! 

"#$ %&' ()

      $) *+,*-.   $%) /,++.,*0/0,-   1 2 1 11!,*-.                               !" #$     %   " "&&! '&(&& )    )    ) *  + ,    -      -+

  .   /+ "&&!+ *        0   1 2   )) ,   + 2       +                    '' + 3" +    + 4.5 6' !!3 $#6# "+

,    -          ))   + 2                0   -      ' "&&  + ,          )   7   8529 )  ))                            + 2  ) 7   )      %     )      )   0    -     )  2    8 + 2    9 0   0          :  +  )     )          )            )+   )      -   0   -     +  )   .;  )          )       )      !  "    #      <        + ,      $$ )  )                    '#   + ,  *     )  :       7   )         '$        , %    -    + ,     -   ) *          %    &     + ,  *      -       &      #3  + '        "  ( &   &   &      ) &                  + , /     )    &           &   + ,-   &      &$      <              +

)* + 0       )     , %  '$ %  *   *   :        .;   0   2    0   

, -      !   "         # $ ./     !012345    *

= /  .   "&&!

45 '$!' $"'3 4.5 6' !!3 $#6# "  (  ((( $'"3 8 (>> ++> ? @ (  ((( $'"39 finns ingen ro i sinnet finns ingen sorg i minnet finns ingen last att bära men så mycket mycket mer att lära himlen blåser upp!

Mats Ronander

Papers included in this thesis

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I B. Oxelman, M. Backlund & B. Bremer. 1999. Relationships of the s.l. investigated using parsimony jackknife and branch support analysis of chloroplast ndhF and rbcL sequence data. Systematic Botany 24(2): 164-182.

II M. Backlund, B. Oxelman & B. Bremer. 2000. Phylogenetic relationships within the based on ndhF and rbcL se- quences, with particular reference to the . American Journal of Botany 87(7): 1029-1043.

III M. Backlund, B. Bremer & M. Thulin. Manuscript. of Paederieae, recognition of Putorieae and expansion of (-). Submitted to .

IV M. Backlund & M. Thulin. Manuscript. Revision of the Mediter- ranean of Plocama (Rubiaceae). Submitted to Taxon. ______

Published articles were reproduced in this thesis with the kind permis- sion of the publishers.

In papers II, III and IV MB was responsible for the writing (with comments and suggestions given by the co-authors), for all analyses, the major part of the labora- tory work and observations of material. In paper I MB contributed some lab-work, comments and suggestions of the writing. All the papers were planned in co-operation with the co-authors.

Important note. Papers III and IV of this thesis are manuscripts that contain new nomenclatural combinations. These papers have been submitted for publication elsewhere. In order to make clear that these names are not validly published in this thesis, the references to the basionyms, necessary according to the International Code of Botanical Nomenclature, are omitted.

Contents

Introduction...... 9 Phylogenetics ...... 9 Molecules and morphology...... 10 Aims ...... 12 Gentianales...... 14 Traditional and modern views...... 14 Relationships of the Buddlejaceae – paper I ...... 16 Interfamilial relationships of the Gentianales – paper II...... 18 Families of the Gentianales ...... 20 Loganiaceae ...... 20 ...... 22 ...... 22 ...... 23 Rubiaceae...... 23 Rubiaceae systematics ...... 24 The tribes Paederieae & Putorieae – paper III ...... 25 Historical outline...... 25 Identifying the problems ...... 26 The Mediterranean Plocama – paper IV...... 30 Historical outline...... 30 Identifying the problems ...... 30 Nomenclatural considerations ...... 30 Conclusions and future prospects ...... 32 Realignment and synopsis of the Gentianales...... 33 Sammanfattning (Swedish summary) ...... 34 Acknowledgements...... 36 References...... 38

Introduction

The classical botanical systematists created classification systems that were utilistic and practical but not natural in a modern sense. After the introduc- tion of Darwin's ideas in the middle of the 19th century, there was a wide acceptance of evolution as a concept among most of the natural scientists. Many workers attempted to set up classifications that included hypotheses of natural groups, showing how were evolutionarily related to each other. However, these classifications were primarily based on personal opin- ions, which were in fact non-testable. As will be pointed out below this prob- lem was prominent in the Gentianales, as understood in the beginning of the 20th century, and forms the basis for many of the issues adressed in this thesis.

