Rediscovery of Malagasy Lathraeocarpa allows determination of its taxonomic position within Rubiaceae

209

Groeninckx amp al bull Taxonomic position of LathraeocarpaTAXON 58 (1) bull February 2009 209ndash226

INTRODUCTIONLathraeocarpa Bremek described by Bremekamp

in 1957 is a genus of Rubiaceae endemic to the south and south-western part of Madagascar It comprises two (sub)shrub species L acicularis Bremek and the type species L decaryi Bremek The generic name refers to the stipular sheath surrounding the ovary that tears open at fruit maturity Within Rubiaceae the genus is character-ized by ternate leaves nearly truncate stipules fused with the petioles heterodistyly calyx lobes that are twice the number of corolla lobes a tri- or tetralocular gynoecium with one basal ovule per locule a drupaceous fruit and pluri-zonocolporate pollen grains (Bremekamp 1957)

Until recently Lathraeocarpa was only known from five herbarium collections the most recent of which was collected in 1955 In spite of extensive fieldwork in Madagascar during the last two decades and although the type localities have been revisited (eg most recently by S Razafimandimbison Bergius Foundation Stock-holm Sweden and A Davis Royal Botanic Gardens Kew UK) Lathraeocarpa had never been relocated until we discovered a population of L acicularis close to the Parc National de Tsimanampetsotsa in February 2007

The presence of raphides valvate corolla aestiva-tion and pluri-zonocolporate pollen grains indicates that Lathraeocarpa belongs to subfamily Rubioideae but its

exact position has long been subject to debate Initially the herbarium material of Lathraeocarpa on which Breme-kamp (1957) based his description was thought to belong to the genus Triainolepis Hook f Homolle annotated all specimens (except Humbert amp Capuron 2958) as Triai-nolepis decaryi Homolle but never published the name Bremekamp (1957) however considered the material to be too different from Triainolepis and transferred it to the new genus Lathraeocarpa which he placed in a tribe of its own ie Lathraeocarpeae because of its unique combination of character states

Capuron (1973) added the monospecific genus Gom-phocalyx Baker another endemic from Madagascar as a second genus to Lathraeocarpeae Piesschaert (2001) even suggested merging Lathraeocarpa with Gomphocalyx a proposal refuted by Dessein amp al (2005a) Gomphocalyx herniarioides Baker is a procumbent to decumbent annual or short-lived perennial herb which in the past was placed in Spermacoceae sensu stricto (sstr hereafter) (Schumann 1891 Robbrecht 1988 1993) However recent molecular studies excluded Gomphocalyx from Spermacoceae sstr and placed it in the Pentanopsis clade (Thulin amp Bremer 2004) of the Hedyotis-Oldenlandia group (Dessein 2003 Dessein amp al 2005a Groeninckx amp al in press) where it was shown to be sister of the Afro-Madagascan genus Phylohydrax Puff Presently the former tribe Sperma-coceae sstr and the Hedyotis-Oldenlandia group are

Rediscovery of Malagasy Lathraeocarpa allows determination of its taxonomic position within Rubiaceae

Inge Groeninckx1 Petra de Block2 Franck Rakotonasolo3 Erik Smets14 amp Steven Dessein2

1 1 Laboratory of Plant Systematics KU Leuven Kasteelpark Arenberg 31 PO Box 2437 3001 Leuven Belgium ingegroeninckxbiokuleuvenbe (author for correspondence)

2 2 National Botanic Garden of Belgium Domein van Bouchout 1860 Meise Belgium3 3 Parc Botanique et Zoologique de Tsimbazaza BP 4096 Tsimbazaza Antananarivo 101 Madagascar4 4 National Herbarium of the Netherlands Leiden University Branch PO Box 9514 2300 RA Leiden

The Netherlands

Lathraeocarpa acicularis a small woody Rubiaceae endemic to Madagascar was rediscovered after more than 50 years A phylogenetic reconstruction based on four plastid markers (atpB-rbcL rps16 trnL-trnF petD) proves that its previous position within the monogeneric tribe Lathraeocarpeae can no longer be supported Our data clearly show that Lathraeocarpa acicularis has its closest relatives among taxa of the Hedyotis-Oldenlandia group of the herbaceous tribe Spermacoceae sensu lato The taxon falls within the Pentanopsis clade and is sister to a group comprising the Madagascan genus Gomphocalyx and the Afro-Madagascan genus Phylohydrax A detailed survey of the morphology and anatomy of the genus based on our recently collected material of Lathraeocarpa acicularis is presented providing additional arguments for the new taxonomic position

KEYWORDS comparative morphology and anatomy Lathraeocarpa Madagascar molecular phylogeny plastid DNA Rubiaceae

210

TAXON 58 (1) bull February 2009 209ndash226Groeninckx amp al bull Taxonomic position of Lathraeocarpa

united forming Spermacoceae sensu lato (sl hereafter) (Robbrecht amp Manen 2006 Groeninckx amp al in press) If Capuronrsquos decision (1973) to place Gomphocalyx and Lathraeocarpa together was correct Lathraeocarpa too would be related to Phylohydrax and should therefore also be placed in Spermacoceae sl

Several morphological characters distinguish Lath-raeocarpa from Gomphocalyx some of which might even point to an affinity with Triainolepis First of all the (sub)shrubby habit of Lathraeocarpa is much closer to the shrubby habit of Triainolepis than to the herbaceous habit of Gomphocalyx Furthermore the pyrene of L decaryi is surrounded by eight strands of thin-walled cells a condi-tion very similar to that observed in some Triainolepis species (Bremekamp 1957 Piesschaert 2001) Likewise Lathraeocarpa and Trianolepis have a plurilocular ovary and fleshy fruits whereas Gomphocalyx has a bilocular ovary and dry fruits which has prompted some authors (Karingrehed amp Bremer 2007) to believe that Lathraeocarpa should be regarded as a member of the emended tribe Knoxieae rather than a member of Spermacoceae sl

To date no molecular data have been available for Lathraeocarpa so its taxonomic position has remained controversial (Robbrecht amp Manen 2006) This paper presents the results of a multidisciplinary study based on morphological and molecular data illuminating the relationships of this enigmatic genus

MATERIALS AND METHODSMolecular study mdash A few preliminary analyses

of rps16 intron sequences of representatives of the ma-jor groups within subfamily Rubioideae have shown that Lathraeocarpa acicularis belongs to Spermacoceae sl We thus used the matrix of Groeninckx amp al (in press) based on the chloroplast markers atpB-rbcL rps16 and trnL-trnF as a basis for an initial analysis presented in this paper The chloroplast dataset was enlarged by the addition of three atpB-rbcL sequences (Gomphocalyx her-niarioides Lathraeocarpa acicularis and Pentanopsis gracilicaulis (Verdc) Thulin amp Bremer) three rps16 se-quences (Lathraeocarpa acicularis Oldenlandia biflora (L) Lam Pentanopsis gracilicaulis) and five trnL-F sequences (Gomphocalyx herniarioides Lathraeocarpa acicularis Oldenlandia rosulata K Schum Pentanopsis gracilicaulis Phylohydrax carnosa (Hochst) Puff) Our dataset included a total of 132 species representing 35 of the 60 genera within Spermacoceae To improve reso-lution a second more focused analysis was performed with petD included as an extra chloroplast marker using a smaller set of 25 taxa including Lathraeocarpa and 21 closely related taxa as well as the pentamerous Dentella dioeca Airy Shaw D repens (L) JR Forst amp G Forst

and Pentodon pentandrus (K Schum amp Thonn) Vatke as outgroup taxa The Appendix lists all taxa included in this study with author names voucher information and GenBank accession numbers

Methods for DNA extraction PCR amplification se-quencing sequence assembly and alignment are as de-scribed in Groeninckx amp al (in press) The petD region was amplified with the forward primer PIpetB1365F and the reverse primer PIpetD738R as described by Loumlhne amp Borsch (2005)

Equally weighted maximum parsimony (MP) anal-yses were performed using Nona 20 (Goloboff 1993) launched through WinClada 10008 (Nixon 2002) Par-simonious informative gaps were coded manually ac-cording to the conservative lsquosimple indel codingrsquo method described by Simmons amp Ochoterena (2000) The four plastid regions were first analyzed separately and given congruence between the individual topologies then com-bined using a total evidence approach Heuristic searches for the shortest trees were performed using the parsimony ratchet (Nixon 1999) Ratchet runs of 200 iterations each holding 1 tree per iteration and randomly weighting 10 of the potentially informative characters were carried out until the most parsimonious trees (MPTs) were repeat-edly found A strict consensus tree was calculated using the trees obtained in the parsimony ratchet analyses In order to evaluate the relative support of the clades jack-knife (JS) and bootstrap (BS) analyses were executed us-ing 1000 replicates with 100 initial trees holding 1 tree per random addition performing TBR (Tree-Bisection and Reconnection) to hold 1000 trees and calculating a consensus on each repetition Frequency values ( gt 65) were plotted onto the consensus of the MPTs

Bayesian inference (BI) analyses were only carried out for the two combined datasets A substitution model was selected for each DNA region with Modeltest 37 (Posada amp Crandall 1998) under the Akaike Informa-tion Criterion (AIC) (Table 1) Indels were not included in the BI analyses In the combined analysis a mixed-model approach was used (Ronquist amp Huelsenbeck 2003) The combined dataset was partitioned and the same models of evolution were used on the partitions as selected for the single analyses The BI analyses were conducted with Mr-Bayes 3b4 (Huelsenbeck amp Ronquist 2001) as described by Groeninckx amp al (in press) We selected a GTR + I + G a GTR + G and a GTR + I substitution model as being analogous respectively to the TVM + I + G TVM+G and TVM + I models of substitution selected by Modeltest but not implemented in MrBayes The TVM model is similar to the GTR model except for having equal probabilities of change of transitions Four Markov chains (one cold three heated) starting with a random tree were run simultane-ously for three million (Analysis 1) and for one million (Analysis 2) generations sampling trees at every 1000th

211


generation Of the first sampled trees 25 were regarded as lsquoburn-inrsquo and discarded PAUP version 4b10 (Swof-ford 2002) was used to calculate a 50 majority rule consensus tree and to report the posterior probabilities for each clade Only posterior probabilities (PP) above 095 have been considered (Suzuki amp al 2002)

Morphological study mdash Herbarium and alcohol material of the recently collected specimen of Lathraeo-carpa acicularis (Madagascar Toliara Ankarampona zone peacuteripheacuterique du Parc National Tsimanampetsotsa Fokontany Ambola [Efoetse] Commune Beheloka Dis-trict Toliara II 24deg07prime358Prime S 43deg40prime204Prime E 04022007 De Block amp al 2316 [BR G K MO P TAN]) was studied along with all known herbarium collections of L decaryi (Madagascar 18061924 Decary 2803 [holotype P] Madagascar Ambovombe Mahatomotsy 09121931 De-cary 9508 [BR P] Madagascar Ambovombe 02021931 Decary 8457 [P] Madagascar North-East of Cap Sainte Marie in the direction of Lavanono 08031955 Humbert amp Capuron 29308 [P] and L acicularis (Madagascar Toliara dunes of Befanamy 15021921 Poisson 144 [ho-lotype P])

Material of L acicularis preserved in 70 ethanol was dissected under a Wild M3 stereomicroscope (Wild Heerbrugg Ltd Heerbrugg Switzerland) The dissected material (flowers young fruits leaves stipules) was washed repeatedly in 70 ethanol and dehydrated in a 1 1 mixture of ethanol and dimethoxymethan (DMM or formaldehyde-dimethylacetal) for 20 minutes and then in pure DMM for another 20 minutes After critical-point drying (CPD 030 BAL-TEC AG Balzers Liechtenstein) the dried material was mounted on aluminum stubs using Leit-C and coated with gold (SPI Module Sputter Coater Spi Supplies West Chester Pennsylvania USA) prior to observation with a JEOL JSM-6360 scanning electron microscope (SEM Jeol Ltd Tokyo Japan)

Anatomical sections of leaves and young fruits pre-served in 70 ethanol were made from tissues dehydrated through a graded ethanol series Young fruits were embed-ded in KULZERrsquos Technovit 7100 (based on hydroxyethyl-

methacrylate HEMA) as detailed by Igersheim (1993) Leaves were gradually infiltrated with LR white resin (hard grade) (London Resin) using 1 3 1 1 3 1 and 1 0 solu-tions of resin and 100 ethanol for at least 5 hours each The leaf samples were placed in closed capsules filled with fresh resin and polymerized at 60degC for 48 hours

Fruit and leaf sections of 2ndash3 μm in thickness were made with a rotary microtome (Microm HM 360 Wall-dorf Germany) and stained with 01 toluidin blue Pho-tographs were taken under a Leitz Dialux 20 microscope (Wetzlar Germany) equipped with an Olympus DP50 camera (Hamburg Germany)

