of over 300 herbaceous to succulent species of Marsdenia R. Br., Micholitzia N. E. Br., Rhyssolobium Which about 40 are commonly found in cultivation E. Mey., Stephanotis Thou., and Telosma Coville have (Forster et al., 1998, 1999, 2002), although many been examined in previous morphological and more are cultivated in specialist collections. Species molecular phylogenetic studies (Liede & Albers, of Hoya are distributed from mainland Asia (China, 1994; Forster, 1995; Endress & Bruyns, 2000; India, Vietnam. Burma, Laos, Cambodia) through Potgieter & Albert, 2001; Sennblad & Bremer, Malaysia, Indonesia, and New Guinea to Australia and 2002; Rapini et al., 2003; Livshultz, 2003; Meve € the western Pacific islands (Forster & Liddle, 1996). Liede, 2004; Wanntorp et al., 2006a). However, only Most species of Hoya are laticiferous wiry climbers,
a few species of each of these genera were included in often epiphytic, with fleshy to succulent leaves of
We thank Prof. S. S. Renner (Ludwig Maximilians University, Munich) for supporting the laboratory work of this study: Bostróm, €. Forsberg-Heikkilá, D. J. Liddle, and T. Nyhuus for sharing flower material: E. Facher, M. Vosyka, and A. Kocyan (Ludwig Maximilians University, Munich) for laboratory assistance; and D. Goyder (Royal Botanic Gardens. Kew) for kind help
with the herbarium specimen of Hoya spartioides. This paper is part of a research project by the first author supported by the
Swedish Research Council.
Ludwig Maximilians University Munich, Institute of Systematic Botany, Menzinger Strasse 67 D-80638 Munie h, ES rmany. 0
: dress: Department of Botany, Stockholm University, Lilla Frescativ. — 10691
Correspondence and current a Stockholm, Swedenlivia.wanntorpObotan.su.se.
"Queensland Herbarium, Environmental Protection Ageney, Brisbane Botanic Gardens, Mt. Coot-tha Road, Toowong Qld 4066, Australia.
ANN. Missouri Bor. GARD. 94: 36-55. PUBLISHED ON 26 APRIL 2007.
Volume 94, Number 1
Wanntorp & Forster Phylogenetic Relationships of Hoya
various size, color, and venation. A minority of species are non-climbing and with foliage that is either erect or pendent. The sympetalous flowers are clustered in umbelliform extra-axillary inflorescences mostly with perennial peduncles ( 1998). The complex, fleshy flowers of Hoya comprise a pentamer- ous corolla that is highly variable in shape, color, and
dimensions; a staminal corona with inner processes
Forster et a
..
adnate to the anther connective and outer processes with revolute lobes; a gynostegium, standing on a short or long column above the corolla and including five
fused anthers, the ovaries, and the style head (Forster
et al., 1998); and two pollinia from adjacent anthers, united by a translator apparatus secreted. from the
style head to a pollinarium (Omlor, 1998; Kleijn & van Donkelaar, 2001).
A morphological revision of the Marsdenieae placed Hoya, Dischidia, and the "EU. genera Absolmsia Kuntze, Madangia P. I. , Liddle & I. M. Liddle, and Micholitzia 1 in a 1 sharing an epiphytic habit, fleshy to succulent leaves (in Absolmsia only present at an early age stage), generally persistent extra-axillary inflorescences, and comose and fusiform seeds without prominent wings but with keel-shaped thickenings on the seed coats (Omlor, 1998; Livshultz, 2003). In addition to Omlor's work, a possible close relationship between Micho- litzia, Madangia, Absolmsia, and Hoya was discussed in morphological papers specific to each of these genera (Goyder & Kent, 1994; Forster et al., 1997; Kloppenburg, 2001). While the first two of these studies discussed the similarity of Madangia inflata P. I. Forst, Liddle € I. M. Liddle and Micholitzia obcordata N. E. Br. to Hoya without taking position egarding the relationship of these species to Hoya, Kloppenburg (2001) concluded that A. spartioides Kuntze should be included in Hoya based on its morphological similarities in the corolla and corona. Kloppenburg therefore proposed a change of name for the single species of Absolmsia to H. spartioides (Benth.) Kloppenb. None of these studies, however, examined relationships using a phylogenetic ap- Another species has been referred to the This taxon
—
0
proach. genus Absolmsta, namely was, however, recently shown to belong to the genus Tylophora R. Br. (Gil 1995).
Recently, two molecular phylogenetic studies based on chloroplast gene regions of the m intron, trnL- trnF spacer, and atpB-rbcL spacer, as well as on nuclear sequences of the ribosomal ITS region, were conducted to examine the phylogeny of Hoya and its taxonomic delimitation (Wanntorp et al., 2006a, b). three species of Dis- Madangia inflata, and together with
oligophylla. I
vert et al.,
—
Thirty-five species of Hoya, chidia, Absolmsia spartioides, Micholitzia obcordata were analyzed,
species representing subfamilies Periplocoideae and Secamonoideae, as well as tribes Fockeae, Marsde- nieae, Ceropegieae, and Asclepiadeae (sensu Endress & Stevens, 2001; 2003). A major result of these studies was that Hoya is paraphyletic without the inclusion. of the Absolmsia, Madangia, and Micholitzia, which are all found clearly nested among the 35 species of Hoya examined ( 2006a, b).
The aim of the present study is to examine the flower morphology of Absolmsia spartioides, Madangia inflata, Micholitzia obcordata, of closely related species of Hoya by using light and SEM microscopy. We discuss which morphological
characters bear on the phylogenetic and taxonomic
Verhoeven et al.,
monotypic genera
Wanntorp et al.,
and a selection
positions of these genera within Hoya.
MATERIAL AND METHODS
We examined the floral morphology of the species Hoya caudata Hook. f. H. hypolasia Schltr., H. H. retusa Dalzell,
5
Absolmsia spartioides, curtisii King Gamble, lacunosa Blume, H. patella Schltr.,
H. venusta Schltr., Madangia inflata, and Micholitzia
obcordata. Flower material at anthesis was collected and stored in 70% ethanol at the Botanical De- partment of Stockholm University, except for the
material of Madangia inflata provided by D. J. Liddle (Queensland, Australia), of Absolmsia spartioides by T. Nyhuus (Järfälla, Sweden), of H. curtisii by A. Boström Stockholm, Sweden), and of H. hypolasia and H. venusta by C. Forsberg-Heikkilä (Porvoo, Finland). The identity of all species examined was confirmed with the literature or by comparison with original type reports voucher information for the
—
* Ae
material. Table specimens examined.