Phylogenetics Acceptance of a single origin of life on Earth, results in a single, albeit com- plex history reflecting its development and evolution (e.g. Martin & Embley, 2004). To address the evolutionary relationships in a scientific way, one requires a method by which different hypotheses can be tested and com- pared. In the middle of the 20th century this became possible and many sys- tematists focused on these questions to develop computational tools. In the 1950's and 60's Hennig (1950; 1966) and others began to develop the theo- retical framework leading to parsimony analysis. This is based on the princi- ple often attributed to William of Ockham (*1285 †1349?), the so-called Ockham's razor:

PLURALITAS NON EST PONENDA SINE NECCESITATE i.e., plurality should not be posited without necessity. In the case of parsi- mony analysis this is fulfilled by searching for the simplest explanation of the data provided, with the result often represented as a branching diagram, commonly referred to as a cladogram. In systematic studies this diagram is subsequently often interpreted as indicative of evolutionary relationships. A number of other methods for phylogenetic reconstruction have been developed with different theoretical backgrounds. Most notable and widely used being neighbor joining (NJ, Kimura, 1983; Nei, 1987), maximum like-

9 lihood (ML, Edwards & Cavalli-Sforza, 1964; Felsenstein, 1973), and Bayesian inference (BI, Li & al., 1996; Mau & Newton., 1997; Yang & Rannala, 1997; Huelsenbeck & Ronquist, 2001). My reasons for the consis- tent use of parsimony analysis throughout this thesis can be summarised in the following points: • use of an explicit optimality criterion (in contrast to NJ) • avoiding a model (not the case in ML and BI) Parsimony analysis works without the use of an evolutionary model. Some people would consider this a problem, but for me it appears un- intuitive to invoke an evolutionary model to study evolutionary relation- ships, a situation resembling circular evidence. • intuitive construction In parsimony analysis there is an immediate connection between the data and the result of the analysis, as the branches and nodes of the branching diagram correspond directly to character-state changes in the data matrix. After obtaining which then are interpreted as representing a phy- logeny, estimates of reliability, often referred to as support, should be calcu- lated for the different branches in the tree. Different kinds of indices may be calculated for the most parsimonious trees retrieved, as in papers I, II, and III. In paper I jackknife and Bremer support (BS) values were calculated for the trees. In paper II we also chose to include bootstrap values. In paper III only the bootstrap values were included.

Molecules and morphology The first parsimony studies were based on morphological characters in a wide sense. Later the ability to perform DNA restriction site analyses and subsequently to obtain nucleotide sequences has resulted in vastly larger data sets. When I started in 1996 as a PhD student, the emphasis in the field of angiosperm systematics was on resolving phylogenetic relationships at higher taxonomic levels. Several of these questions were solved quite easily (some are not yet solved), and the focus was then shifted to order and delimitations as demonstrated in a number of theses from the department of Systematic Botany in Uppsala. The ability to obtain more sequences in shorter time and at lower cost has led to an enormous increase in the amount of information included in the analyses. The more data, the better the resolu- tion (usually), and nowadays it is easy to add an extra DNA region to get a better resolution and higher support values. By combining data sets, the sup- port values often increase (e.g. Bremer & al., 1999). The majority of data used in this thesis are obtained from nucleotide se- quences of different regions in the chloroplast genome. The regions selected for papers I, II, and III are rbcL, ndhF, rps16, trnT-F. They exhibit different evolutionary rates at a specific taxonomic level. The rbcL gene, of about

10 1400 base pairs, codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and is frequently used for molecular plant studies. The ndhF gene of 2300-2400 base pairs, codes for subunit 6 of NADH-dehydrogenase (Sugiura, 1992). The intron of rps16 is a group II intron of 800-900 base pairs. The trnT-F region consists of approximately 1200-1600 base pairs, and is composed of four main sections. Starting with the gene trnT, the trnT-trnL spacer is followed by the gene trnL including the trnL intron, and is completed by the trnL-trnF spacer. Oligonucleotide primers used for amplification and sequencing in papers I, II, and III are shown in Table 1. The results obtained from the molecular data are compared with morphol- ogy of the monophyletic groups since it is difficult to draw any taxonomic conclusions without morphological support. In my opinion a group, on spe- cies level as well as in higher taxonomic levels, has to be recognisable in some way to be of practical use, and therefore I apply a morphological spe- cies concept according to e.g. Stuessy (1990). In paper IV a small group of species within a is revised, based mainly on the study of herbarium specimens.