Pollen grains from herbarium material were acetoly-sed according to Reitsmarsquos (1969) lsquowetting agentrsquo method External features were observed using SEM on grains that had been suspended in 70 ethanol and left to dry Glycerin jelly slides were observed under a light micro-scope Untreated pollen grains from anthers preserved in 70 ethanol were also studied following the method of Halbritter (1998) but using DMM instead of DMP (22-di-methoxypropane) Polar axis length (P) and equatorial diameter (E) were measured on 20 grains from thrum (brevistylous) and 20 from pin (longistylous) flowers us-ing the software programme Carnoy (Schols amp al 2002) Pollen terminology follows Punt amp al (2007)

Table 1 Substitution models selected with Modeltest 37 (Posada amp Crandall 1998) for each DNA region used in Analysis 1 and for each DNA region used in Analysis 2

Analysis 1a Analysis 2b

atpB-rbcL TVM + I + G TVM + Grps16 GTR + I + G TVM + ItrnL-F TVM + G TVM + GpetD GTR + GaatpB-rbcL rps16 trnL-FbatpB-rbcL rps16 trnL-F petDThe models can ignore rate variation or include invariable sites ( + I) rate variation among sites ( + G) or both ( + I + G)GTR = General time reversible TVM = Transversion model

Table 2 Characteristics of each data matrix used in the phylogenetic analysis in Spermacoceae sl (Analysis 1) and the corresponding tree statistics

No of taxa

No of characters

No of PI characters

No of PI indels

No of MPT

MPT length CI RI

atpB-rbcL 106 1292 187 38 4502 415 057 085rps16 111 664 176 22 147 474 056 083trnL-trnF 117 914 172 33 678 396 061 088Combined 132 2870 525 93 47571 1352 054 083CI = Consistency Index (Kluge amp Farris 1969) MPT = Most Parsimonious Tree(s) PI = Potentially-Informative RI = Retention Index (Farris 1989)

212


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213


RESULTSMolecular evidence mdash For the initial study involv-

ing samples of 132 taxa sequence data from the aligned atpB-rbcL rps16 and trnL-trnF regions were analyzed separately (results not presented) and in combination us-ing a total evidence approach (Table 2) Only the results from the MP and the BI analysis of the combined matrix are presented (Fig 1A B) because they resulted in more resolved trees The MP tree and the BI tree are congruent with those published by Groeninckx amp al (in press) In this analysis Lathraeocarpa falls within the Pentanopsis clade and comes out as sister to a clade comprising Gom-phocalyx and Phylohydrax (JS = 100 BS = 100 PP = 100) This well-supported clade comprising Lathraeocarpa Gomphocalyx and Phylohydrax (the LGP clade) shares a common ancestor with Manostachya ternifolia E Sampaio Martins and Oldenlandia rosulata (JS = 100 BS = 100 PP = 093) In the BI analysis the latter two species are sister to the LGP clade whereas in the MP analysis their relationship to the LGP clade is unresolved

Results of the second more focused analysis of 25 taxa based on the three markers indicated above plus petD are presented in Fig 2A B and in Tables 3 and 4 The petD region has never been used previously in phylogenetic studies of Rubiaceae The chloroplast region includes the petB-petD intergenic spacer the petD 5prime exon and the petD 5prime intron (Loumlhne amp Borsch 2005) Our study shows that the amount of sequence variability in this chloroplast marker is relatively high suggesting that it may be useful for fur-ther studies in the family After exclusion of regions that could not be aligned with confidence the total length of the aligned sequences was 1190 characters with 106 vari-able characters of which 80 were potentially parsimony informative Gap coding generated an extra 26 parsimony informative characters which means that about 245 of the total potential phylogenetic information within petD is present as indel characters This particularly high number of (phylogenetically informative) indels explains the con-siderable length variability in the petD marker

For the second analysis the pentamerous genera Den-tella and Pentodon were chosen as outgroup taxa As in the larger analysis Lathraeocarpa comes out as sister to Gomphocalyx + Phylohydrax (JS = 100 BS = 100 PP = 1) comprising the LGP clade which also shares a common ancestor with Manostachya ternifolia and Oldenlandia rosulata (JS = 99 BS = 99 PP = 1) In the MP analysis Oldenlandia rosulata is placed as sister to the LGP clade but without significant jackknife and bootstrap support In the BI analysis a clade comprising Manostachya terni-folia and Oldenlandia rosulata is sister to the LGP clade but the relationship between Manostachya ternifolia and Oldenlandia rosulata has low Bayesian posterior prob-ability (PP lt 090)

878

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214


A

1

1

Kohautia amatymbica

Kohautia caespitosaKohautia coccinea

Kohautia cynanchicaKohautia subverticillata

11

Kohautia senegalensis

Dentella dioecaDentella repens

Pentodon pentandrus 11

11

Phylohydrax madagascariensisPhylohydrax carnosaGomphocalyx herniarioides

Lathraeocarpa acicularis

Oldenlandia rosulata

1

1Amphiasma benguellenseAmphiasma luzuloides

Oldenlandia affinisPentanopsis fragrans

1

1

Conostomium natalenseConostomium quadrangulare

Conostomium zoutpansbergenseOldenlandia herbacea var goetzei

Oldenlandia herbacea var herbacea

Manostachya ternifolia

Pentanopsis gracicaulis

B

Kohautia amatymbica





Pentodon pentandrus


Amphiasma benguellenseAmphiasma luzuloidesOldenlandia affinis

Pentanopsis fragrans


Conostomium natalenseConostomium quadrangulareConostomium zoutpansbergenseOldenlandia herbacea var goetzeiOldenlandia herbacea var herbacea

100100


100100

100100100100

9999

95949186

Phylohydrax madagascariensisPhylohydrax carnosa

Gomphocalyx herniarioidesLathraeocarpa acicularis

99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1

Fig 2 Results of phylogenetic Analysis 2 A strict consensus tree of the MPTs from the combined plastid analysis using atpB-rbcL rps16 trnL-trnF and petD sequences for 25 taxa (L = 540 CI = 069 RI = 084) Jackknife (left) and bootstrap (right) values ( gt 65) are indicated above branches B bayesian tree based on combined atpB-rbcL rps16 trnL-trnF and petD data Posterior probabilities are indicated above branches

Table 3 Characteristics of atpB-rbcL rps16 trnL-trnF and petD sequences in the focused analysis of taxa in the Pentanopsis clade (Analysis 2)

No of characters(unaligned)

PIcharacters

PI indels vs total no of characters

(unaligned)

PI indels vs no of PI characters

(unaligned)atpB-rbcL 679ndash734 (av 698) 8 2 220rps16 466ndash482 (av 476) 12 1 110trnL-trnF 327ndash384 (av 350) 24 6 290petD 941ndash994 (av 964) 11 3 245PI = Potentially Informative

Table 4 Characteristics of each data matrix and the corresponding tree statistics

No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI

atpB-rbcL 23 879 58 13 2 83 075 088rps16 23 531 56 6 1 92 082 091trnL-trnF 26 645 83 21 5 143 074 088petD 24 1216 106 26 2 168 077 089Combined 26 3271 303 66 12 531 070 084CI = Consistency Index (Kluge amp Farris 1969) MPT = Most Parsimonious Tree(s) PI = Potentially-Informative RI = Retention Index (Farris 1989)

215


Individual plastid sequence MP analyses were to-pologically congruent except for the sister group of the LGP clade which differs depending on the molecular marker used As in the combined MP analysis the trnL-trnF analysis places Oldenlandia rosulata as sister to the LGP clade although with rather moderate support (JS = 77 BS = 79) The rps16 intron analysis on the other hand shows a clade with O rosulata and M ternifolia as sister to the LGP clade as in the combined BI analysis although the sister relationship of these two species lacks jackknife and bootstrap support In the petD analysis both O rosulata and M ternifolia are unresolved In the atpB-rbcL analysis a clade in which M ternifolia is sister to Conostomium (Stapf) Cufod and O herbacea (L) Roxb is placed as sister to the LGP clade but without significant jackknife or bootstrap support

Morphology and anatomy mdash Since Bremekamp (1957) originally described Lathraeocarpa not a single study has been devoted to the morphology and anatomy of the genus except for some pollen observations of L decaryi by Dessein amp al (2005a) The collection of L acicularis now makes it possible to present a more detailed description of the genus expanding the work of Bremekamp (1957) and Dessein amp al (2005a) with our new (macro- and micro-) morphological and anatomical observations

Growth form ndash Lathraeocarpa acicularis individuals are (sub)shrubs up to ca 25 cm with woody stems and well-developed woody taproots (Figs 3A 4A B) In the protologue Bremekamp (1957) describes L decaryi as a subshrub (ldquofruticulusrdquo) of 12 m or more However the herbarium labels only tell the species is a subshrub and give no details about size In our opinion the plants are

Fig 3 Line drawing of Lathraeocarpa acicularis A habit B ovary and ca-lyx surrounded by three leaves C brevistylous flower D longistylous flower E young fruit

216


much smaller than 12 m and probably not much larger than those of L acicularis Both species have woody stems with grey or brown bark which are much branched (Fig 4A B) In L acicularis the branches are subterete greyish-brown erect-ascending or horizontal and bear numerous short branchlets (Fig 4A B D) that are ini-tially densely pubescent up to 5 cm long but often much shorter with short internodes and terminated by a flower (Fig 4CndashE)

Leaves and stipules ndash The somewhat succulent leaves on the branchlets of Lathraeocarpa are ternate sessile and connate with the stipule base forming a sheath around the stem The leaf-blades are narrowly elliptic or sometimes slightly narrowly ovate 3ndash14 mm long and 03ndash16 mm wide Leaves are pubescent on both surfaces or almost glabrous below and have revolute margins (Fig 5A B) In both species there seems to be a great deal of variation in the density and the length of the trichomes (eg very short hairs in Humbert amp Capuron 29308) When fresh the lamina are green above and reddish-brown below (Fig 4CndashE) Venation is indistinct with the veins sunk in the mesophyll (Fig 5A B D) and only the mid-vein somewhat prominent in L decaryi The leaf apices are mucronate and the bases are slightly narrowed In L acicularis a row of colleters is associated with the inner side of the leaf bases (Fig 6A) The stipular sheaths are cup-shaped 04ndash14 mm long pubescent outside and appear to be

truncate to the naked eye while under high magnification a single short stipular tooth is visible (Fig 5C)

Leaf anatomy ndash Leaf sections of Lathraeocarpa acicu-laris show that both the upper and lower epidermis consist of a single layer of relative large cells covered by a cuticle (Fig 5D H) The leaves are amphistomatic but with a higher density of paracytic stomata on the upper surface (Fig 5A B D) A mesophyll comprised of 2ndash4 layers of palisade-like parenchyma forms a ring from the base towards the rest of the leaf (Fig 5D H) and contains raphide idioblasts (Fig 5D) Inside the ring of mesophyll there is a rather large-celled parenchymatous tissue without chloroplasts which presumably serves to store water surrounding the median vascular bundle and some smaller lateral bundles (Fig 5D) The vascular bundles contain mainly sclerenchy-matic cells which we interpret as metaxylem fibres (Fig 5D E) Phloem is present on the abaxial and lateral sides of the vascular bundles (Fig 5E F) On the adaxial side of the metaxylem fibres a few protoxylem spiral tracheids can be observed (Fig 5E GndashI) Both median and lateral vascular bundles have conspicuous parenchymatic bundle sheaths (Fig 5D E) The parenchymatic bundle sheath from the central vascular bundle extends to the lower epidermis disrupting the otherwise equifacial character of the leaves (Fig 5D) The central vascular bundle is crescent-shaped in cross section and has an inconspicuous sclerenchymatic cap at its abaxial side (Fig 5E)

Fig 4 Photographs of Lathraeocarpa acicularis AndashB growth form C flower bud D longistylous flower E brevistylous flower Photographs by Steven Dessein

217


Inflorescence ndash The inflorescences of Lathraeocarpa are one-flowered and terminal on lateral branchlets Pe-duncles are very short or absent Each flower is subtended by a whorl of three leaves (Fig 3B 4CndashE)

Flowers ndash Lathraeocarpa has white heterodistylous 4-merous flowers (Fig 3C D 4D E) The short calyx tubes are crowned by 8 ciliate calyx lobes (Fig 3B 6A) which are triangular to narrowly triangular and up to 06 mm long in L acicularis (Fig 6B) and up to 15 mm long in L decaryi In L acicularis the corolla tubes are fun-nel-shaped 41ndash64 mm long 07ndash1 mm wide at the base and 17ndash23 mm wide at the throat (Figs 3C D 6A) The corolla tubes are covered with relatively long trichomes on the outer surface (Fig 6A) and with shorter trichomes within (Fig 6C) The corolla lobes are elliptic 22ndash34 mm

long 2ndash23 mm wide and glabrous on both surfaces or papillose inside (Fig 6A D) In L decaryi the corolla tubes are also funnel-shaped ca 7 mm long 04ndash06 mm wide at the base 1ndash15 mm wide at the throat pubescent outside and pubescent in the lower half inside The corolla lobes are ca 2 mm long and sparsely pubescent outside

The four stamens are inserted just below the throat of the corolla tube The white anthers are dorsimedifixed elliptic in outline 05ndash12 mm long and dehisce with lon-gitudinal slits In brevistylous flowers the anthers are exserted for 12ndash26 mm (Fig 3C) whereas in longistylous flowers they are completely included within the corolla tube (Fig 3D)