SEM and light microscopic studies were performed on all species mentioned above with the exception of Hoya hypolasia and H. venusta, which were only examined by SEM microscopy, due to scarce material. For SEM microscopy, all flowers were dehydrated in an alcohol-acetone series, critical-point dried, and sputter-coated with gold. SEM microscopy was done with a LEO 438 VP scanning electron microscope (Zeiss, Jena, Germany) at the Department of System- atic Botany, Ludwig Maximilians University, Munich. For sectioning, microtome sections were made using 2-hydroxyethyl methacrylate embedding (Igersheim & Cichocki, 1996), cut at 10 um, and stained with toluidine blue and ruthenium red. When discussing the morphology of a single corona lobe, we use the term corona lobe, while we define the inner and outer parts of each lobe by using the terms inner (ip) and
—
outer (op) processes, respectively.
Annals of the Missouri Botanical Garden
Table 1.
Herbariorum (Holmgren et al., 1990).
Voucher information on the specimens used
the present study. Herbarium. abbreviations. follow Index
Species
Voucher information
Vosolmsia spartioides Kuntze
Hoya caudata Hook. f.
Hoya curtisii King & Gamble Hoya hypolasta Schltr.
Hoya lacunosa Blume Hoya patella Schltr. Hoya retusa Dalzell
Hoya venusta Schltr
Madangia inflata P. V. Forst., Liddle & J. M. Liddle
Micholitzia obcordata N. E. Br
592 (5), Sipitang, Borneo
593 (S), ex hort. Department of Botany, Stockholm University
597 (S). 594 (S) 571 (S), Stoc kholm University
Wanntorp L. Wanntorp L. Wanntorp L. ex hort. Wanntorp L. y ex hort. Wanntorp L. ex hort. Department of Botany, Wanntorp L. 575 (S), ex hort. Department of Botany, EM niversity
380 (8). 1998-3127, Rosendal Uppsala,
Ippsala pups al Garden
Wanntorp L.
; 1 L. 595 (S), ex hort. ). J. Liddle 75 1076 (BRI), Madang Province, 596 (S), ex hort.
New Guinea
l anntorp L.
ResULTS
1. Hoya spartioides (Benth.) Kloppenb., Fraterna 14: 8. 2001. Basionym: Benth. in Hook., le. Pl. spartioides (Benth.) Kuntze, as 418. 1891. TYPE: 4 Burbidge s.n. fl). 2 of 2 (t)] not
Astrostemma spartioides 1880. Absolmsia “spartoides,” [Malaysia]. (lectotype,
Revis. Gen. PI. 2: North
designated here, K [1 of 2 (
Borneo, s«
seen).
Hoya spartioides is an epiphytic, sparsely branched, non-twining subshrub with fleshy leaves from Borneo. Inflorescences pedunculate, comprising concave um- bels with 4
6 flowers; pedicels ca. 1 mm long,
glabrous, rough. Calyx lobes obtuse-triangular, ca.
0.02 0.02 mm. Corolla shortly campanulate to salver-shaped, orange-yellow, glabrous, ca. 10 mm diam., with 5 triangular oval lobes, ca. 3 X 3 mm (Fig. 1760 Gynostegium on a short hairy column (Fig. IC). Corona flat, star-shaped, white, smooth,
covered by thick hairs on the upright part (Fig. LA—C,
Harrow,
L
E, G); inner processes of lobes spathulate,
appressed to the anther appendages (Fig. 1D); outer
lobes with deeply revolute processes (Fig. 1D, E);
—
anther appendages lanceolate-acute, approximately as long as the corona lobes (Fig. IF. C); style head flat
(Fig. 1H).
nected by thickened winged caudicles to a corpuscu-
Pollinarium consisting of 2 pollinia con- lum: pollinia square in outline, almost as long as wide. ca. 0.3 X 0.2 mm (Fig. 2E, G, H), the pellucid margin very wide but covering only ca. 1/3 of the dorsal margin of the pollinia (Fig. 2H); corpusculum thick, * 0.2 2 long, spreading appendages, upper part enclosing X 0.1 mm (Fig. 2F
almost round in shape, ca. 0.2 mm, ending with
the caudicles, ca. 0.2
The herbarium specimen chosen as lectotype for Hoya spartioides was selected among the collections available for this plant at the Royal Botanic Gardens, Kew (D. Goyder, pers. comm., 2007)
Brit. Ind.
“Malacca,”
H10): 60. Maingay
2. Hoya caudata Hook. f., Fl. 1883. TYPE: 1128 (holotype, K not seen).
| Malaysia].
Hoya caudata is an epiphytic twiner with leathery- fleshy leaves from the Malay Peninsula and Thailand.
Inflorescences pedunculate, comprising | geotropic,
concave umbels, with up to 12 pendent flowers:
pedicels longer in the periphery of the inflorescence
and shorter in its middle, 2-3 em long, hirsute. Calyx
lobes linear-lanceolate, ca. 2 X ca. 1 mm, glabrous.
Corolla flattened-campanulate, pink-yellow, inside
o»ubescent in the middle, with long caudate hairs al | 8 lobes ovate-
the margins (Fig. 3A, B), ca. 2 em diam.:
—
triangular, acute-cuspidate, 4-7 X 4-5 mm (Fig. 3A). Fig. 3A).
Corona with a dark red ring in the middle but lighter
—
Gynostegium stalked on a short column
at the margins, smooth; inner processes slightly
B
»nnüng up (Fig. 3C); outer processes very wide, } o o / | J
bending upward, not deeply revolute (Fig. 3C); anther and whip-like, very much ex-
3B—D); style head long-
apiculate (Fig. 3D). Pollinarium comprising 2
appendages slender ceeding the corona lobes (Fig. pollinia connected by broadly winged and flattened caudicles to a corpusculum (Fig. 3E); pollinia longer than wide, cylindrical-elongate, ca. 08 X 0.2 mm; pellucid margin covering the dorsal margin of the pollinia; corpusculum. narrow-rhomboid, ca. 0.2 0.1 mm, ending with 2 long, spreading appendages (Fig. JF); caudicles ca. 0.1 X 0.1 mm (Fig. 3E).