11 Aims All papers in this thesis deal with groups that have been associated with the order Gentianales. Some of the main questions addressed are: Which families should be recognised within the Gentianales? What are the relationships of the genera traditionally included in the family Logani- aceae and in the Buddlejeae, and how should these taxa be circum- scribed? How should the apparently paraphyletic tribe Paederieae within the Rubiaceae be circumscribed and reclassified? Which species should be rec- ognised within the Mediterranean members of Paederieae currently treated as the genus Putoria? These questions concern different levels in the taxonomic hierarchy, but common to all of them is the striving for monophyletic as well as recognis- able entities.

12 Table 1. Comprehensive list of oligonucleotide primers used for DNA amplification above the dotted line and sequencing below in papers I, II and III. Amplification primers: rbcL z1 and 895R; rps F and R2; trnL a1, a, d, i, c, and f were also used for sequencing. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Primer label Primer sequence Ref. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– rbcL z1 5'-ATGTCACCACAAACAGAAACTAAAGCAAGT-3' A rbcL 1020R 5'-ATCATCGCGCAATAAATCAACAAAACCTAAAGT-3' B rbcL 895R 5'-ACCATGATTCTTCTGCCTATCAATAACTGC-3' B rbcL 427BS 5'-GCTTATATTAAAACCTTCCAAGGCCCGCC-3' C rbcL 3' 5'-CTTTTAGTAAAAGATTGGGCCGAG-3' A rpsF 5'-GTGGTAGAAAGCAACGTGCGACTT-3' D rpsR2 5'-TCGGGATCGAACATCAATTGCAAC-3' D trnT-F a1 5'-ACAAATGCGATGCTCTAACC-3' C trnT-F a 5'-CATTACAAATGCGATGCTCT-3' E trnT-F d 5'-GGGGATAGAGGGACTTGAAC-3' E trnT-F i 5'-CCAACTCCATTTGTTAGAAC-3' C trnT-F c 5'-CGAAATCGGTAGACGCTACG-3' E trnT-F f 5'-ATTTGAACTGGTGACACGAG-3' E ndhF -47 5'-AGGTAAGATCCGGTGAATCGGAAAC-3' F ndhF 1201 5'-AGGTACACTTTCTCTTTGTGGTATTCC-3' F ndhF 1350R 5'-ATAGATCCGAAACATATAAAATGCGGTT-3' F ndhF 606R 5'-ACCAAGTTCAATGTTAGCCAGATTAGTG-3' F ...... rbcL 234 5'-CGTTACAAAGGACGATGCTACCACATCGA-3' B rbcL 895 5'-GCAGTTATTGATAGACAGAAAAATCATGGT-3' B rbcL 1020 5'-ACTTTAGGTTTTGTTGATTTATTGCGCGATGATT-3' B rbcL 1204R 5'-CCCTAAGGGTGTCCTAAAGTTTCTCCACC-3' B trnT-F b 5'-TCTACCGATTTCGCCATATC-3' E trnT-F e 5'-GGTTCAAGTCCCTCTATCCC-3' E ndhF 172 5'-CTGTCTATTCAGCAAATAAAT-3' *F ndhF 396 5'-TACTTCCATGTTGGGATTAGTTACTAG-3' *F ndhF 590 5'-TTGGATAACGGGGAGTTTCGAATTT-3' *F ndhF 803 5'-CAATGGTAGCAGCGGGAATTTTTC-3' *F ndhF 1024 5'-GCTTTATTTCATTTGATTACTCATGCT-3' *F ndhF 1427 5'-TTCTATTCAATATCTCTATGGGGT-3' *F ndhF 1600 5'-ATCCTTATGAATCGGATAATACTATG-3' *F ndhF 1811 5'-CAGTCAGTATAGCCTCTTTCGGAAT-3' *F ndhF 1955 5'-TATATGATTGGTCATATAATCG-3' *F ndhF 2134 5'-TGGGCGTATTTCTTCTTATCTGTTC-3' *F ndhF 1 5'-ATGGAACAGACATATCAATA(CT)G(CG)(AG)TG-3' *F ndhF 1' 5'-ATGCAACAGACATATCAATACGGGTGGAT-3' F ndhF 1658 5'-TTTGTTCGTTGGAT(CT)(CT)(AT)TAGGAATT-3' F ndhF 355R 5'-GATCATGAGCCATATAATTATCACTAT-3' F ndhF 953R 5'-CCTCTCTTAATGTCTTTTTGAGCAAGAGCT-3' F ndhF 1626R 5'-CATAGTATTGTC(AC)GATTC(AT)(CGT)AAGGAT-3' F ndhF 1947R 5'-CTATGTAAGC(AC)CGATTAT(AC)(CT)ACCAA-3' F –––––––––––––––––––––––––––––––––––––––––––––––––––––––– Primer references: A = Olmstead & al., 1993, B = G. Zurawski (DNAX Research Institute), C = Bremer & al., 2002, D = Oxelman & al., 1997, E = Taberlet & al., 1991, F = Oxelman & al.,1999. Primers marked with * were kindly shared by R.K Jansen and R.G. Olmstead.