The ovaries are cup-shaped and up to 18 mm long densely covered with trichomes except on the ribs formed

Fig 5 Leaf and stipule characters of Lathraeocarpa acicularis A adaxial leaf surface B abaxial leaf surface C stipular sheath with one stipular tooth (arrow) D transverse leaf section stomata indicated by arrows E detail of the median vascular leaf bundle F detail of abaxial phloem G detail of adaxial xylem protoxylem tracheids indicated by arrows H longitudinal leaf section at xylem level spiral tracheids indicated by arrows I detail of longitudinal section with metaxy-lem vessels (blue coloured) and spiral tracheids (arrows) Abbreviations ep epidermis me mesophyll mx metaxylem p phloem pa parenchyma px protoxylem sc sclerenchymatic cap x xylem

218


Fig 6 Flower characters of Lathraeocarpa acicularis A flower bud with one corolla lobe removed ring of colleters associated with the bracts indicated by arrow B outside surface of calyx lobe C inner surface of co-rolla tube D inner surface of corolla lobe E outside sur-face of ovary F locule with one ovule basally attached G cylindrical nectary disc

Fig 7 Pollen characters of Lathraeocarpa acicu-laris A equatorial view of thrum pollen grain B detail of thrum mesocolpium C polar view of thrum pollen grain D detail of thrum apo-colpium E equatorial view of pin pollen grain F detail of pin mesocolpium G polar view of pin pollen grain H detail of pin apocolpium All pollen grains studied following the modified method of Halbritter (1998)

219


by the vascular bundles (Figs 3B 6A E) The ovaries are basically 4-locular although flowers with 3-locular ovaries are sometimes observed Each locule has a single ovule inserted at the base of the septum (Fig 6F) The nectary disc on top of the ovary a circular zone surround-ing the base of the style (Fig 6G) is covered with para-cytic stomata and surrounded by a ring of trichomes (Fig 6G) The white styles have a 3- or 4-lobed papillose stigma (Fig 6A) The styles are included within the corolla tube in brevistylous flowers (Fig 3C) and exserted for ca 2ndash3 mm in longistylous flowers (Fig 3D)

Pollen ndash The pollen grains are 7ndash8-zonocolporate suboblate with an equatorial diameter of 24ndash31 μm in Lathraeocarpa acicularis (Fig 7A C E G) and 41ndash50 μm in L decaryi Detailed observations of additional features are only available for L acicularis The tectum is hetero-brochate and varies from reticulate to micro-reticulate (Fig 7B D F H) The inner nexine surface is granu-lar Differences in pollen structure between brevi- and longistylous forms are limited Brevistylous flowers have slightly larger pollen and an ornate apocolpium (reticu-late ornamentation consisting of broad curved muri and lumina that are often anastomosing) with granules on the muri (Fig 7D) whereas longistylous flowers have a (mi-cro-)reticulate apocolpium without granules on the muri (Fig 7H) Moreover grains of brevistylous flowers have a better developed double reticulum than those of longi-stylous flowers (Fig 7B F)

Fruits ndash The fruits of Lathraeocarpa acicularis are obconical 22ndash28 mm long 18ndash2 mm wide indehis-cent and crowned by persistent calyx lobes (Figs 3E 8A)

Distinct ribs are visible on the outer surface The fruits are beset with long trichomes which are more sparse on the ribs (Fig 8B) The fruit wall comprises three distinct layers (Fig 8CndashE) a one-layered exocarp with some cells developed into trichomes a parenchymatic mesocarp with raphides and a thick sclerenchymatic endocarp The vas-cular traces are embedded in the mesocarp close to the endocarp Fruits of L decaryi are similar in shape but up to 35 mm in diameter and sulcate rather than costate According to Bremekamp (1957) the fruit wall anatomy of L decaryi differs from the one of L acicularis in the presence of eight strands of thin-walled cells surround-ing the pyrene which are thought to enhance the floating capacity of the fruits Seeds could not be studied due to lack of appropriate material

Distribution and habitat mdash Collections of Lath-raeocarpa come from two areas in the southern part of Madagascar L decaryi from Ambovombe and Cap Sainte Marie (South Madagascar) and L acicularis from Toliara (southwest Madagascar) The new locality for L acicu-laris is Lac Tsinamampetsotsa (Toliara) Lathraeocarpa acicularis grows on sandy soils in dunes close to the sea whereas L decaryi seems to be restricted to limestone in the dry forests and scrublands of the South

DISCUSSIONInclusion of Lathraeocarpa acicularis in tribe Sper-

macoceae sl is strongly supported by our molecular data Seeing the morphological similarities between

Fig 8 Fruit characters of Lathraeocarpa acicularis A young fruit B detail of the outside surface of the fruit wall with rib in the middle C cross-section through young fruit D cross-section through young fruit E detail of the fruit wall Ab-breviations en endocarp ex exocarp me mesocarp

220


L acicularis and L decaryi the type species of the ge-nus Bremekamprsquos (1957) tribe Lathraeocarpeae can no longer be recognized A close relationship between Lath-raeocarpa and Knoxieae sl as proposed by Karingrehed amp Bremer (2007) is not supported As Capuron (1973) sug-gested our results confirm that Lathraeocarpa is closely related to Gomphocalyx A sister relationship between Lathraeocarpa and the clade comprising Gomphocalyx and Phylohydrax as suggested by our analyses has not been proposed previously Table 5 gives a summary of the morphological characters of the three genera comprising the LGP clade In the following paragraphs we will dis-cuss these morphological data in the light of our molecular evolutionary hypothesis

Growth form mdash Both Gomphocalyx and Phylo-hydrax are creeping herbs forming dense mats whereas Lathraeocarpa is a (sub)shrub Within the Pentanopsis clade most species are annual or perennial herbs some of which have a woody base (eg Manostachya ternifolia) The (sub)shrubby habit of Lathraeocarpa is thus excep-tional within the group and may be interpreted as a case of secondary woodiness Although Rubiaceae are mainly woody comprising predominantly shrubs and trees some lineages of the family include herbaceous representatives Recent molecular studies have shown that most herbaceous Rubiaceae belong to the subfamily Rubioideae (Robbrecht amp Manen 2006) Outside Rubioideae herbaceousness only occurs in Sabiceeae Virectarieae and Sipaneeae Within Rubioideae Jansen amp al (2002) reported strong

indications for cases of secondary woodiness in the tribes Rubieae and Anthospermeae Within the predominantly herbaceous Spermacoceae sl other possible cases of sec-ondary woodiness may be found in the Hawaiian genus Kadua Cham amp Schltdl the Asian Hedyotis L species the neotropical genus Arcytophyllum Willd ex Schult amp Schult f and in some genera of the former tribe Sper-macoceae sstr (Diodella Small Ernodea Sw Galianthe Griseb Spermacoce L)

Phylohydrax is further characterized by a distinct dif-ferentiation into longer vegetative and shorter reproduc-tive stems This kind of shoot differentiation is not found in Gomphocalyx or Lathraeocarpa but does character-ize several other taxa such as Hydrophylax maritima L f and Diodia vaginalis Benth both belonging to Spermac-oceae sstr (Puff 1986 Dessein amp al 2005a) The simi-lar growth form observed in Phylohydrax Hydrophylax maritima and Diodia vaginalis can be explained as an adaptation to similar growth conditions

Leaves and stipules mdash Lathraeocarpa Gomphoca-lyx and Phylohydrax all have amphistomatic leaves (Puff 1986 Dessein amp al 2005a) an uncommon character in Rubiaceae (Robbrecht 1988) A stipular sheath with very short appendage(s) is also a feature shared by the three taxa (Puff 1986 Dessein amp al 2005a) Anatomically the leaves of Lathraeocarpa correspond best with those of Phylohydrax (Puff 1986) Both genera have mesophyll that is not differentiated into spongy and palisade paren-chyma as in Gomphocalyx (Dessein amp al 2005a) but

Table 5 Selected morphological and anatomical characters and character states observed in Lathraeocarpa Gompho-calyx and Phylohydrax

Character Lathraeocarpa Gomphocalyx PhylohydraxGrowth form Subshrubs with terminal flow-

ers on short branchletsProcumbent to decumbent annual or short-lived perennial herbs

Perennial herbs with differentia-tion into longer vegetative stems and short erect flowering stems

Leaf Amphistomatic Amphistomatic Amphistomatic Leaf mesophyll Not differentiated Differentiated Not differentiatedHeterostyly Present Present PresentCalyx tube Reduced Reduced Well-developed Number of calyx lobes 8 8 4 Corolla tube Funnel-shaped Narrowly cylindrical Funnel-shapedOvule position Attached near base of septum Attached near base of septum Attached near base of septumNectary disc Ring-shaped 2-lobed Ring-shaped Stigma 3- or 4-lobed 2-lobed 2-lobedFruit type Drupe Dry and indehiscent Dry and indehiscentSeed Unknown Obovoid ellipsoid or pyriform

without ventral grooveEllipsoid without ventral groove

Pollen type Pluri-zonocolporate Pluri-zonocolporate Pluri-zonocolporatePollen sexine (Micro-)reticulate with granules (Micro-)reticulate with granules (Micro-)reticulate with granules

221


instead consists only of palisade-like parenchyma So far undifferentiated mesophyll has not been reported within Spermacoceae sl Results thus support the hypothesis that the loss of mesophyll differentiation has occurred at least once within the tribe The main difference between Lathraeocarpa and Phylohydrax is the presence of an extension of parenchymatic cells running from the central vascular bundle to the lower epidermis in the leaves of Lathraeocarpa

Inflorescence mdash Lathraeocarpa is characterized by terminal inflorescences The inflorescences of Gom-phocalyx are terminal initially but are pushed aside dur-ing anthesis by the developing shoot in one of the axils making them pseudo-axillary (Dessein amp al 2005a) The inflorescences of Phylohydrax are by contrast truly ax-illary Phylohydrax has single-flowered inflorescences with subsessile flowers (Puff 1986) similar to those in Lathraeocarpa whereas in Gomphocalyx inflorescences are few- to many-flowered

Flowers mdash Based on the four herbarium specimens in which he only observed flowers with an exserted style and included anthers Bremekamp (1957) concluded that Lathraeocarpa is most likely to be isostylous However in L acicularis we clearly observed heterodistyly with longistylous and brevistylous flowers occurring on dif-ferent individuals within the population The collection Humbert amp Capuron 29308 unknown to Bremekamp proves that also L decaryi is truly heterostylous Hetero-styly is very common in Rubiaceae especially in genera of Psychotrieae and Spermacoceae sl (Robbrecht 1988) Gomphocalyx Phylohydrax and all other taxa in the Pen-tanopsis clade (except Conostomium) are heterostylous (Bremekamp 1952 Puff 1986 Dessein amp al 2005a) The sister group of the Pentanopsis clade Kohautia subgenus Kohautia is not heterostylous Taxa of subgenus Kohau-tia are characterized by a flower morphology in which anthers and stigma are always included with the stigma well below the anthers or occasionally just touching them (Mantell 1985) This monomorphic short-styled condition is with exception of a few individuals of Conostomium (Bremekamp 1952) unique among the African members of Spermacoceae It probably evolved as a modification of the dimorphic condition (Robbrecht 1988)

Both Lathraeocarpa and Gomphocalyx have a re-duced calyx tube with eight calyx lobes whereas Phy-lohydrax has a well-developed calyx tube with only four lobes reduced to colleter-tipped outgrowths Flowers of Gomphocalyx have a narrowly cylindrical corolla tube and a bilobed nectary disc in contrast with those of Lathraeo-carpa and Phylohydrax which have funnel-shaped corolla tubes and an annular nectar disc surrounding the base of the style (Puff 1986 Dessein amp al 2005a) The bilobed nectary disc is an autapomorphy in Gomphocalyx not yet observed within other taxa of the Pentanopsis clade

In Gomphocalyx and Phylohydrax the ovary is biloc-ular whereas in Lathraeocarpa it is tri- or tetralocular the 2-locular condition reported by Capuron (1973) could not be confirmed The three taxa all have a single ovule per locule Almost all taxa within Spermacoceae sl have pluri-ovulate ovaries (Robbrecht 1988) Only representa-tives of the former tribe Spermacoceae sstr have uni-ovulate ovaries In Spermacoceae sstr the ovules are however attached to the middle of the septum (Dessein 2003) which is not the case for members of the LGP clade where the ovules are attached near the base of the septum (Puff 1986 Dessein amp al 2005a) Within Sper-macoceae uni-ovulate ovaries with basal attachment of the ovules can thus be considered as a synapomorphy for the LGP clade