Wanntorp & Forster 39
Volume 94, Number 1 Phylogenetic Relationships of Hoya
2007
Hoya a —A. B. on views of flowers showing remains of the corolla (el), corona (co), guide rail (gr). Side view of flower l (c pm . Details of coronas showing inner processes (ip aa). and Mn ulum (cq "n 1 il sec oy of f lowe T E. the postca and UNE appendages (aa . Detail of flower (top view) with Light mic eh section (longitudinal view)
= | mm (A, C), 4 mm (B), 100 um (F, H
Figure !. and aa ulum (cp). — ouler processes (op), Ear 'r appe ndages { of inner corona processes (ip), guide rail (gr), Ae us corona (co) with hairs (h), anther appendages (aa), : of flower showing the style head (st) and part of s corpusculum (cp). Scale ER
200 um (E). 300 um (D, C).
orpusculum (cp).
=>
Annals of the Missouri Botanical Garden
SS US o EX
XQ N
(AN
. pollinia and
anther append: wes (ad).
Hoya inflata. —A. Upper part of flower seen from above with inner (ip) and outer (op) corona proce showing the pos sitions of coronas (co).
(ca). and pollinia (po) with > ue id margins (pe) and one
l . Side view of pollinaria
E Pollinarium seen from
re 2. B. Gross section of flower D. Pollinaria with corpusculum ( cp). caudicle D). Hoya spartioides. —F. I ged caudicles. —H. Pollinia (po) with
(B). 100 Um (C.
Figu
(po). and St tyle ies (sl).
pollinia (po). —C. D.
anther appe om ge (aa). Scale bars = 1 mm (A). 4 mm
formed by the corpuscula (ep), the caudicles (ca), and the pollinia ( | (po) with pe Nue id margin (pe). \rrows denote long appendages from the corpusculum partly covering the
bread: pellucid margin (pe) and caudicles (ea). Seale bars = 30 um (F-H), 100 155 (E)
be low
Volume 94, Number 1 Wanntorp & Forster 41 2007 Phylogenetic Relationships of Hoya
ir Raa ate Figure 3. Hoya caudata. —A, B. F lowers showing remains of corolla (cl) D 80 (h), corona (co) with inner (ip) and ouler (op) processes on a column (c 10 anther appendages (aa). and style head (s . Detail of flower showing 1 0 ri ails (gr). —D. Upper part of flower with anther appendages (aa). style head (st), and im (po) with falcate caudicles (ca). — Details of a pollinarium: pollinia (po), caudicles (ca), and corpusculum (cp). —F. Detail of flower showing the rM 5
guide rails (gr), corpusculum (ep), and style head (st). Seale bars = 1 mm (A-C), 200 um 100 30 um (E). 100 um (F).
Asiat. Soc. narrowly triangular, ca. 2 X | mm. Corolla rotate,
—
3. Hoya curtisii King & Gamble, | Bengal, Pt. 2, Nat. Hist. 742): 563. 1907. TYPE: creamy white, inside completely pubescent, except on Malay Peninsula, Curtis 2894 (holotype, SING!). the margins, ca. 0.7 mm diam. (Fig. 4A); lobes
oblong- oblanceolate, acute at the lips, reflexed,
Hoya curtisii is an epiphyte with non-twining 7 X 4mm. Gynostegium borne on an annular
pendent stems and succulent leaves from the Malay structure (annulus) on a high column (Fig. 4A, B,
me
Peninsula. Inflorescences pedunculate, comprising D). Corona red in the middle and white at the margins: convex, globose umbels with up to 30 flowers; lobes sulcate, with a central depression or an umbo
pedicels ea. 22 mm long, densely hairy. Calyx lobes (Fig. 4A); inner processes blunt (Fig. 4E); outer
45 Annals of the Missouri Botanical Garden
má ` yr Je 5
Figure 4. Hoya curtisii. —A. D. Flowers with calyx (ex). Ea ui corona (co) with inner ed (op) processes, annulus (an), guide i (
s (gr). tyle ena . and pollinia (po). — B. ¿nlarge ment * E position f the annulus (an). „ Part of f Mos Y bonne ini rails (gr), corpusculum (cp). 2 style head (s -E. Single coron 85 lying on annulus (an) with inner (ip) and outer 1 9 processes 7. Part of flower showing coronas (co), ed rails (gr), “pollini 1 (po), and style head (st). Scale bars = Iı (A, B), 2 2n mm (D), 100 um (C), 300 um (E, F
processes made of 3 parts, of which 2 wide and bent lum ca. 1 X 0.3 mm, ending with 2 long. spread- against each other, and the third part bending upon ing appendages: caudicles ca. 0.1 X 0.04 mm these 2 and touching them (Fig. 4E): anther appen- (Fig. SA, D). | Pl 8 dages lanceolate, blunt, exe 'eeding the corona lobes "ig. OC); style head flat (Figs. F, 5 ollinar- (Fig. 5€); style head flat (Figs. 4C, F, 5C). Polli
2 MH 4. dn a Schltr., Bot. Jahrb. Syst. 50: 123. ium comprising 2 pollinia attached by flattened and
1913. TYPE: New Guinea. R. Schlechter 18075
winged caudicles to a corpusculum: pollinia spindle- e r ] l | mes vom B not seen
shaped, much longer than wide, ca. 10 X 0.3 mm (Figs. dF. 5A, B. D): pellucid margin covering 1/2 of Hoya hypolasia is an epiphytic twiner with fleshy
the dorsal margin of the pollinia (Fig. 5D): corpuscu- leaves from New Guinea. Inflorescences pedunculate.
Volume 94, Number 1 2007
Wanntorp & Forster 43
Phylogenetic Relationships of Hoya
H
" * * id Ex ^ da LE à ^ afe es r
Figure 5.