13 Gentianales

The order Gentianales forms together with , , , and four families of uncertain ordinal classification the euasterids I in the classification of flowering plants (APG II, Angiosperm Phylogeny Group, 2003). The euasterids I is in turn the of euasterids II (Fig. 1). The Gentianales consists of approximately 17 200 species, of which the ma- jority are woody plants found in tropical and subtropical areas. Characteristic features of the order are opposite, entire , often with stipules and col- leters (multicellular, glandular hairs). are mostly regular and pen- tamerous, with superior or, in the Rubiaceae, inferior ovaries. Endosperm formation is nuclear, in contrast to cellular in Lamiales and Solanales, inter- nal phloem is present (except in Rubiaceae), and often complex indole alka- loids (Jensen, 1991; 1992).

Traditional and modern views Taxa with contorted aestivation were by Bartling (1830) placed in a group named Contortae, and the families recognised in that entity were Apocyna- ceae, Asclepiadaceae, Gentianaceae, and Spigeliaceae (Gentianales of Lind- ley, 1833). The circumscription of Gentianales in Genera Plantarum (Ben- tham & Hooker, 1862-1883) included the six families Apocynaceae, Ascle- piadaceae, Gentianaceae, Loganiaceae, , and Salvadoraceae. The family Rubiaceae was first included in Gentianales by Wagenitz (1959), an opinion followed by, e.g., Dahlgren (1983) and Thorne (1983), but not by Cronquist (1983). Molecular systematic studies in the early 1990's indicated that Gentianales formed a monophyletic group (Bremer & Struwe, 1992; Downie & Palmer, 1992; Olmstead & al., 1993) comprising the families Apocynaceae, Asclepiadaceae, Gentianaceae, Rubiaceae, and parts of Lo- ganiaceae, although some years later Asclepiadaceae was merged with Apo- cynaceae (Struwe & al., 1994; Sennblad & Bremer, 1996; Endress & al., 1996). In the classification by Struwe & Albert (in Struwe & al., 1994) the seven families Apocynaceae s.lat., Gelsemiaceae, Geniostomataceae, Gen- tianaceae, Loganiaceae, Rubiaceae, and Strychnaceae were recognised in Gentianales.

14 Takhtajan (1997) on the other hand, not prioritizing the recognition of mo- nophyletic taxa, included nine families, Antoniaceae, Gelsemiaceae, Genio- stomataceae, Gentianaceae, Loganiaceae, Plocospermataceae, Saccifoli- aceae, Spigeliaceae, and Strychnaceae, while Rubiaceae and Apocynaceae were excluded. Currently, the classification by Backlund & al. (2000, paper II) based on comprehensive phylogenetic analyses is the most widely ac- cepted, and the order Gentianales here contains the five families Apocyna- ceae (including Asclepiadaceae), Gelsemiaceae, Gentianaceae, Loganiaceae s.str., and Rubiaceae.

Gunnerales Aextoxicaceae Berberidopsidaceae Dilleniaceae Caryophyllaceae core Myrtales

Fabales eurosids I eurosids II Garryales Gentianales euasterids I Lamiales Solanales euasterids II

Figure 1. Cladogram showing the relationships of the core eudicots according to APG II (Angiosperm Phylogeny Group, 2003).