Pollen mdash Lathraeocarpa has pluri-zonocolporate pollen as previously reported in Gomphocalyx and Phy-lohydrax (Puff 1986 Dessein amp al 2005a) The presence of this type of pollen was one of the main reasons why the latter two genera were previously included in Spermac-oceae sstr where it is more common than in the rest of Spermacoceae sl in which 3-colporate pollen predomi-nates (Dessein amp al 2002 2005b Dessein 2003) The Asian genus Neanotis WH Lewis is a notable exception in having pluri-zonocolporate pollen grains The genus also shows a trend towards reduction in the number of seeds per locule In mature fruits only one or two seeds are present (Lewis 1966) However with no molecular se-quence data available for the genus it would be premature to hypothesize a close relationship between Neanotis and the LGP clade Nevertheless Neanotis confirms that there is an evolutionary tendency in Spermacoceae to develop pluri-aperturate pollen grains and uni-ovulate ovaries

In contrast to Gomphocalyx and Phylohydrax pol-len grains of Lathraeocarpa acicularis have a double reticulum a feature that also occurs in the tribes Cocco-cypseleae (Piesschaert amp al 2000) and Pavetteae (De Block amp Robbrecht 1998) in the genus Metabolos Blume (Puff amp Igersheim 1994) and in several other members of Spermacoceae sl (Dessein 2003 Dessein amp al 2005b Groeninckx 2005 Pire 1997 Pire amp Cabral 1992) Within the Pentanopsis clade a double reticulum has also been observed in Amphiasma Bremek Oldenlandia af-finis (Roem amp Schult) DC and O herbacea (Scheltens 1998) In Lathraeocarpa the double reticulum is better de-veloped in pollen of brevistylous flowers than in pollen of longistylous flowers A similar dimorphism was observed in the genus Galianthe Griseb of Spermacoceae sstr (Pire amp Cabral 1992) and in several species of Cocco-cypseleae (Piesschaert amp al 2000) Lathraeocarpa pollen further differs in having 8ndash10 relatively long ectocolpi whereas Gomphocalyx and Phylohydrax are characterized by pollen with short colpi (8ndash10 colpi in Gomphocalyx and 10ndash12 colpi in Phylohydrax) (Puff 1986 Dessein amp al

222


2005a) Other members of the Pentanopsis clade as far as known all have pollen with long colpi except for the African genus Conostomium The presence of short ec-tocolpi would support a relationship between the African genus Conostomium and the Gomphocalyx-Phylohydrax clade as suggested by our atpB-rbcL data but without sig-nificant jackknife or bootstrap values However besides short ectocolpi not a single morphological character is known by us that would support a close relationship be-tween Conostomium and the LGP clade Conostomium is characterized by multiovulate placentas and 3-zonocolpo-rate pollen grains The length of the colpi is very variable within Spermacoceae but sometimes demarcates genera or groups of related species as shown by Dessein amp al (2002) for the African Spermacoce

Fruits mdash Fruits of Lathraeocarpa are drupes and their fleshiness stands in contrast with the dry fruits of Gomphocalyx and Phylohydrax Most Spermacoceae have dry fruits but fleshy fruits are also observed In all three taxa the fruit wall consists of three distinct layers (exo- meso- endocarp) but transverse sections through young fruits show that the relative thickness of meso- and endo-carp as well as the position of the vascular traces in the fruit wall differ among the three taxa In Phylohydrax a relatively thick sclerenchymatous endocarp and a thin parenchymatous mesocarp are present and the vascu-lar traces are situated just underneath the exocarp (Puff 1986) Gomphocalyx on the other hand has a relatively thin endocarp a more extensive mesocarp and the vascu-lar traces lie close to the endocarp (Dessein amp al 2005a) The fruit wall anatomy of Lathraeocarpa shows an af-finity with that of both Phylohydrax and Gomphocalyx As in Phylohydrax the endocarp is relatively thick in comparison to the mesocarp but the vascular traces are situated closer to the endocarp as in Gomphocalyx Fruit wall anatomical characters support a close relationship between the three taxa of the LGP clade However too little is known about the fruit wall anatomy of other taxa within Spermacoceae to assess fruit anatomical synapo-morphies to the LGP clade

Morphology of taxa in the LGP clade mdash The op-timization of morphological characters on the molecular tree implies that the common ancestor of Lathraeocarpa Phylohydrax and Gomphocalyx likely had all character states shared by the three taxa amphistomatic leaves with paracytic stomata a stipular sheath with very short appendage(s) heterostyly one basal ovule per locule and pluri-zonocolporate pollen grains The last two character states are synapomorphies for the LGP clade with respect to the Pentanopsis clade Most taxa of Spermacoceae sl have multi-ovulate ovaries and the number of apertures of the pollen grains rarely exceeds five Uni-ovulate ova-ries and pluri-colporate pollen grains are however also observed in the former tribe Spermacoceae sstr This

supports the hypothesis that the development towards uni-ovulate ovaries combined with pluri-aperturate pollen grains has evolved at least two times within Spermaco-ceae sl and that these character states are the result of convergent evolution between the LGP clade and Sperma-coceae sstr More detailed morphological and anatomical research within the Pentanopsis clade and the Sperma-coceae tribe is needed however to assess whether the amphistomatic leaves and the stipular sheath with short appendage(s) can also be considered as synapomorphies for the LGP clade

In all molecular analyses except for the individual atpB-rbcL analysis the LGP clade shares a common ancestor with the African taxa Manostachya ternifolia and Oldenlandia rosulata These two species are erect herbs respectively perennial and annual Despite the high support in the combined MP and BI analysis (JS = 100 BS = 100 PP = 093) not a single morphological or ana-tomical character has so far been found to support a rela-tionship between these two taxa and the LGP clade

Taxonomic implications mdash Molecular data are conclusive to place Lathraeocarpa acicularis within Spermacoceae sl With this new taxonomic position the monogeneric tribe Lathraeocarpeae can no longer be rec-ognized Relationships discovered within the LGP clade can be translated into three different classifications (1) re-duction of Phylohydrax to the synonymy of Gomphocalyx with Lathraeocarpa as sister (2) merging the three taxa of the LGP clade into Gomphocalyx or (3) recognition of all three taxa of the LGP clade as distinct genera

The fusion of Lathraeocarpa and Gomphocalyx as proposed by Piesschaert (2001) is not corroborated by our study Merging Gomphocalyx and Phylohydrax based on morphological similarities (succulent leaves with a ring of palisade-like parenchyma one-flowered inflorescences with subsessile funnel-shaped flowers and a ring-like nec-tar disc) can also be rejected when listing all differences between the two taxa habit (herb vs shrub) number of calyx lobes (four vs eight) fruit type (dry vs fleshy) and pollen ectocolpi (short vs long) In conclusion it is clear that morphology does not provide unambiguous evidence to merge taxa of the LGP clade Therefore it is best to main-tain three separate genera especially because each genus is characterized by a unique set of character states

Environmental adaptation mdash Several morpho-logical characters shared by members of the LGP clade can be regarded as adaptations to a dry habitat Both Phylo hydrax and Lathraeocarpa acicularis are maritime plants species of Phylohydrax are beach pioneers grow-ing just above the high water mark whereas L acicularis occurs in dunes close to the sea Gomphocalyx on the other hand grows further inland on sandy or laterite soils in dry spiny forests up to 850 m high but also on dunes and beaches The most striking environmental adaptation

223


are the amphistomatic leaves which are somewhat suc-culent especially in Lathraeocarpa and Phylohydrax The absence of metaxylem vessels and the abundance of metaxylem fibres in the vascular leaf bundles observed in L acicularis can also be interpreted as an environmen-tal adaptation to xeric conditions Water transport in L acicularis is most likely to be symplastic with the large parenchymatic bundle sheat cells functioning as bulliform cells that absorb and release water to allow the leaf blade to curl or roll up This unusual high amount of metaxylem fibres is not found in the allied genera Gomphocalyx and Phylohydrax and could thus represent an autapomorphy for Lathraeocarpa

CONCLUSIONSSequence data from four plastid markers (atpB-rbcL

rps16 trnL-trnF petD) strongly support the inclusion of the Madagascan endemic genus Lathraeocarpa within the tribe Spermacoceae sl sister to Phylohydrax and Gom-phocalyx As a consequence the tribe Lathraeocarpeae can no longer be recognized and instead Lathraeocarpa must be included within Spermacoceae Some morpho-logical and anatomical characters support this molecular evolutionary hypothesis The clade comprising Lathraeo-carpa Gomphocalyx and Phylohydrax is supported by four apparently independent morphological characters All three genera have amphistomatic leaves a stipular sheath with very short appendage(s) a single basal ovule per locule and pluri-zonocolporate pollen grains

ACKNOWLEDGEMENTSWe thank P Baas from the National Herbarium of the Neth-

erlands and F Lens from the Laboratory of Plant Systematics for helpful discussions and A Fernandez from the National Botanic Garden of Belgium for the botanical line drawing We acknowledge the reviewers for their great effort and effective-ness in improving the quality of the manuscript Research in Madagascar was facilitated by the following Malagasy govern-mental institutions Association Nationale pour la Gestion des Aires Proteacutegeacutees (ANGAP) Ministegravere des Eaux et Forecircts (MEF) and Parc Botanique et Zoologique de Tsimbazaza (PBZT) We thank P Lowry Head of the Africa amp Madagascar Department of Missouri Botanical Garden for the opportunity to perform field work within the framework of MBGrsquos Madagascar Re-search and Conservation Program We also thank the members of the MBG staff in Madagascar for their hospitality and help This research was supported by grants from the Fund for Sci-entific Research Flanders (FWO G020505 and G026804) I Groeninckx holds a PhD research grant from the FWO Flanders

Andersson L amp Rova JHE 1999 The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae) Pl Syst Evol 214 161ndash186

Andersson L Rova JHE amp Alzate FG 2002 Relation-ships circumscription and biogeography of Arcytophyllum (Rubiaceae) based on evidence from cpDNA Brittonia 54 40ndash49

Bremekamp CEB 1952 The African species of Oldenlandia L sensu Hiern et K Schumann Verh Kon Ned Akad Wetensch Afd Natuurk Sect 2 48 1ndash297

Bremekamp CEB 1957 Les Lathraeocarpeacutees tribu nouvelle des Rubioiumldeacutees (Rubiaceacutees) Bull Jard Bot Etat Bruxelles 27 159ndash166

Capuron R 1973 Reacutevision des Rubiaceacutees de Madagascar et des Comores Unpublished manuscript notes regroupeacutees et mises en forme par J Bosser dactylographieacutees de F Chauvet Laboratoire de Phaneacuterogamie Paris

De Block P amp Robbrecht E 1998 Pollen morphology of the Pavetteae (Rubiaceae Ixoroideae) and its taxonomic significance Grana 37 260ndash275

Dessein S 2003 Systematic Studies in the Spermacoceae (Rubiaceae) PhD thesis Katholieke Universiteit Leu-ven Leuven

Dessein S Huysmans S Robbrecht E amp Smets E 2002 Pollen of African Spermacoce species (Rubiaceae) mor-phology and evolutionary aspects Grana 41 69ndash89

Dessein S Andersson L Geuten K Smets E amp Robbrecht E 2005a Gomphocalyx and Phylohydrax (Rubiaceae) sister taxa excluded from the Spermacoceae ss featuring a re-markable case of convergent evolution Taxon 54 91ndash107

Dessein S Ochoterena H De Block P Lens F Rob-brecht E Schols P Smets E Vinckier S amp Huys-mans S 2005b Palynological characters and their phylo-genetic signal in Rubiaceae Bot Rev 71 354ndash414

Farris JS 1989 The retention index and the rescaled consis-tency index Cladistics 5 417ndash419

Goloboff PA 1993 Nona vers 20 Program and documenta-tion distributed by the author Tucuman Argentina

Groeninckx I 2005 Zoektocht naar de taxonomische positie van Mitrasacmopsis (Rubiaceae) op basis van moleculaire en morfologische data Licentiate thesis Katholieke Uni-versiteit Leuven L euven

Groeninckx I Dessein S Ochoterena H Persson C Motley T Karingrehed J Bremer B Huysmans S amp Smets E In press Phylogeny of the herbaceous tribe Sper-macoceae (Rubiaceae) based on plastid DNA data Ann Missouri Bot Gard 96

Halbritter H 1998 Preparing living pollen material for scan-ning elektron microscopy using 22-dimethoxypropane (DMP) and critical-point drying Biotech Histochem 1052 137ndash143

Huelsenbeck J amp Ronquist F 2001 MRBAYES Bayes-ian inference of phylogenetic trees Bioinformatics 17 754ndash755

Igersheim A 1993 The character states of the Caribbean mo-notypic endemic Strumpfia (Rubiaceae) Nord J Bot 13 545ndash559

Jansen S Robbrecht E Beeckman H amp Smets E 2002 A survey of the systematic wood anatomy of the Rubiaceae IAWA J 23 1ndash67

LITERATURE CITED

224


Karingrehed J amp Bremer B 2007 The systematics of Knoxieae (Rubiaceae) ndash molecular data and their taxonomic conse-quences Taxon 56 1051ndash1076

Kluge AG amp Farris JS 1969 Quantitative phyletics and the evolution of anurans Syst Zool 18 1ndash32

Lewis WH 1966 The Asian genus Neanotis nomen novum (Anotis) and allied taxa in the Americas (Rubiaceae) Ann Missouri Bot Gard 53 32ndash46