Cross section). showing inner (ip) and outer (op) corona (co) p T | mm (C), 2 mm (B), 100
corpusculum (cp), winged caudicles (ca), and pollinia (po) with pellucid margins (pe).
jars =
and style head (st). Scale
Hoya curtisii. —A, D. Pollinaria with pollinia (po), corpuscula (cp). and caudicles (ca). n
—B, C. Flowers (B. ocesses, anther appendages (aa). guide rails (gr), pollinia (po um (D), 200 um (A). Hoya lacunosa —E. Pollinarium with -F. Detail of a pollinarium with
corpusculum (ep) and part of caudicles (ca). Scale bars = 10 um (F), 100 um (E).
comprising concave umbels of ca. 10 flowers, ca. 2 cm diam.; pedicels ca. 40 mm long, glabrous. Calyx lobes obtuse, ca. 0.1 X 0.1 mm. Corolla flattened-campan- ulate, creamy white, outside glabrous, inside hairy, ca. 1.5 em diam. (Fig. 6A, B, D); lobes ovate-triangular, ca. 0.8 X 0.6 mm. Inner processes of corona lobe very short and obtuse (Fig. 6F, H), not covering the anther appendages and the style head (Fig. 6C); outer processes revolute (Fig. 6F, H); anther appendages lobes
lanceolate, acute, exceeding the corona
(Fig. 6F); style head flat (Fig. 6C, D). Pollinarium
comprising 2 pollinia attached by broadly winged and flattened caudicles to a corpusculum; pollinia oblong- elongate, basal part protruding outward, ca. 0
0.3 mm (Fig. 6E, G); pellucid margin covering the entire dorsal margin (Fig. 6G); corpusculum ca. 0.2 X ] mm, ending with 2 short appendages (Fig. 6G): caudicles very short, ca. 0.1 X 0.03 mm (Fig. 6E, C).
5. Hoya lacunosa Blume, Bijdr. Fl. Ned. Ind. 16: 1063. 1826. (Blume)
Otostemma lacunosum
44
Annals of the Missouri Botanical Garden
—D. Parts of flowers seen from s (aa), pollinia
0).
») processes, guide ra uls (gr). an ithe zd B en idage
Figure 6. Hoya hypolasia. . Flower from side showing corollas (el) and corona (c above with corolla (cl). inner T ane Pollinaria with num (po). es scula (cp). caudi single corona lobe ee inner (ip) — outer (op) processes, anth
d outer (« op) corona (c
(5 , cles (ca), and dpud Apo) i pe ‘lucid margins (pe). —F, 170 a views of : T appendages (aa and pollinia (po). Se ale bars = 1 mm (A-D), 0 um (E
and stvle head
300 um (F,
Volume 94, Number 1 2007
Wanntorp & Forster Phylogenetic Relationships of Hoya
Indone-
Blume in Rumphia 4: 30. 1849. TYPE: Horsfield s.n. (holotype, K not seen
sla. Hoya lacunosa is a twiner from Thailand and the Malay
prising pendent, convex umbels, with up to 30 flowers.
Peninsula. Inflorescences pedunculate, com-
^
'a. 5 mm diam.; pedicels of outer flowers longer than
10-20 mm long, elabrous.
central ones, g.g Calvx lobes
triangular, ca. 1 X 0.5 mm. Corolla rotate, white, ca 5 mm diam.: lobes lanceolate, acute-cuspidate. totally reflexed giving the flower a globose shape. very hairy. X 2 mm (Fig. 7A). uous column (Fig. 7G). Corona lobes elliptical. basal
ca. 4 Gynostegium on a conspic-
part forming a continuous structure looking like a skirt
(Fig. 7B. D. F. C): inner processes acutely ascending.
outer processes obtuse, slightly ascending (Fig. 7A— F); be appendages lanceolate, acute, exceeding
the corona lobes (Fig. 7F); style head shortly apiculate (Fig. 7E, H). Pollinarium
attached by broadly winged and flattened
comprising 2 pollinia caudicles
to a corpusculum; pollinia elongate-falcate. ca. 0.4 X
0.1 mm (Fig. 5E); corpusculum ca. 0.1 X 0.1 mm. ending with 2 short appendages (Fig. 5E, F); caudicles thick, broadly winged, 0.1 X 0.1 mm (Fig. 5E).
6. Hoya inflata (P. I. Forst, Liddle & I. M. Liddle) L. Wanntorp & P. I. Forst., comb. nov. Basionym: Madangia inflata P. Forst., Liddle € I. M.
Liddle. Austrobaileya 5: 54. 1997. TYPE: Papua
New Guinea. Madang A ince: Headwaters Dom
River, 20 Nov. 1995, M. Liddle IMLIO76 (holotype. BRI!). Hoya inflata is an epiphytic twiner with fleshy
leaves from New Guinea. Inflorescences pedunculate, to 9
flowers. ca. | X ca. 2 em; pedicels sparsely hirsute,
comprising pendent, convex umbel, with up
30-45 mm long. Calyx lobes triangular, ca. 1 2 mm. Corolla urceolate-globose, fleshy, creamy
calyx (Fig. 8D), glabrous basal inside part covered by long, ca. | X 2 em (Fig.
reflexed, ca.
white, basally hiding the on the outside, 3A); lobes X mm.
columnar
slender, woolly hairs,
triangular, somewhat
Gynostegium on a conspicuous hairy continuous
skirt,
structure (Fig. BA). Corona made of
lobes forming a membranous smooth whose lower part builds up a continuous rim that is bent inward (Fig. 8A, C-E). white: lobes free at the style head (Figs. 8E, acute, protruding above the corona lobes (Fig. 8B, E); style head flat (Fig. 8B, F,
2 pollinia connected by unwinged and flattened
F, 2A); anther appendages lanceolate,
—
G). Pollinarium comprising
caudicles to a corpusculum; pollinia with oblong- round apical part, c: X 0.3 mm (Figs. 8G, 21
D); pellucid margin covering the entire dorsal margin
—
of the pollinia and slightly protruding outward at the corpusculum oblong-ovate, ca. 0.4 X D): 0.1 X 0.7 mm
basal part; 0.2 mm (Fig. 2C. (Fig. 2C).
caudicles ca
Hoya yuennanensis Hand.-Mazz.. Symb. Sin. 7(4): 1001. 1936. TYPE: China. Yunnan: betw. Ota-Tanschan & Tsedjrong. 4 Oct. 1915. Han-
del-Mazzetti 7971 (holotype. W not seen).
iano tna obcordata N. E. Br., Bull. Misc. Inform. . 1909. Syn. nov., non Hoya obcordata
s = Fl. Brit. India 4: 56. 885 . Dischidia obcordata (N. E. Br.) J. F. Maxwell & R. van Donkelaar, Nat. Hist. Bull. Siam. Soc. 39: 78. 1991. TYPE: *India & Burma." cult. Roval Bot. Gard. Glasnevin, Dublin, 1909, Micholitz s.n.