15 Relationships of the Buddlejaceae – paper I The family Buddlejaceae is generally congruent with the circumscription of the tribe Buddlejeae, and is currently regarded as a group of the Scrophulari- aceae. It comprises about 110 species in the genera , often including Chilianthus and Nicodemia, Emorya, and . The taxa in Buddlejeae are woody plants occupying the tropics and warm areas of the world, and several are grown as ornamentals. They were for a long time included in the family Loganiaceae, where Bentham (1856) placed them in the tribe Loganieae. In the classification of Leeuwenberg & Leenhouts (1980) the tribe Buddlejeae comprised , Buddleja (incl. Nicodemia), Emorya, Gomphostigma, , , and . Several systema- tists (e.g. Wagenitz, 1959; Cronquist, 1981; Dahlgren, 1983; Takhtajan, 1987; Thorne, 1992) have suggested that Buddlejeae is close to Scrophulari- aceae, and the genus Buddleja was already placed there by Jussieu (1789). This is supported by chemical data (e.g. Jensen & al., 1975; Jensen, 1992; Tomas-Barberan & al., 1988; Scogin 1992), palynology (Punt, 1980), anat- omy (Carlquist, 1992), and nucleotide sequence data (Bremer & al., 1994; Olmstead & Reeves, 1995). The aim of paper I was to clarify the relationships of the taxa of Buddle- jaceae (Buddlejeae sensu Leeuwenberg & Leenhouts, 1980), by using DNA sequence data from the chloroplast genes rbcL and ndhF. The results from paper I (Fig. 2) clearly show that Buddlejeae sensu Leeuwenberg & Leenhouts (1980) is an unnatural group with members scat- tered in Lamiales (in paper I called Scrophulariales). Other taxa previously placed in the Loganiaceae and included in the study of paper I are Desfon- tainia, , , and . Our results support the placement of in euasterids II (Bremer & al., 1994; in the Dip- sacales by Backlund & Bremer, 1997). Plocosperma is, with weak support, most closely related to Borago, outside the Lamiales (Scrophulariales) . Polypremum is included in and Retzia in , fami- lies belonging to Lamiales (). Buddlejaceae s.str. forms a well supported, monophyletic group including the genera Buddleja, Emorya, Gomphostigma, and Nicodemia (no represen- tatives of Chilianthus were included). No firm decision was reached in paper I regarding the relationships and generic delimitations within Buddlejaceae s.str. The sister group relationships within the family are poorly supported, and in the combined tree (Fig. 2) Buddleja is unresolved. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Figure 2. Strict consensus tree from combined data of rbcL and ndhF. • = earlier included in Buddlejeae (Loganiaceae) according to Leeuwenberg & Leenhouts (1980). * = earlier included in other tribes in Loganiaceae according to Leeuwen- berg & Leenhouts (1980). Thick branches indicate jackknife support • 95 % and Bremer support • 5. Results from paper I (Oxelman & al., 1999).

16 Barnadesia ASTERALES Menyanthes Desfontainia * Viburnum DIPSACALES Lonicera Borago Juanulloa Lycopersicon SOLANALES Nicotiana Luculia Alstonia GENTIANALES Plocosperma * Nyctanthes OLEACEAE Olea Polypremum * Peltanthera • Sanango • Antirrhinum Hippuris Globularia Digitalis SCROPH II Plantago Veronica Nuxia • Retzia * STILBACEAE Androya • Myoporum Hebenstretia Selago SCROPH I Scrophularia Verbascum Buddleja davidii • Buddleja asiatica • BUDDLEJACEAE Emorya • Nicodemia • Gomphostigma • Sesamum Thunbergia Barleria ACANTHACEAE Ruellia Hypoestes Justicia Lindenbergia Congea Tectona Vitex LAMIACEAE Ajuga Caryopteris Catalpa Martinella BIGNONIACEAE Tabebuia Schlegelia Stachytarpheta Rhaphithamnus Verbena

17 Data from DNA sequences by Wallick and co-workers (NCBI accession numbers AF380843-77) have been referred to as "Wallick & al., 2000 in American Journal of Botany vol 6, suppl.", but that study was never pub- lished in a refereed journal. In a more recent study by Oxelman & al. (2005), additional data from ndhF, rps16, and trnL-F nucleotide sequences were added and the results also showed that Buddleja is paraphyletic. Additional, unpublished information from a graduation thesis by K. Kaunulainen (2005, personal communication) confirms that conclusion. The remaining taxa previously placed in the Buddlejeae sensu Leeuwen- berg & Leenhouts (1980), are now with the aid of nucleotide sequence data and phylogenetic analyses firmly positioned within the Lamiales (Oxelman & al., 1999).