Loumlhne C amp Borsch T 2005 Molecular evolution and phylo-genetic utility of the petD group II intron a case study in basal angiosperms Molec Biol Evol 22 317ndash332

Mantell DE 1985 The Afro-South-west Asiatic Genus Ko-hautia Cham amp Schlechtd (Rubiaceae ndash Rubioideae ndash Hedyotideae) Morphology Anatomy Taxonomy Phyto-geography and Evolution PhD dissertation Universitaumlt Wien Vienna

Nixon KC 1999 The parsimony Ratchet a new method for rapid parsimony analysis Cladistics 15 407ndash414

Nixon KC 2002 WinClada (beta) vers 10008 Published by the author Ithaca

Piesschaert F 2001 Carpology and Pollen Morphology of the Psychotrieae (Rubiaceae-Rubioideae) Towards a New Tribal and Generic Delimitation PhD thesis Katholieke Universiteit Leuven Leuven

Piesschaert F Huysmans S Jaimes I Robbrecht E amp Smets E 2000 Morphological evidence for an extended tribe Coccocypseleae (Rubiaceae-Rubioideae) Pl Biol 2 536ndash546

Pire SM 1997 Geacutenero Galianthe subg Ebelia (Rubiaceae Spermacoceae) estudio palinoloacutegico Ann Missouri Bot Gard 84 878ndash887

Pire SM amp Cabral EL 1992 El valor del polen en la re-validacioacuten de Galianthe (Spermacoceae-Rubiaceae) Dar-winiana 31 1ndash10

Posada D amp Crandall KA 1998 Modeltest testing the model of DNA substitution Bioinformatics 14 817ndash818

Puff C 1986 Phylohydrax (Rubiaceae-Spermacoceae) ndash a new genus to accommodate the African and Madagascan ldquoHydrophylaxrdquo species Pl Syst Evol 154 343ndash366

Puff C amp Igersheim A 1994 The character states and taxo-nomic position of Metabolos Bl (syn Allaeophania Thw) (Rubiaceae) Bull Jard Bot Natl Belg 63 241ndash262

Punt W Hoen PP Blackmore S Nilsson S amp Le Thomas A 2007 Glossary of pollen and spore terminol-ogy Rev Paleobot Palynol 143 1ndash81

Reitsma T 1969 Size modifications of recent pollen grains under different treatments Rev Paleobot Palynol 9 175ndash202

Robbrecht E 1988 Tropical woody Rubiaceae Characteristic features and progressions Contributions to a new subfa-milial classification Opera Botanica Belgica 1 National Botanic Garden of Belgium Meise

Robbrecht E 1993 Supplement to the 1988 outline of the clas-sification of the Rubiaceae Index to genera Pp 173ndash196 in Robbrecht E (ed) Advances in Rubiaceae Macro-systematics Opera Botanica Belgica 6 National Botanic Garden of Belgium Meise

Robbrecht E amp Manen JF 2006 The major evolutionary lineages of the coffee family (Rubiaceae angiosperms) Combined analysis (nDNA and cpDNA) to infer the posi-tion of Coptosapelta and Luculia and supertree construc-tion based on rbcL rps16 trnL-trnF and atpB-rbcL data A new classification in two subfamilies Cinchonoideae and Rubioideae Syst Geogr Pl 76 85ndash146

Ronquist F amp Huelsenbeck JP 2003 MRBAYES 3 Bayes-ian phylogenetic inference under mixed models Bioinfor-matics 19 1572ndash1574

Scheltens A 1998 Pollenmorfologische studie van de Afri-kaanse Hedyotideae (Rubiaceae) Licentiate thesis Katho-lieke Universiteit Leuven Leuven

Schols P Dessein S DrsquoHondt C Huysmans S amp Smets E 2002 CARNOY a new digital measurement tool for palynology Grana 41 124ndash126

Schumann K 1891 Rubiaceae Pp 1ndash156 in Engler A amp Prantl K (eds) Die natuumlrlichen Pflanzenfamilien vol 4 Engelmann Leipzig

Simmons MP amp Ochoterena H 2000 Gaps as characters in sequence-based phylogenetic analyses Syst Biol 49 369ndash381

Suzuki Y Glazko GV amp Nei M 2002 Over credibility of molecular phylogenies obtained by Bayesian phylogenet-ics Proc Natl Acad Sci USA 99 16138ndash16143

Swofford DL 2002 PAUP Phylogenetic Analysis Using Parsimony (and Other Methods) vers 40b10 Sinauer Sunderland

Thulin M amp Bremer B 2004 Studies in the tribe Sperma-coceae (Rubiaceae-Rubioideae) the circumscriptions of Amphiasma and Pentanopsis and the affinities of Phylo-hydrax Pl Syst Evol 247 233ndash239

Appendix List of taxa used in the phylogenetic analyses with voucher information (geographic origin collector collector number herbarium) accession numbers and literature citations for previouslz published sequences for the four plastid markers atpB-rbcL rps16 trnL-trnF and petD (1) = Andersson amp Rova 1999 (2) = Andersson amp al 2002 (3) = Dessein amp al 2005 (4) = Groeninckx amp al in press New sequences are marked with

Agathisanthemum Klotzsch A bojeri Klotzsch Zambia Dessein amp al 671 (BR) EU542917(4) EU543018(4) EU543077(4) A globosum (Hochst ex A Rich) Klotzsch Zambia Dessein amp al 201 (BR) EU542918(4) EU543019(4) EU543078(4) Amphiasma Bremek A benguellense (Hiern) Bremek Angola Kers 3350 (S) EU542919(4) AF002753(1) EU543079(4) EU557680 A luzu-loides (K Schum) Bremek Zambia Dessein amp al 1167 (BR) EU542920(4) EU543020(4) EU543080(4) EU557681 Arcytophyl-lum Willd ex Schult amp Schult f A aristatum Standl Ecuador Hekker amp Hekking 10335 (GB) AF333348(2) AF333349(2) A ciliolatum Standl Ecuador Oslashllgaard amp al 58395 (NY) AF333350(2) AF333351(2) A ericoides (Willd ex Roem amp Schult) Standl unknown Edwin amp al 3624 (S) AF333352(2) AF333353(2) A lavarum K Schum Costa Rica Cronquist 8827 (NY) AF333354(2) AF333355(2) A macbridei Standl Peru Wurdack 1073 (NY) AF333356(2) AF333357(2) A muticum (Wedd) Standl Colombia Andersson amp al 2195 (GB) EU542921(4) AF002754(1) EU543081(4) A nitidum (Kunth) Schltdl Venezuela Pipoly amp al 6467 (GB) AF333359(2) A rivetii Danguy amp Cherm Ecuador Harling amp Andersson 22232 (GB) EU542922(4) AF333362(2) AF333363(2)

225


A serpyllaceum (Schltdl) Terrell Mexico Stafford amp al 203 (MO) AF333364(2) A setosum (Ruiz amp Pav) Schltdl Colombia Andersson amp al 2196 (GB) AF002755(1) AF333365(2) A thymifolium (Ruiz amp Pav) Standl Ecuador Staringhl 4481 (GB) EU542923(4) AF333366(2) EU543082(4) Batopedina Verdc (outgroup Analysis 1) B pulvinellata Robbrecht Zambia Dessein amp al 264 (BR) EU542924(4) EU543021(4) EU543083(4) Bouvardia Salisb B glaberrima Engelm cult Forbes sn (S) EU542925(4) EU543022(4) EU543084(4) B ternifolia (Cav) Schltdl unknown Van Caekenberghe 264 (cult at BR) AF002758(1) Mexico Spencer amp al 363 (NY) EU642537(4) Carphalea Juss (outgroup Analysis 1) C madagascariensis Lam Madagascar De Block amp al 578 (BR) EU542926(4) EU543023(4) Conostomium (Stapf) Cufod C natalense (Hochst) Bremek South Africa Dahlstrand 1346 (GB) AF002760(1) EU543085(4) EU557687 South Africa Bremer amp al 4341 (UPS) EU542927(4) C quadrangulare (Rendle) Cufod Ethiopia Puff amp Kelbessa 821222 (UPS) EU542928(4) EU543024(4) EU543086(4) EU557688 C zoutpansbergense (Bremek) Bremek South Africa Bremer amp al 4331 (UPS) EU542929(4) EU543087(4) EU557689 Crusea Cham amp Schltdl C calocephala DC Guatemala Gustafsson amp al 215 (GB) EU542930(4) EU543088(4) C megalocarpa (A Gray) S Watson Mexico Pringle 3852 (S) EU542931(4) EU543025(4) EU543089(4) Dentella JR Forst amp G Forst (outgroup Analysis 2) D dioeca Airy Shaw Australia Harwood 1559 (BR) EU543090(4) EU557692 D repens (L) JR Forst amp G Forst Australia Andersson 2262 (GB) EU542932(4) AF333370(2) EU543091(4) EU557693 Dibrachionostylus Bremek D kaessneri (S Moore) Bremek Kenya Strid 2598 (GB) EU542933(4) AF002761(1) Diodella Small D sarmentosa (Sw) Bacigalupo amp Cabral ex Borhidi French Guiana Anderson amp al 2071 (GB) AF002762(1) Diodia L D aulacosperma K Schum Kenya Luke 9029 (UPS) EU542934(4) EU543026(4) EU543092(4) D spicata Miq French Guiana Anderson amp al 1961 (GB) EU542935(4) EU543027(4) EU543093(4) Emmeorhiza Pohl ex Endl E umbellata (Spreng) K Schum Trinidad Hummel 4 (GB) EU542936(4) AY764289(3) EU543094(4) Ernodea Sw E littoralis Sw Cuba Rova amp al 2286 (GB) EU542937(4) AF002763(1) EU543095(4) Galianthe Griseb G brasiliensis (Spreng) EL Cabral amp Bacigalupo Argentina Vanni amp Radovancick 996 (GB) EU542938(4) AY764290(3) EU543096(4) G eupatorioides (Cham amp Schltdl) EL Cabral Argentina Schinini amp Cristobal 9811 (GB) EU542939(4) EU543028(4) EU543097(4) Gomphocalyx Baker G herniarioides Baker Madagascar De Block amp al 569 (BR) AY764291(3) Madagascar Groeninckx amp al 125 (BR) EU542940 EU567466 EU567461 Hedyotis L H consanguinea Hance Hong Kong Shiu Ying Hu 10821 (S) EU542941(4) H fruticosa L Sri Lanka Larsson amp Pyddoke 22 (S) EU542942(4) EU543098(4) H korrorensis (Valeton) Hosok The Caroline Islands Fosberg 47697 (S) EU542943(4) EU543099(4) H lawsoniae Wight Sri Lanka Wambeek amp Wanntorp 2996 (S) EU542944(4) H lessertiana var lassertiana Thwaites Sri Lanka Klackenberg 413 (S) EU542945(4) EU543029(4) EU543100(4) H lessertiana var marginata Thwaites amp Trimen Sri Lanka Fagerlind 3668 (S) EU542946(4) EU543030(4) EU543101(4) H macrostegia Stapf Malaysia Sabah Wallander 6 (GB) EU542947(4) AF002767(1) EU543102(4) H quinquenervis Thwaites Sri Lanka Bremer amp al 163 (S) EU542948(4) EU543103(4) H rhinophylla Thwaires ex Trimen Sri Lanka Fagerlind 5082 (S) EU542949(4) EU543104(4) H swertioides Hook f South India Klackenberg amp Lundin 3 (S) EU542950(4) EU543031(4) EU543105(4) Hedythyrsus Bremek H spermacocinus (K Schum) Bremek Zambia Dessein amp al 1017 (BR) EU542951(4) EU543032(4) EU543107(4) Hemidiodia K Schum H ocymifolia (Willd ex Roem amp Schult) K Schum French Guiana Andersson amp al 2040 (GB) EU542952(4) EU543108(4) Houstonia L H caerulea L USA Vincent amp Lammers sn (GB) EU542953(4) AF333379(2) EU543109(4) H longifolia Gaertn USA Yatskievych 96-49 (MO) EU542954(4) AF002766(1) USA Weigend 9963 (NY) EU642536(4) Kadua Cham amp Schltdl K acuminata Cham amp Schltdl USA Hawaii cult at BR EU542955(4) EU543110(4) K affinis Cham amp Schltdl USA Hawaii Motley 1733 (NY) EU642523(4) EU642538(4) K axillaris (Wawra) WL Wagner amp Lorence USA Hawaii Harrison-Gagne sn (GB) AF002765(1) USA Hawaii Maul Motley 1724 (NY) EU642524(4) EU642535(4) K centran-thoides Hook amp Arn USA Hawaii Skottsberg 6788 (S) EU542956(4) EU543033(4) EU543111(4) K cordata Cham amp Schltdl cult Lorence 8021 (PTBG) EU542957(4) AF333376(2) EU543112(4) K coriacea (JE Smith) WL Wagner amp Lorence USA Hawaii Motley 1703 (NY) EU642525(4) EU642539(4) K degeneri (Fosberg) WL Wagner amp Lorence cult Wood 5062 (PTGB) EU542958(4) AF333371(2) EU543113(4) K elatior (H Mann) WL Wagner amp Lorence USA Hawaii Kauai Wagner 6350 (BISH) EU642526(4) EU642540(4) K fluviatilis CN Forbes USA Hawaii Oahu Motley 1747 (NY) EU642527(4) EU642541(4) K flynnii (WL Wagner amp Lorence) WL Wagner amp Lorence USA Hawaii Kauai Perlman 15631 (BISH) EU642528(4) EU642542(4) K foggiana (Fosberg) WL Wagner amp Lorence USA Hawaii Sparre 27 (S) EU542959(4) EU543114(4) K fosbergii (WL Wag-ner amp DR Herbst) WL Wagner amp Lorence USA Hawaii Oahu Motley 1677 (NY) EU642529(4) EU642543(4) K laxiflora H Mann USA Hawaii Molokai Perlman 6647 (BISH) EU642530(4) EU642544(4) K littoralis Hillebr USA Hawaii Molokai Kiehn amp Luegmayr 920823 (WU) EU542960(4) EU543034(4) EU543115(4) K parvula A Gray cult Perlman 12783 (GB) EU542961(4) AF333375(2) EU543116(4) K rapensis F Br Rapa Is French Polynesia Perlman 17953 (NY) EU642531(4) EU642545(4) Kohautia Cham amp Schltdl K amatymbica Eckl amp Zeyh South Africa Bremer amp al 4307 (UPS) EU542962(4) EU543035(4) EU543117(4) EU557721 K caespitosa Schnizl Zambia Dessein amp al 432 (BR) EU542963(4) EU543036(4) EU543118(4) EU557722 K coccinea Royle Zambia Dessein amp al 751 (BR) EU542964(4) EU543037(4) EU543119(4) EU557723 K cynanchica DC Zambia Dessein amp al 469 (BR) EU542965(4) EU543038(4) EU543120(4) EU557724 K microcala Bremek Zambia Dessein amp al 1149 (BR) EU542966(4) EU543039(4) EU543121(4) K obtusiloba Schnizl Kenya Luke 9035 (UPS) EU542967(4) EU543040(4) EU543122(4) K senegalensis Cham amp Schltdl Burkina Faso Madsen 5940 (NY) EU642546(4) K subverticillata (K Schum) D Mantell Zambia Dessein amp al 432 (BR) EU542968(4) EU543041(4) EU543123(4) EU557727 K virgata (Willd) Bremek Madagascar De Block amp al 539 (BR) EU542969(4) EU543124(4) Lathraeocarpa Bremek L acicularis Bremek Madagascar De Block amp al 2316 brevistylous (BR) EU642516 EU642521 EU642532 EU642519 Madagascar De Block amp al 2316 longistylous (BR) EU642517 EU642522 EU642533 EU642520 Leyla Bremek L osteocarpa Bremek Tanzania Gereau 2513 (BR) EU542970(4) EU543125(4) Manettia Mutis ex L M alba (Aubl) Wernh French Guiana Andersson amp al 1917 (GB) EU542971(4) AF002768(1) M lygistum (L) Sw Colombia Andersson amp al 2128 (GB) EU542972(4) AF002769(1) EU543126(4) Manostachya Bremek M ternifolia ES Martins Zambia Dessein amp al 265 (BR) EU542973(4) EU543042(4) EU543127(4) EU557731 Mitracarpus Zucc ex Schult amp Schult f M frigidus (Willd ex Roem amp Schult) K Schum French Guiana Andersson amp al 1995 (GB) EU542974(4) AF002770(1) EU543128(4) M microspermus K Schum Guiana Jansen-Jacobs