(holotype, K not seen).
Hoya lantsangensis Tsiang & P. T. Li, Acta Phytotax. Sin. 12: 126. 1974. SP nov. Antiostelma lantsangense (Tsiang & P. T. Li) P. T. La, Novon 2: 218. 1992. TYPE: China. Yunnan: Mo-Jiang, Talang, A. Henry 13589 [cited by Tsiang & P. T.
Li as A. Henry 13689] (holotype, NY not seen: isotypes, IBSC not seen, K not seen). Hoya manipurensis Deb, J. Indian Bot. Soc. 34: 50.
955. Antiostelma manipurense (Deb) P. T. Li. Novon 2: 219. 1992. TYPE: India. Manipur: Litan, 1 Sep. 1953, D. B. Deb 1081 (holotype.
CAL not seen).
Hoya yuennanensis is an epiphytic vine with fleshy
leaves from China (Yunnan). India. Burma, and
Thailand. Inflorescences pedunculate. comprising
racemes of up to 7 flowers, ca. 1.4 ca. 0.8 mm;
pedicels ca. 2 mm long, hirsute. Calyx lobes obtuse-
triangular, ca. 0.2 0.3 mm, slightly pubescent.
Corolla narrowly urceolate, greenish with reddish lobular margins uniformly densely pilose on the outside: tube wider at the base and gradually
narrowing up to the rim, inside covered by uniformly 1.2 X 0.6 mm (Fig. 9A):
densely
distributed long hairs. ca. lobes triangular, slightly reflexed. x 0.1 mm (Fig. 9A).
fleshy sulcate lobes, white; inner processes short and
inside
pilose, ca. 0. Corona with 5
Fig. 9C); outer processes with
—
slightly bent upward a deep groove (Fig. 9C, D); anther appendages much exceeding the corona lobes and ending with a tri- angular structure that (Fig. 9A—C, E); style head short, conical. Pollinarium comprising 2 pollinia connected by flattened, un-
touches the style head
winged caudicles to a corpusculum; pollinia lying in an almost horizontal position in regard to the corona, 0.2 mm (Fig. OF, G); pellucid margin covering the entire dorsal margin of
the pollinia (Fig. 9F, G):
almost squared, ca.
corpusculum rhomboid, c:
46 Annals of the Missouri Botanical Garden
quei LP, M z., A
fact d aN
SA isles
+ J^ — [
gum 8
Figure 7. Hoya lacunosa. —A. Flower with corolla (el) and inner (ip) and outer (op) corona (co) proce D.
2I Different views of coronas with inner un and outer (op) processes, anther ¿ appendages (aa). and guide rails (gr). pus shows the fused outer corona processes. —E. Section of flower showing pollinia (po) and style head (st). —F. Single corona b» with inner (ip) and outer (op) paa and adnate xd appe ndi age (aa). Arrow shows the fused outer corona processe G. ( F | (cm). H. Cross section of flower 1 8 guide rails (gr). corpuse ulum (cp). E
head (st). Scale bars = 1 mm (A), LOO um (400. 20 200 um (F), 300 um (B-E,
Volume 94, Number 1 2007
Wanntorp & Forster Phylogenetic Relationships of Hoya
Figure 8. Hoya inflata. —A. and outer (op) corona (c ʻo) proces and style heac
3
st). —
inner (1p) ) and outer (op) corona proce (cp). Arrow in E
s, column (cm), and pollinia (po).
sses, euide rails
shows the fused processes of the corona. Scale ba
ower showing the remainder of the corolla (cl) with hairs (h), corona (co) with inner (ip
—
pper part of flowers with anther appendages Flower seen from side with corona (co) and TA rest of corolla (cl) with hairs (h). —D. Fl from below with the rem: 11 of the corolla (cl) and corona process
| mm (A, C-).
(aa)
. Flower seen s bent inward. —E-G. her appe Dus 5
per part of flowers showing aa), pollinia (bol silo head (st)
JA un ec Mc
—
100 mm (B, G).
48 Annals of the Missouri Botanical Garden
N
fr YR fh f
Figure O. Hoya yuennanensis. — . D. Longitudinally sectioned flowe isi Ones el) A hairs (h). corona (co) with revolute outer. processes (op). guide r is (gr), and anther appendages (aa). pper par of flower with inner corona processes (ip), revolute outer processes (op), anther appendages (aa), and pollini 1 (po). "m: . Pollinaria with equa (cp). ie les (ca), and pollinia (no) with pellue idn margins (pe). Scale bars mm (A). 4 mm ps 30 um (C). 100 um (E. F), 200 um (B, C).
Volume 94, Number 1 2007
Wanntorp 4 Forster 49 Phylogenetic Relationships of Hoya
0.1 X 0.08 mm, appendages (Fig. 9F, C).
(Fig. OF, G).
with 2 short and straight Caudicles ca. 0.1 X 0.1 mm
ending
8. Hoya patella Schltr., Bot. 1913. TYPE: New
(holotype, B not seen).
Jahrb. Syst. 50: 132.
Guinea. Schlechter 16375
Hoya patella is an epiphyte with pendent or weakly
twining stems and fleshy leaves from New Guinea.
Inflorescences pedunculate, comprising racemes with
| to 5 flowers; pedicels ca. 50 mm long, glabrous.
Calyx lobes obtuse-triangular, ca. | mm.
^
Corolla campanulate, light pink, externally glabrous, hirsute, internally with reticulate nervature, ca. 3 cm diam.: lobes triangular-ovate, reflexed, ca. 5 X 2 mm, with hairy margins (Fig. 10A). Gynostegium on a shorl
column (Fig. LOA).
corolla.