Appendix Continued

226


amp al 4785 (GB) EU542975(4) EU543044(4) Mitrasacmopsis Jovet M quadrivalvis Jovet Zambia Dessein amp al 1273 (BR) EU542976(4) EU543045(4) EU543129(4) Nesohedyotis (Hook f) Bremek N arborea (Roxb) Bremek cult Chase 2915 (K) AF003607(1) Oldenlandia L O affinis (Roem amp Schult) DC Zambia Dessein amp al 627 (BR) EU542977(4) EU543046(4) EU543130(4) EU557734 O angolensis K Schum Zambia Dessein amp al 932 (BR) EU542978(4) EU543047(4) EU543131(4) O biflora (L) Lam Japan Van Caekenberghe 63 (cult at BR) EU542979(4) EU543132(4) O capensis L f var capensis Zambia Dessein amp al 843 (BR) EU542980(4) EU543048(4) EU543133(4) O capensis L f var pleiosepala Bremek Tanzania Kayombe amp al 4 (BR) EU542981(4) EU543049(4) EU543134(4) O corymbosa L Zambia Dessein amp al 487 (BR) EU542982(4) EU543050(4) EU543135(4) O echinulosa K Schum Zambia Dessein amp al 928 (BR) EU542983(4) EU543051(4) EU543136(4) O echinulosa K Schum var pellicida (Hiern) Verdc Tanzania Kayombo amp Kahemela 1993 (BR) EU542984(4) EU543137(4) O fastigiata Bremek Zambia Dessein amp al 1019 (BR) EU542985(4) EU543052(4) EU543138(4) O galioides (F Muell) F Muell Australia Harwood 1511 (BR) EU542986(4) EU543053(4) EU543139(4) O geophila Bremek Zambia Dessein amp al 935 (BR) EU542987(4) EU543054(4) EU543140(4) O goreensis (DC) Summerh Zambia Dessein amp al 1286 (BR) EU542988(4) EU543055(4) EU543141(4) O herbacea (L) Roxb var goetzei (DC) Summerh Zambia Dessein amp al 442 (BR) EU542989(4) EU543056(4) EU543142(4) EU557746 O herbacea (L) Roxb var herbacea Zambia Dessein amp al 463 (BR) EU542990(4) EU543057(4) EU543143(4) EU557747 O lancifolia (Schumach) DC Zambia Dessein amp al 1356 (BR) EU542991(4) EU543058(4) EU543144(4) O mi-crotheca (Cham amp Schltdl) DC Mexico Froumldestroumlm amp Hulteacuten 681 (S) EU542992(4) EU543059(4) EU543145(4) O mitrasacmoides F Muell Australia Harwood 1516 (BR) EU542993(4) EU543146(4) O nematocaulis Bremek Zambia Dessein amp al 924 (BR) EU542994(4) EU543060(4) O nervosa Hiern Gabon Andersson amp Nilsson 2326 (GB) AF333382(2) O robinsonii Pit Zambia Dessein amp al 346 (BR) EU543061(4) EU543147(4) O rosulata K Schum Zambia Dessein amp al 1197 (BR) EU543043(4) EU567467 EU567465 O salzmannii (DC) Benth amp Hook f ex BD Jacks Brazil Harley 15514 (UPS) EU542995(4) AY764294(3) EU543148(4) O taborensis Bremek Tanzania Bidgood amp al 4015 (BR) EU542996(4) EU543149(4) O tenelliflora (Blume) Kuntze Japan Van Caekenberghe 70 (cult at BR) EU542997(4) EU543062(4) EU543106(4) O tenuis K Schum Guyana Jansen-Jacobs amp al 41 (UPS) EU542998(4) AY764293(3) O uniflora L USA Godfrey 57268 (GB) EU542999(4) AY764295(3) EU543150(4) O wiedemannii K Schum Kenya Luke amp Luke 8362 (UPS) EU543000(4) EU543063(4) EU543151(4) Paraknoxia Bremek (outgroup Analysis 1) P parviflora (Stapf ex Verdc) Verdc ex Bremek Zambia Dessein amp al 678 (BR) EU543001(4) EU543064(4) EU543152(4) Pentanopsis Rendle P fragrans Rendle Ethiopia Gilbert amp al 7458 (UPS) EU543065(4) EU543153(4) EU557758 P gracilicaulis (Verdc) Thulin amp Bremer Somalia Thulin amp al 10512 (UPS) EU567458 EU567460 EU567468 Pentodon Hochst (outgroup Analysis 2) P pentandrus (K Schum amp Thonn) Vatke Zambia Dessein amp al 598 (BR) EU543002(4) EU543066(4) EU543154(4) EU557759 Phylohydrax Puff P carnosa (Hochst) Puff South Africa Bremer 3783 (UPS) EU543003(4) EU543067(4) EU642534 EU557760 P madagascariensis (Willd ex Roem amp Schult) Puff Madagascar De Block amp al 640 (BR) EU543004(4) AY64292(3) EU543155(4) EU557761 Richardia L R scabra L Colombia Andersson amp al 2073 (GB) EU543005(4) AF003614(1) EU543156(4) R stellaris L Australia Egerod 85343 (GB) EU543006(4) EU543068(4) EU543157(4) Spermacoce L S capitata Ruiz amp Pav French Guiana Andersson 1908 (GB) EU543007(4) EU543069(4) EU543158(4) S confusa Rendle ex Gillis Colombia Andersson amp al 2074 (GB) AF003619(1) S erosa Harwood Australia Harwood 1148 (BR) EU543008(4) EU543070(4) EU543159(4) S filituba (K Schum) Verdc Kenya Luke 9022 (UPS) EU543009(4) EU543071(4) EU543160(4) S flagelliformis Poir Madagascar De Block amp al 794 (BR) EU543010(4) EU543072(4) EU543161(4) S hispida L Sri Lanka Wanntorp amp al 2667 (S) EU543011(4) EU543073(4) EU543162(4) S prostrata Aubl Colombia Andersson amp al 2078 (GB) EU543012(4) EU543163(4) S remota (Lam) Bacigalupo amp Cabral French Guiana Andersson amp al 2016 (GB) EU543013(4) EU543164(4) S ruelliae DC Gabon Andersson amp Nilsson 2296 (GB) EU543014(4) EU543074(4) EU543165(4) Stenaria (Raf) Terrell S nigricans (Lam) Terrell USA Yatskievych 96-92 (MO) EU543015(4) AF333373(2) EU543166(4) Synaptantha Hook f S tillaeacea (F Muell) Hook f Australia Lazarides amp Palmer 272 (K) EU543016(4) EU543075(4) EU543167(4) Thecorchus (Hiern) Bremek T wauensis (Hiern) Bremek Ethiopia Friis amp al 2560 (UPS) EU543017(4) EU543076(4) EU543168(4)

Appendix Continued

210


united forming Spermacoceae sensu lato (sl hereafter) (Robbrecht amp Manen 2006 Groeninckx amp al in press) If Capuronrsquos decision (1973) to place Gomphocalyx and Lathraeocarpa together was correct Lathraeocarpa too would be related to Phylohydrax and should therefore also be placed in Spermacoceae sl

Several morphological characters distinguish Lath-raeocarpa from Gomphocalyx some of which might even point to an affinity with Triainolepis First of all the (sub)shrubby habit of Lathraeocarpa is much closer to the shrubby habit of Triainolepis than to the herbaceous habit of Gomphocalyx Furthermore the pyrene of L decaryi is surrounded by eight strands of thin-walled cells a condi-tion very similar to that observed in some Triainolepis species (Bremekamp 1957 Piesschaert 2001) Likewise Lathraeocarpa and Trianolepis have a plurilocular ovary and fleshy fruits whereas Gomphocalyx has a bilocular ovary and dry fruits which has prompted some authors (Karingrehed amp Bremer 2007) to believe that Lathraeocarpa should be regarded as a member of the emended tribe Knoxieae rather than a member of Spermacoceae sl

To date no molecular data have been available for Lathraeocarpa so its taxonomic position has remained controversial (Robbrecht amp Manen 2006) This paper presents the results of a multidisciplinary study based on morphological and molecular data illuminating the relationships of this enigmatic genus

MATERIALS AND METHODSMolecular study mdash A few preliminary analyses

of rps16 intron sequences of representatives of the ma-jor groups within subfamily Rubioideae have shown that Lathraeocarpa acicularis belongs to Spermacoceae sl We thus used the matrix of Groeninckx amp al (in press) based on the chloroplast markers atpB-rbcL rps16 and trnL-trnF as a basis for an initial analysis presented in this paper The chloroplast dataset was enlarged by the addition of three atpB-rbcL sequences (Gomphocalyx her-niarioides Lathraeocarpa acicularis and Pentanopsis gracilicaulis (Verdc) Thulin amp Bremer) three rps16 se-quences (Lathraeocarpa acicularis Oldenlandia biflora (L) Lam Pentanopsis gracilicaulis) and five trnL-F sequences (Gomphocalyx herniarioides Lathraeocarpa acicularis Oldenlandia rosulata K Schum Pentanopsis gracilicaulis Phylohydrax carnosa (Hochst) Puff) Our dataset included a total of 132 species representing 35 of the 60 genera within Spermacoceae To improve reso-lution a second more focused analysis was performed with petD included as an extra chloroplast marker using a smaller set of 25 taxa including Lathraeocarpa and 21 closely related taxa as well as the pentamerous Dentella dioeca Airy Shaw D repens (L) JR Forst amp G Forst

and Pentodon pentandrus (K Schum amp Thonn) Vatke as outgroup taxa The Appendix lists all taxa included in this study with author names voucher information and GenBank accession numbers