Corona much smaller than the dark red, smooth; inner processes slightly
ascending (Fig. 10B), shorter than anther appendages (Fig. 10D, E):
margins that do not
processes revolute but with (Fig. 10C. G): appendages lanceolate, acute, exceeding the corona
(Fig. 10D, E);
(Fig. 10D, E, H). Pollinarium comprising 2
ouler
anther
touch
lobes style head shortly apiculate pollinia connected by short, flattened, winged caudicles to
a corpusculum: pollinia longer than wide, cylindrical- elongate, protruding outward at the basal part, ca. 0.7 X 0.3 mm (Fig. I0F): pellucid margin covering the of the pollinia (Fig. 10F); X 0.] mm, with 2 short appendages (Fig. 10F); caudicles ca. 0.1 X 0.03 mm (Fig. 10F).
entire dorsal margin
corpusculum rhomboid, ca. 0.2 ending
9. Hoya retusa Dalzell, Kew J. Bot. 4: 294. 1852. Hoya retusa Warb. Repert. Spec. Nov. Regni Veg. 3: 344. 1907, nom. illeg. TYPE: Ind. Or.
Dalzell s.n. (holotype, K not seen).
Hoya retusa is an epiphyte with non-twining pendent stems and fleshy foliage from India. Inflor- escences pedunculate, comprising a much-reduced a single flower; pedicels ca. a. 0.33 X
white, ca.
raceme carrying only 10 mm long. Calyx lobes elliptic-oblong, <
0.2 mm. Corolla flattened-campanulate,
15 mm diam.; lobes triangular-acute, pubescent, ca. 5 X 2 mm. Corona outer processes keel-shaped, dark aculely ascending, outer pro- (Fig. 11A-C, E);
appendages lanceolate, acute, noticeably exceeding
red; inner processes
cesses deeply revolute anther
the corona lobes (Fig. 11B, C); style head shortly apiculate (Fig. 11A, D).
pollinia connected by broadly f.
Pollinarium comprising 2
attened, unwinged caudicles to a corpusculum; pollinia oblong-elongate,
ca. 0.4 X 0.2 mm (Fig. 11F); corpusculum ca. 0.3 X
O. Umm (Fig. LIF); caudicles ending with short
).04. X 0.02 mm (Fig. 11F).
appendages, ca. (
10. Hoya venusta Schltr., 1913. TYPE: New (holotype, B not seen).
Bot. Jahrb. Syst. 50: 128.
Guinea. Schlechter 18767
Hoya venusta is an epiphytic climber with fleshy leaves from New Guinea. Inflorescences pedunculate, comprising umbels with 10 to 15 flowers, ca. 1.5 em
diam. Calyx lobes obtuse, 1-2.5 X 0.5 mm, glabrous.
Corolla campanulate, white to dark pink, outside elabrous, internally papillose-hairy; lobes obovate, 5— 7 X 5-6 mm. Gynostegium on a short column
(Fig. 12A). Corona creamy white with light red center, striate; inner processes ascending, covering the style head (Fig. 12B—D), central part of the lobes with an
umbo (Fig. 12B, F); outer processes deeply revolute
Fig. 12E); anther appendages lanceolate-acute, much exceeding the corona lobes (Fig. 12C, D); style head
shortly apiculate (Fig. 12B—D). Pollinarium compris-
ing 2 pollinia connected by broadly flattened, unwinged caudicles to a corpusculum; pollinia
0.4 X 0.2 mm (Fig. 126, H);
rhomboid,
obovoid-oblong, ca.
corpusculum small, size not measured
(Fig. 12G, H); caudicles ca. 0.1 X 0.04 mm (Fig. 12G, H).
DISCUSSION
Omlor (1998) considered the genera Absolmsia, Dischidia,
In his generic revision of the Marsdenieae, Hoya, Madangia, and Micholitzia to form a group (the Hoya-group) characterized by flowers having coronas with revolute outer processes, pollinia with pellucid
margins, and fusiform seeds without prominent wings.
g
Likewise, a close relationship between the genera Hoya, Dischidia, and Micholitzia has been reported in several molecular phylogenetic studies (e.g., Potgieter & Albert, 2001; Livshultz, 2002, 2003; Rapini et al., 2003; Meve & Liede,
studies resolved the phylogenetic
2004). However, none of these relationship. be- tween these genera. In order to explore the boundaries of Hoya, (2006a. b) species of this genus and close relatives of the Ma- dangia, and Micholitzia were found nested within 2006a, b).
Absolmsia
Wanntorp et al. analyzed
Marsdenieae. The monotypic genera Absolmsia, Hoya with strong support (Wanntorp et al.,
Below, we discuss the position of spartioides, Madangia inflata, and Micholitzia obcor- data nested within Hoya by comparing their flower morphology with that of closely related species of
Hoya.
50 Annals of the
Missouri Botanical Garden
i 5h n " JD 1
.
REG > tt um A NS NS
e A Err he
ami
PON
—
Figure. 10. He oya patella. . Side (A) and above (B, C) views of flowers with corolla (el), corona ia revolute outer processes (op). inner processes (ip), anther appendages (aa), guide rails (gr), pollinia (po), and style head (s part of flower s inner 5 corona proce
). E. Upper (cp). and slyle head (st). —
sses, anther appendages (aa), pollinia (po) with pellucid margins es . corpusculum Pollinarium with pollinia (po), corpusculum (cp), and caudicles (ca) art of flower showing ovary (ov outer corona (op) processes. —H. | l 8 D p) I
G. Cross section of lower guide rails (gr), corpuscula (ep), and style head (st). Scale bars
ongitudinal section of flower showing position of
mm (A-C), 1.5 mm (G), 100 um (F, H), 200 um (D, E).
Volume 94, Number 1 Wanntorp € Forster 51 2007 Phylogenetic Relationships of Hoya
Fig 11. Hoya retusa. A. Sectioned flower showing inner (ip) and outer (op) revolute corona processes, guide rails (gr), em Reni head (st). —B, C. Corona seen from side (B) and from above (C) showing inner (ip) and outer (op) revolute corona processes, guide rails (gr), and anther appendages (aa). —D. Longitudinal section of upper p of flower with guide rails (gr). corpusculum (ep), and style head (st). —E. Longitudinal section of flower showing calyx (cx), corolla (cl), inner (ip) and outer (op) corona processes, guide rails (gr). style he: d (st). and y (po). —F. Pollinarium with colla i pe elluc ‘id margins (pe). corpusculum (ep), and caudicles (ca). Scale bars = n (A). 3 mm (B), 100 um (D, F), 300 um (C.
THE GENERIC STATUS OF ABSOLMSIA almost entirely found in the section Eriostemma Schltr.
At first sight, mature plants of Absolmsia look Absolmsta spartioides was first described by Ben- very unlike Hoya because they lack the leaves tham in 1880 as Astrostemma spartioides Benth., but characteristic of that genus. Leaves are only present this name was later changed to Absolmsia by Kuntze, in very young plants of Absolmsia; in mature plants, because he regarded it as too similar to the name of photosynthesis is instead carried out mainly by long, another genus of tribe Marsdenieae, Asterostemma green, perennial peduncles of the inflorescences. In Decne. (Omlor, 1998). The similarity between most species of Hoya, the peduncles are also — Absolmsia and Hoya was first pointed out by perennial; only a few Hoya species have annual Schlechter (1916: 2), who stated that “Absolmsia,
peduncles, a derived character state in this genus and placed by K. Schumann within Cynanchinae, belongs
52 Annals of the Missouri Botanical Garden
Figure 12. oya venusta. \—D. Details of flowers showing the corona a inner (ip) and outer (op) revolute processes, "sr (em). anther appendages (aa). style head (st). and pollinia (po). —E. F. Coronas seen from side showing guide rails (gr). —G, H. Pollinaria adnate to anther appendage (aa). VV nd caudicles (ea). Scale bars = 1 mm (B). 100 um (C, G, H), 200 um (Dr 300 Um (A, E. F)
Volume 94, Number 1 2007
Wanntorp 4 Forster 53 Phylogenetic Relationships of Hoya
doubts near to Hoya.” Omlor (1998
included Absolmsia in a Hoya-group based on several
without any
morphological characters common to both of these
genera: a corona with inner processes that cover the
anthers and outer revolute processes (Fig. 1D, E):
pollinia with a dorsal pellucid margin (Fig. 2H): and
almost round seeds with thickenings on the lateral
walls of cells in their seed coats. Based on
morphological similarities in the shapes of the corona Kloppenburg reduced Absolmsia to
and corolla,
synonymy under Hoya, transferring the name as H. spartioides (Kloppenburg, 2001) Hoya
indicates that the sister group of Absolmsia spartioides
The available molecular phylogeny for
is H. curtisii and that these two species are part of
a weakly supported clade also including H. lacunosa and a well-supported subclade a 5 imbricata and
H. caudata (Wanntorp et al., 2006b). Absolmsia
e
H. curtisii differ EUR in flower latter pletely reflexed. lobes and a corona lying on an annulus (Fig. 4A,
and a
spartioides anc morphology. the having a corolla with com- B), while Absolmsia has a campan-
ulate corolla corona without any annular structure (Fig. LA, €). Because the sample of Hoya species included in the molecular studies of Wanntorp
et al. (2006a, b)
species of Hoya, it is probable that the retrieved sister
—
was limited to only 35 out of over 300
relationship between A. spartioides and H. curtisii will change by the inclusion of additional species of Hoya in the molecular phylogeny. Although it is not possible at this stage to point to a particular species of Hoya as sister to Absolmsia, the inclusion of this genus in Hoya was nevertheless clearly shown in the (2006a, b) concordance with previous morphological
(Omlor, 1998; Kloppenburg, 2001).
The shape of the corolla varies extensively in Hoya
and is i
tree of Wanntorp et al.
studies
from rotate with revolute lobes (e.g.. H. lacunosa, Fig. 7A), (Fig. Kloppenb. 1996).
in M. curtisii. Corolla characters (especially the shape)
flattened-campanulate in H. caudata 3A), or urceolate to globose in H. heuschkeliana
and H. Omlor (Omlor,
The corolla lobes are commonly reflexed
telosmoides R.
—
Hoya seem highly labile and have undoubtedly
evolved numerous times. The shape of the corona, on
the other hand, seems to be a
phylogenetic value for linking Absolmsta to Hoya, as both have coronas with deeply revolute outer pro- 1998; Fig. 1D, E). In
eynostegium of Absolmsia, as in many Hoya, 1s
a column that varies in the degree of indument and in
cesses to the lobes (Omlor, addition, the species of raised above the corolla on conspicuousness. This column is short in Absolmsia (Fig. IC). H. patella (Vig. LOA), anc
(Fig. 3B) and not as conspicuous as in H. curtisii
—
H. caudata
character of higher
(Fig. 4D). Pollinia of Absolmsta, Hoya, Micholitzia, and Madangia have a pellucid dorsal margin in which pollen tubes germinate (Omlor, 1998: this study). This trait is unknown in the genus Dischidia (Meve, 2001) and all other species of the Marsdenieae (Omlor, 1998). These
molecular results (Wanntorp et al., 2006a. b) support
morphological characters and the
the inclusion of Absolmsia in Hoya, transferred. by Kloppenburg (2001) as H. spartioides (Benth.) Klop-
penb.
THE GENERIC STATUS OF VADANGIA
Forster et al. (1997) collected a plant in the Madang province of New Guinea that very much resembled species of Hoya in its epiphytic habit and. fleshy
leaves. The plant was assigned to the monotypic genus
Madangia on the basis of floral morphological characters unique for this species. One of these
characters was the elobose shape of the corolla, which
is unusual Hoya as discussed by Forster et al. (1997
species with similar corolla shape. Omlor (1996) also
. who mentioned H. heuschkeliana as the only
mentioned a species from Sabah, H. telosmoides, with
a similar corolla shape, although this species has
^
corollas internally covered. with long recurved hairs. Madangia inflata is well supported as sister species to the New Guinean H. hypolasia, both being part of a larger clade, including other species from Australia H. patella, H. venusta, and ex J. Traill and A. Wanntorp et al., 2006a, b).
associ-
and New Guinea (e.g., a subelade with H. australis R. Br. albiflora Zipp. ex Blume) on the other
—
Hoya heuschkeliana, hand, was ated with species from the Acanthostemma Blume group, Which have pubescent corollas and completely reflexed lobes that give the flowers a globular shape. Hoya telosmoides was not found close to M. inflata in 2006b).
the corolla in Hoya and
the tree (Wanntorp et al., These results again support the plasticity of underline the hazard of establishing too much reliance upon single corolla characters in the recognition of genera in the group.