Methods for DNA extraction PCR amplification se-quencing sequence assembly and alignment are as de-scribed in Groeninckx amp al (in press) The petD region was amplified with the forward primer PIpetB1365F and the reverse primer PIpetD738R as described by Loumlhne amp Borsch (2005)

Equally weighted maximum parsimony (MP) anal-yses were performed using Nona 20 (Goloboff 1993) launched through WinClada 10008 (Nixon 2002) Par-simonious informative gaps were coded manually ac-cording to the conservative lsquosimple indel codingrsquo method described by Simmons amp Ochoterena (2000) The four plastid regions were first analyzed separately and given congruence between the individual topologies then com-bined using a total evidence approach Heuristic searches for the shortest trees were performed using the parsimony ratchet (Nixon 1999) Ratchet runs of 200 iterations each holding 1 tree per iteration and randomly weighting 10 of the potentially informative characters were carried out until the most parsimonious trees (MPTs) were repeat-edly found A strict consensus tree was calculated using the trees obtained in the parsimony ratchet analyses In order to evaluate the relative support of the clades jack-knife (JS) and bootstrap (BS) analyses were executed us-ing 1000 replicates with 100 initial trees holding 1 tree per random addition performing TBR (Tree-Bisection and Reconnection) to hold 1000 trees and calculating a consensus on each repetition Frequency values ( gt 65) were plotted onto the consensus of the MPTs

Bayesian inference (BI) analyses were only carried out for the two combined datasets A substitution model was selected for each DNA region with Modeltest 37 (Posada amp Crandall 1998) under the Akaike Informa-tion Criterion (AIC) (Table 1) Indels were not included in the BI analyses In the combined analysis a mixed-model approach was used (Ronquist amp Huelsenbeck 2003) The combined dataset was partitioned and the same models of evolution were used on the partitions as selected for the single analyses The BI analyses were conducted with Mr-Bayes 3b4 (Huelsenbeck amp Ronquist 2001) as described by Groeninckx amp al (in press) We selected a GTR + I + G a GTR + G and a GTR + I substitution model as being analogous respectively to the TVM + I + G TVM+G and TVM + I models of substitution selected by Modeltest but not implemented in MrBayes The TVM model is similar to the GTR model except for having equal probabilities of change of transitions Four Markov chains (one cold three heated) starting with a random tree were run simultane-ously for three million (Analysis 1) and for one million (Analysis 2) generations sampling trees at every 1000th

211













No of taxa

No of characters

No of PI characters

No of PI indels

No of MPT

MPT length CI RI


212


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fe (l

eft)

and

boot

stra

p (r

ight

) val

ues

(gt 65

) are

indi

cate

d ab

ove

bran

ches

B b

ayes

ian

tree

bas

ed o

n co

mbi

ned

atpB

-rbc

L

rps1

6 an

d tr

nL-t

rnF

data

Pos

teri

or p

roba

bilit

ies

gt 0

9 ar

e in

dica

ted

abov

e br

anch

es

214


A

1

1

Kohautia amatymbica



11




11




1



1

1






B

Kohautia amatymbica





Pentodon pentandrus






100100


100100

100100100100

9999

95949186



99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1




PIcharacters


(unaligned)




No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI


215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

211













No of taxa

No of characters

No of PI characters

No of PI indels

No of MPT

MPT length CI RI


212


969

6

949

5

848

493

95

747

4

989

6

999

8

100

100

878

6

949

410

010

0

918

6

Kad

ua s

pp (

16)

Old

enla

ndia

bifl

ora

928

5

999

9

999

9

959

510

099

Den

tella

dio

eca

Den

tella

repe

ns

Pen

todo

n pe

ntan

drus

100

100

100

100 10

010

095

95

887

7

Bat

oped

ina

pulv

inel

lata

(out

grou

p)C

arph

alea

mad

agas

carie

nsis

(out

grou

p)P

arak

noxi

a pa

rvifl

ora

(out

grou

p)

100

100

726

9

Pentanopsis clade

Koh

autia

am

atym

bica

Koh

autia

cae

spito

saK

ohau

tia c

occi

nea

Koh

autia

cyn

anch

ica

Koh

autia

sub

verti

cilla

ta

Koh

autia

sen

egal

ensi

sM

anos

tach

ya te

rnifo

liaO

lden

land

ia ro

sula

ta

Dib

rach

iono

styl

us k

aess

neri

Hed

ythy

rsus

spe

rmac

ocin

usM

itras

acm

opsi

s qu

adriv

alvi

s

Old

enla

ndia

ech

inul

osa

Old

enla

ndia

ech

inul

osa

var

pelli

cida

Old

enla

ndia

fast

igia

ta

Old

enla

ndia

geo

phila

Old

enla

ndia

ner

vosa

999

7

938

7

979

6

Koh

autia

obt

usilo

ba

Koh

autia

virg

ata

Old

enla

ndia

cap

ensi

s va

r ca

pens

isO

lden

land

ia c

apen

sis

var

plei

osep

ala

Old

enla

ndia

nem

atoc

aulis

Old

enla

ndia

robi

nson

ii

Old

enla

ndia

tabo

rens

is

Old

enla

ndia

wie

dem

anni

i

Thec

orch

us w

auen

sis

Koh

autia

mic

roca

la

Old

enla

ndia

cor

ymbo

sa

Hou

ston

ia c

aeru

lea

Hou

ston

ia lo

ngifo

lia

Old

enla

ndia

mic

roth

eca

Ste

naria

nig

rican

sA

rcyt

ophy

llum

spp

(10

)93

92

868

3

Old

enla

ndia

mitr

asac

moi

des

Hed

yotis

tene

lliflo

raO

lden

land

ia g

alio

ides

Old

enla

ndia

lanc

ifolia

Syn

apta

ntha

tilla

eace

a

888

3

Aga

this

anth

emum

boj

eri

Aga

this

anth

emum

glo

bosu

m

Lely

a os

teoc

arpa

Old

enla

ndia

ang

olen

sis

Old

enla

ndia

gor

eens

is

Old

enla

ndia

uni

flora

868

710

099

989

799

99

100

99

Hed

yotis

spp

(10

)10

010

0

Am

phia

sma

beng

uelle

nse

Am

phia

sma

luzu

loid

es98

99

Old

enla

ndia

affi

nis

Pen

tano

psis

frag

rans

Pen

tano

psis

gra

cica

ulis

100

100

959

6

Con

osto

miu

m n

atal

ense

Old

enla

ndia

her

bace

a va

r go

etze

iO

lden

land

ia h

erba

cea

var

herb

acea

100

100

Con

osto

miu

m q

uadr

angu

lare

Con

osto

miu

m z

outp

ansb

erge

nse

999

999

99

939

598

98

Gom

phoc

alyx

her

niar

ioid

esP

hylo

hydr

ax c

arno

saP

hylo

hydr

ax m

adag

asca

riens

is

Lath

raeo

carp

a ac

icul

aris

100

100

1

Arc

ytop

hyllu

m s

pp (

10)

Kad

ua s

pp (

16)

Hed

yotis

spp

(10

)

Bat

oped

ina

pulv

inel

lata

(out

grou

p)C

arph

alea

mad

agas

carie

nsis

(out

grou

p)P

arak

noxi

a pa

rvifl

ora

(out

grou

p)

Den

tella

dio

eca

Den

tella

repe

ns

Pen

todo

n pe

ntan

drus

Koh

autia

am

atym

bica

Koh

autia

cae

spito

saK

ohau

tia c

occi

nea

Koh

autia

cyn

anch

ica

Koh

autia

sub

verti

cilla

ta

Koh

autia

sen

egal

ensi

sM

anos

tach

ya te

rnifo

liaO

lden

land

ia ro

sula

ta0

93

Gom

phoc

alyx

her

niar

ioid

esP

hylo

hydr

ax c

arno

saP

hylo

hydr

ax m

adag

asca

riens

is

Lath

raeo

carp

a ac

icul

aris

11

11

1

1

1

1

111

1

11

1

095

1

1

1

1 1

1 1

Aga

this

anth

emum

boj

eri

Aga

this

anth

emum

glo

bosu

m

Lely

a os

teoc

arpa

Old

enla

ndia

ang

olen

sis

Old

enla

ndia

gor

eens

is

Old

enla

ndia

uni

flora

Dib

rach

iono

styl

us k

aess

neri

Old

enla

ndia

ech

inul

osa

Old

enla

ndia

ech

inul

osa

var

pelli

cida

Old

enla

ndia

geo

phila

Old

enla

ndia

ner

vosa

11

11 1

Hed

yotis

tene

lliflo

raO

lden

land

ia g

alio

ides

Old

enla

ndia

lanc

ifolia

Syn

apta

ntha

tilla

eace

a1

1

Old

enla

ndia

bifl

ora

Old

enla

ndia

mitr

asac

moi

des

1

1

1

Hou

ston

ia c

aeru

lea

Hou

ston

ia lo

ngifo

lia

Old

enla

ndia

mic

roth

eca

Ste

naria

nig

rican

s

1

1

1

Koh

autia

mic

roca

laK

ohau

tia o

btus

iloba

Koh

autia

virg

ata

1

Old

enla

ndia

cap

ensi

s va

r ca

pens

isO

lden

land

ia c

apen

sis

var

plei

osep

ala

Old

enla

ndia

nem

atoc

aulis

Old

enla

ndia

robi

nson

ii

Old

enla

ndia

tabo

rens

is

Old

enla

ndia

wie

dem

anni

i

Thec

orch

us w

auen

sis

Old

enla

ndia

cor

ymbo

sa

1

1

092

1

Am

phia

sma

beng

uelle

nse

Am

phia

sma

luzu

loid

es

Old

enla

ndia

affi

nis

Pen

tano

psis

frag

rans

Pen

tano

psis

gra

cica

ulis

11

1 1

Con

osto

miu

m n

atal

ense

Con

osto

miu

m q

uadr

angu

lare

Con

osto

miu

m z

outp

ansb

erge

nse

Old

enla

ndia

her

bace

a va

r go

etze

iO

lden

land

ia h

erba

cea

var

herb

acea

Hed

ythy

rsus

spe

rmac

ocin

usM

itras

acm

opsi

s qu

adriv

alvi

s

Old

enla

ndia

fast

igia

ta

213






878

7

828

391

88

949

584

84

939

274

71

949

4

100

99

858

799

99

100

100

878

7N

esoh

edyo

tis a

rbor

ea

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

um

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Spe

rmac

oce

flage

llifo

rmis

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Hem

idio

dia

ocym

ifolia

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

capi

tata

Spe

rmac

oce

conf

usa

Spe

rmac

oce

eros

a

Spe

rmac

oce

filitu

ba

Spe

rmac

oce

pros

trata

Spe

rmac

oce

rem

ota

Spe

rmac

oce

ruel

liae

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Dio

dia

spic

ata

Em

meo

rhiz

a um

bella

ta

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Ern

odea

litto

ralis

Spe

rmac

oce

hisp

ida

Spe

rmac

oce

flage

llifo

rmis

Hem

idio

dia

ocym

ifolia

Spe

rmac

oce

rem

ota

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

umN

esoh

edyo

tis a

rbor

eaE

mm

eorh

iza

umbe

llata

Dio

dia

spic

ata

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Spe

rmac

oce

conf

usa

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Spe

rmac

oce

capi

tata

Spe

rmac

oce

eros

aS

perm

acoc

e pr

ostra

ta

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

filitu

baS

perm

acoc

e ru

ellia

e

Spe

rmac

oce

hisp

ida

Ern

odea

litto

ralis

1

1

1 1

099

1

1

1 1

1

11

1

10

98

AB

Fig

1 R

esul

ts o

f phy

loge

netic

Ana

lysi

s 1

A s

tric

t con

sens

us tr

ee o

f the

MPT

s fr

om th

e co

mbi

ned

plas

tid a

naly

sis

usin

g at

pB-r

bcL

rps1

6 an

d tr

nL-t

rnF

sequ

ence

s fo

r 13

2 ta

xa (L

= 1

952

CI =

05

4 R

I = 0

83)

Jac

kkni

fe (l

eft)

and

boot

stra

p (r

ight

) val

ues

(gt 65

) are

indi

cate

d ab

ove

bran

ches

B b

ayes

ian

tree

bas

ed o

n co

mbi

ned

atpB

-rbc

L

rps1

6 an

d tr

nL-t

rnF

data

Pos

teri

or p

roba

bilit

ies

gt 0

9 ar

e in

dica

ted

abov

e br

anch

es

214


A

1

1

Kohautia amatymbica



11




11




1



1

1






B

Kohautia amatymbica





Pentodon pentandrus






100100


100100

100100100100

9999

95949186



99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1




PIcharacters


(unaligned)




No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI


215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

212


969

6

949

5

848

493

95

747

4

989

6

999

8

100

100

878

6

949

410

010

0

918

6

Kad

ua s

pp (

16)

Old

enla

ndia

bifl

ora

928

5

999

9

999

9

959

510

099

Den

tella

dio

eca

Den

tella

repe

ns

Pen

todo

n pe

ntan

drus

100

100

100

100 10

010

095

95

887

7

Bat

oped

ina

pulv

inel

lata

(out

grou

p)C

arph

alea

mad

agas

carie

nsis

(out

grou

p)P

arak

noxi

a pa

rvifl

ora

(out

grou

p)

100

100

726

9

Pentanopsis clade

Koh

autia

am

atym

bica

Koh

autia

cae

spito

saK

ohau

tia c

occi

nea

Koh

autia

cyn

anch

ica

Koh

autia

sub

verti

cilla

ta

Koh

autia

sen

egal

ensi

sM

anos

tach

ya te

rnifo

liaO

lden

land

ia ro

sula

ta

Dib

rach

iono

styl

us k

aess

neri

Hed

ythy

rsus

spe

rmac

ocin

usM

itras

acm

opsi

s qu

adriv

alvi

s

Old

enla

ndia

ech

inul

osa

Old

enla

ndia

ech

inul

osa

var

pelli

cida

Old

enla

ndia

fast

igia

ta

Old

enla

ndia

geo

phila

Old

enla

ndia

ner

vosa

999

7

938

7

979

6

Koh

autia

obt

usilo

ba

Koh

autia

virg

ata

Old

enla

ndia

cap

ensi

s va

r ca

pens

isO

lden

land

ia c

apen

sis

var

plei

osep

ala

Old

enla

ndia

nem

atoc

aulis

Old

enla

ndia

robi

nson

ii

Old

enla

ndia

tabo

rens

is

Old

enla

ndia

wie

dem

anni

i

Thec

orch

us w

auen

sis

Koh

autia

mic

roca

la

Old

enla

ndia

cor

ymbo

sa

Hou

ston

ia c

aeru

lea

Hou

ston

ia lo

ngifo

lia

Old

enla

ndia

mic

roth

eca

Ste

naria

nig

rican

sA

rcyt

ophy

llum

spp

(10

)93

92

868

3

Old

enla

ndia

mitr

asac

moi

des

Hed

yotis

tene

lliflo

raO

lden

land

ia g

alio

ides

Old

enla

ndia

lanc

ifolia

Syn

apta

ntha

tilla

eace

a

888

3

Aga

this

anth

emum

boj

eri

Aga

this

anth

emum

glo

bosu

m

Lely

a os

teoc

arpa

Old

enla

ndia

ang

olen

sis

Old

enla

ndia

gor

eens

is

Old

enla

ndia

uni

flora

868

710

099

989

799

99

100

99

Hed

yotis

spp

(10

)10

010

0

Am

phia

sma

beng

uelle

nse

Am

phia

sma

luzu

loid

es98

99

Old

enla

ndia

affi

nis

Pen

tano

psis

frag

rans

Pen

tano

psis

gra

cica

ulis

100

100

959

6

Con

osto

miu

m n

atal

ense

Old

enla

ndia

her

bace

a va

r go

etze

iO

lden

land

ia h

erba

cea

var

herb

acea

100

100

Con

osto

miu

m q

uadr

angu

lare

Con

osto

miu

m z

outp

ansb

erge

nse

999

999

99

939

598

98

Gom

phoc

alyx

her

niar

ioid

esP

hylo

hydr

ax c

arno

saP

hylo

hydr

ax m

adag

asca

riens

is

Lath

raeo

carp

a ac

icul

aris

100

100

1

Arc

ytop

hyllu

m s

pp (

10)

Kad

ua s

pp (

16)

Hed

yotis

spp

(10

)

Bat

oped

ina

pulv

inel

lata

(out

grou

p)C

arph

alea

mad

agas

carie

nsis

(out

grou

p)P

arak

noxi

a pa

rvifl

ora

(out

grou

p)

Den

tella

dio

eca

Den

tella

repe

ns

Pen

todo

n pe

ntan

drus

Koh

autia

am

atym

bica

Koh

autia

cae

spito

saK

ohau

tia c

occi

nea

Koh

autia

cyn

anch

ica

Koh

autia

sub

verti

cilla

ta

Koh

autia

sen

egal

ensi

sM

anos

tach

ya te

rnifo

liaO

lden

land

ia ro

sula

ta0

93

Gom

phoc

alyx

her

niar

ioid

esP

hylo

hydr

ax c

arno

saP

hylo

hydr

ax m

adag

asca

riens

is

Lath

raeo

carp

a ac

icul

aris

11

11

1

1

1

1

111

1

11

1

095

1

1

1

1 1

1 1

Aga

this

anth

emum

boj

eri

Aga

this

anth

emum

glo

bosu

m

Lely

a os

teoc

arpa

Old

enla

ndia

ang

olen

sis

Old

enla

ndia

gor

eens

is

Old

enla

ndia

uni

flora

Dib

rach

iono

styl

us k

aess

neri

Old

enla

ndia

ech

inul

osa

Old

enla

ndia

ech

inul

osa

var

pelli

cida

Old

enla

ndia

geo

phila

Old

enla

ndia

ner

vosa

11

11 1

Hed

yotis

tene

lliflo

raO

lden

land

ia g

alio

ides

Old

enla

ndia

lanc

ifolia

Syn

apta

ntha

tilla

eace

a1

1

Old

enla

ndia

bifl

ora

Old

enla

ndia

mitr

asac

moi

des

1

1

1

Hou

ston

ia c

aeru

lea

Hou

ston

ia lo

ngifo

lia

Old

enla

ndia

mic

roth

eca

Ste

naria

nig

rican

s

1

1

1

Koh

autia

mic

roca

laK

ohau

tia o

btus

iloba

Koh

autia

virg

ata

1

Old

enla

ndia

cap

ensi

s va

r ca

pens

isO

lden

land

ia c

apen

sis

var

plei

osep

ala

Old

enla

ndia

nem

atoc

aulis

Old

enla

ndia

robi

nson

ii

Old

enla

ndia

tabo

rens

is

Old

enla

ndia

wie

dem

anni

i

Thec

orch

us w

auen

sis

Old

enla

ndia

cor

ymbo

sa

1

1

092

1

Am

phia

sma

beng

uelle

nse

Am

phia

sma

luzu

loid

es

Old

enla

ndia

affi

nis

Pen

tano

psis

frag

rans

Pen

tano

psis

gra

cica

ulis

11

1 1

Con

osto

miu

m n

atal

ense

Con

osto

miu

m q

uadr

angu

lare

Con

osto

miu

m z

outp

ansb

erge

nse

Old

enla

ndia

her

bace

a va

r go

etze

iO

lden

land

ia h

erba

cea

var

herb

acea

Hed

ythy

rsus

spe

rmac

ocin

usM

itras

acm

opsi

s qu

adriv

alvi

s

Old

enla

ndia

fast

igia

ta

213






878

7

828

391

88

949

584

84

939

274

71

949

4

100

99

858

799

99

100

100

878

7N

esoh

edyo

tis a

rbor

ea

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

um

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Spe

rmac

oce

flage

llifo

rmis

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Hem

idio

dia

ocym

ifolia

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

capi

tata

Spe

rmac

oce

conf

usa

Spe

rmac

oce

eros

a

Spe

rmac

oce

filitu

ba

Spe

rmac

oce

pros

trata

Spe

rmac

oce

rem

ota

Spe

rmac

oce

ruel

liae

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Dio

dia

spic

ata

Em

meo

rhiz

a um

bella

ta

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Ern

odea

litto

ralis

Spe

rmac

oce

hisp

ida

Spe

rmac

oce

flage

llifo

rmis

Hem

idio

dia

ocym

ifolia

Spe

rmac

oce

rem

ota

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

umN

esoh

edyo

tis a

rbor

eaE

mm

eorh

iza

umbe

llata

Dio

dia

spic

ata

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Spe

rmac

oce

conf

usa

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Spe

rmac

oce

capi

tata

Spe

rmac

oce

eros

aS

perm

acoc

e pr

ostra

ta

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

filitu

baS

perm

acoc

e ru

ellia

e

Spe

rmac

oce

hisp

ida

Ern

odea

litto

ralis

1

1

1 1

099

1

1

1 1

1

11

1

10

98

AB

Fig

1 R

esul

ts o

f phy

loge

netic

Ana

lysi

s 1

A s

tric

t con

sens

us tr

ee o

f the

MPT

s fr

om th

e co

mbi

ned

plas

tid a

naly

sis

usin

g at

pB-r

bcL

rps1

6 an

d tr

nL-t

rnF

sequ

ence

s fo

r 13

2 ta

xa (L

= 1

952

CI =

05

4 R

I = 0

83)

Jac

kkni

fe (l

eft)

and

boot

stra

p (r

ight

) val

ues

(gt 65

) are

indi

cate

d ab

ove

bran

ches

B b

ayes

ian

tree

bas

ed o

n co

mbi

ned

atpB

-rbc

L

rps1

6 an

d tr

nL-t

rnF

data

Pos

teri

or p

roba

bilit

ies

gt 0

9 ar

e in

dica

ted

abov

e br

anch

es

214


A

1

1

Kohautia amatymbica



11




11




1



1

1






B

Kohautia amatymbica





Pentodon pentandrus






100100


100100

100100100100

9999

95949186



99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1




PIcharacters


(unaligned)




No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI


215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

213






878

7

828

391

88

949

584

84

939

274

71

949

4

100

99

858

799

99

100

100

878

7N

esoh

edyo

tis a

rbor

ea

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

um

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Spe

rmac

oce

flage

llifo

rmis

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Hem

idio

dia

ocym

ifolia

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

capi

tata

Spe

rmac

oce

conf

usa

Spe

rmac

oce

eros

a

Spe

rmac

oce

filitu

ba

Spe

rmac

oce

pros

trata

Spe

rmac

oce

rem

ota

Spe

rmac

oce

ruel

liae

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Dio

dia

spic

ata

Em

meo

rhiz

a um

bella

ta

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Ern

odea

litto

ralis

Spe

rmac

oce

hisp

ida

Spe

rmac

oce

flage

llifo

rmis

Hem

idio

dia

ocym

ifolia

Spe

rmac

oce

rem

ota

Old

enla

ndia

sal

zman

nii

Old

enla

ndia

tenu

isA

rcyt

ophy

llum

ser

pylla

ceum

Bou

vard

ia g

labe

rrim

aB

ouva

rdia

tern

ifolia

Man

ettia

alb

aM

anet

tia ly

gist

umN

esoh

edyo

tis a

rbor

eaE

mm

eorh

iza

umbe

llata

Dio

dia

spic

ata

Gal

iant

he b

rasi

liens

is

Gal

iant

he e

upat

oriid

es

Spe

rmac

oce

conf

usa

Cru

sea

calo

ceph

ala

Cru

sea

meg

aloc

arpa

Ric

hard

ia s

cabr

aR

icha

rdia

ste

llaris

Spe

rmac

oce

capi

tata

Spe

rmac

oce

eros

aS

perm

acoc

e pr

ostra

ta

Dio

dia

aula

cosp

erm

aD

iode

lla s

arm

ento

sa

Mitr

acar

pus

frigi

dus

Mitr

acar

pus

mic

rosp

erm

us

Spe

rmac

oce

filitu

baS

perm

acoc

e ru

ellia

e

Spe

rmac

oce

hisp

ida

Ern

odea

litto

ralis

1

1

1 1

099

1

1

1 1

1

11

1

10

98

AB

Fig

1 R

esul

ts o

f phy

loge

netic

Ana

lysi

s 1

A s

tric

t con

sens

us tr

ee o

f the

MPT

s fr

om th

e co

mbi

ned

plas

tid a

naly

sis

usin

g at

pB-r

bcL

rps1

6 an

d tr

nL-t

rnF

sequ

ence

s fo

r 13

2 ta

xa (L

= 1

952

CI =

05

4 R

I = 0

83)

Jac

kkni

fe (l

eft)

and

boot

stra

p (r

ight

) val

ues

(gt 65

) are

indi

cate

d ab

ove

bran

ches

B b

ayes

ian

tree

bas

ed o

n co

mbi

ned

atpB

-rbc

L

rps1

6 an

d tr

nL-t

rnF

data

Pos

teri

or p

roba

bilit

ies

gt 0

9 ar

e in

dica

ted

abov

e br

anch

es

214


A

1

1

Kohautia amatymbica



11




11




1



1

1






B

Kohautia amatymbica





Pentodon pentandrus






100100


100100

100100100100

9999

95949186



99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1




PIcharacters


(unaligned)




No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI


215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

214


A

1

1

Kohautia amatymbica



11




11




1



1

1






B

Kohautia amatymbica





Pentodon pentandrus






100100


100100

100100100100

9999

95949186



99999999

100100

9999

10099

100100

100100

100100100100

78776772

1

1

11

1

098

1




PIcharacters


(unaligned)




No oftaxa

No ofcharacters

No of PI characters

No of PI indels

No ofMPT

MPTlength CI RI


215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

215






216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

216







217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

217








218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

218




219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

219










220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

220














221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

221









222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

222










223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

223

























LITERATURE CITED

224































225



Appendix Continued

226



Appendix Continued

224































225



Appendix Continued

226



Appendix Continued

225



Appendix Continued

226



Appendix Continued

226



Appendix Continued

Documents

Rediscovery of Malagasy Lathraeocarpa allows determination of its taxonomic position within Rubiaceae