The corona of Madangia inflata has a remarkable shape, which contributed to the decision of Forster et
al. (1997) to establish a new genus for this species.
-
The five inner corona processes are free at the apical part near the style head as in Hoya, but their outer parts, corresponding to the outer processes of the corona lobes in Hoya, form a continuous rim that is folded inward (Fig. 8D). which has not been previously discussed, is found in
A similar corona shape,
H. lacunosa (Vig. 7B). In this species also, the five
outer corona lobes are fused at their basal part,
forming a skirt that somewhat resembles the membra- Madangia (Fig. 8D).
nous corona of According to
Annals of the Missouri Botanical Garden
Wanntorp et al. (2006b). H. lacunosa, however, Hoya
separated from M. inflata. Vhis corona tvpe there
groups
with many other species of and is clearly
ore seems to have arisen independently several times in Hoya, paralleling the variation in corolla morphology.
The remaining flower parts of Madangia are very similar to Hoya. The column between the corolla and the gynostegium is very conspicuous in Madangía as Hoya, such as H. curtisii. H. venusta (Figs. 4D, 7G, 12A). The D) have a clear 6G). Another
interesting character not previously noted in Madan-
species of and H.
Madangia (Fig. 2C.
in many lacunosa, pollinia of pellucid margin as in Hoya (e.g... Fig. gia 1s the dimerous style head that is flat and shortly both and H. (Figs. 8B, OC) and shortly apiculate-conical in
macgillivrayt V. Bailey, H. venusta (Fig. 12€, D). and H. patella (Fig. 10D) of the same clade. The
shape of the style head and the morphology. of the
apiculate i Madangia hypolasia
pollinaria are in agreement with the molecular results (2006b). that
Madangia should be included in Hoya as H. inflata.
of Wanntorp et al. and we propose
THE GENERIC STATUS OF VICHOLITZIA
During an expedition to India and Burma, the
orchid collector W. Micholitz discovered a new taxon,
later described as Micholitzia obcordata (Brown, 1909). Micholitzia was again collected China by
Handel-Mazzetti and respectively, H.
in India by Deb and named, Hand.-Mazz. and H.
Tsiang and Li collected
yuennanensis manipurensis Deb. In 1974, the same plant in China, thought it to be a new species of Hoya that they called H. lantsangensis Tsiang & P. T. Li. and erected a new section Antiostelma for i
0 . rred 1 9 ia to Dischidia 17
and P. i (1992: 218) argued that the cylindric shape É ^n corollas and the bud aestivation, which “twisted leftward.” were different enough from those of Hoya and Dischidia,
rotate and urceolate corollas, respectively, to
with valvate aestivation and ralse section Antiostelma in Hoya to generic rank. Accord- i 2006a),
nested within Hoya, even though its
ing to molecular evidence (Wanntorp et al., Micholitzia is sister relationship to any of the species of Hoya is less Absol- : Madangia. In these Micholitzia Absolmsia, adventitious roots are produced at the leaf nodes (Goyder & Kent, 1994). The corolla of Micho-
litzia is of a similar urceolate or tubular shape to that
Micholitzia is more similar to Dischidia. ot
but not in
clear. In habit, msia than to Hoya. last three genera, and
of a few species of Hoya (e.g., H. heuschkeliana) but is wider at the lower part of the corollas (Goyder & Kent, 1994).
many species of Dischidia, but in this genus usually
This shape superficially resembles that of
only the upper internal part of the corolla is
pubescent. while in Micholitzia the corollas are uniformly pubescent on the outside and inside (Goyder & Kent, 1994). The corona of Micholitzia has been considered to be different from that of
Dischidia and Hoya because its five fleshy and almost round lobes do not end as in Hoya with five pointed inner processes on the anther appendages. lts inner corona processes are instead blunt-ended and appear almost as if they. were united to the five conspicuous anther appendages hiding the flat and short style head. Despite the different inner corona processes of
Micholitzia, the outer processes of the corona lobes
have revolute margins (Fig. 9C) similar to those of Hoya (e. g., H. retusa) (Omlor, 1998: Fig. 11B). In addition, the pollinaria of Micholitzia have clear
; Fig. 9F). Clear pellu- 2H),
5E) but are
pellucid margins (Omlor, 1998 cid margins are also present in Absolmsia (Fig. Madangia (Vig. 2C), and Hova (e.g.. Fig. Dischidia.
margin of Micholitzia, however, is superficially differ-
absent in The position of the pellucid
ent from Hoya because it is apparently situated at the
ower part of the pollinia instead of at the outer edge 1994),
this is due to the twisting of the pollinia of Micholitzia.
(Fig. OF), but as observed by Goyder and Kent By rotating the pollinia at 90 , the pellucid margin takes the same position as in the pollinia of Hoya. Based on the molecular evidence for Hoya and on the the outer
similarity of the pollinia and of corona
processes, Micholitzia should be included in a broadly defined Hoya. Due to the prior use of the epithet obcordata in Hoya, inclusion of Micholitzia obcordata in Hoya necessitates a change of epithet for the spe- cies, based on the next available name of the several synonymous species, which is H. yuennanensis.
CONCLUSION
A recurring theme in recent molecular-based
phylogenies within the Apocynaceae, Asclepiadoideae
is that morphological classifications based on single,
— pe
albeit unusual, floral features (especially of the corolla or coronas) are not corroborated by molecular data (e. g.
Meve & Liede, 2001, 2002, 2004).
the molecular phylogeny to date (Wanntorp et al..
In the Hoya group,
2006b) indicates relationships that follow geographical
patterns, as shown by the clade including almost
exclusively Australian and New Guinean species, but
—
also partly reflect morphological similarity, as in
Madangia, Absolmsia, and Micholitzia.
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