Interfamilial relationships of the Gentianales – paper II Even if the of Gentianales was generally accepted in the 1990's, the interfamilial relationships were not fully understood, and this was one of the aims of paper II. Representative genera from the families Apocynaceae (including Asclepiadaceae), Gelsemiaceae, Gentianaceae, Rubiaceae, and all the genera (except Norrisia) in Loganiaceae as recognised by Leeuwenberg & Leenhouts (1980) were selected. The results (Fig. 4) indicated strong sup- port for Rubiaceae as sister group to the rest of the families in the order. The placement of Gentianaceae as sister to Apocynaceae, Gelsemiaceae, and Loganiaceae s.str. is moderately supported. The relationships among the latter three families have somewhat weaker support. The small family Gel- semiaceae is resolved as sister to Apocynaceae, but with a moderate support. Also the relationship of Apocynaceae and Gelsemiaceae as sister group to Loganiaceae is not that highly supported. All of the included families are monophyletic, after the exclusion of various taxa previously placed in Lo- ganiaceae (see above and below).

Figure 3. (from Leeuwenberg & Leenhouts, 1980)

18 Barnadesia Menyanthes MENYANTHACEAE Desfontainia • Viburnum ADOXACEAE Lonicera CAPRIFOLIACEAE

Nicotiana Borago Plocosperma • PLOCOSPERMATACEAE Nyctanthes OLEACEAE Polypremum • TETRACHONDRACEAE Peltanthera • Sanango • GESNERIACEAE Streptocarpus Schlegelia SCROPHULARIACEAE Ajuga LAMIACEAE Verbena VERBENACEAE Sesamum PEDALIACEAE Barleria ACANTHACEAE Antirrhinum Veronica SCROPHULARIACEAE Nuxia • Stilbe STILBACEAE Retzia • Androya • MYOPORACEAE Verbascum SCROPHULARIACEAE Buddleja • Gomphostigma • Nicodemia • BUDDLEJACEAE Emorya • Oldenlandia Ophiorrhiza Rondeletia Chiococca Cinchona RUBIACEAE Pinckneya Mussaenda Gardenia Vangueria Exacum • GENTIANACEAE • • Alstonia Kopsia Wrightia APOCYNACEAE Periploca Stephanotis Gelsemium • • GELSEMIACEAE • • • • Neuburgia • • LOGANIACEAE • Mitreola • • • • •

Figure 4. One of the most parsimonious trees from combined rbcL and ndhF se- quence data. • = taxa included in Loganiaceae by Leeuwenberg & Leenhouts (1980). Thick branches indicate jackknife and bootstrap support > 90 %. Results from paper II (Backlund & al., 2000).

19 Families of the Gentianales The five families of Gentianales are further discussed below.

Loganiaceae Comprising 13 genera and about 400 species (paper II), Loganiaceae is now a comparatively small family. They are generally woody plants, widely dis- tributed in tropical areas of the world. Best known is probably the genus Strychnos, from which the poisonous alkaloid is derived. Several other taxa in the family also contain biologically active alkaloids, which sometimes have been used as drugs or poisons (e.g. curare). Interpetiolar stipules are a characteristic of the family, with colleters often present, in particular on the stipules. The gynoecium has two carpels, and the are often septicidal capsules, sometimes berries, often containing winged . Some anatomical features such as superficial cork development and simple hairs are also characteristics for the family. Loganiaceae was first described by de Martius (1827), but the circum- scription has varied considerably (Table 2). Part of the scope of paper II was to investigate the relationships and systematic positions of the genera in Lo- ganiaceae sensu Leeuwenberg & Leenhouts (1980). Although some of them had been studied previously (Bremer & Struwe, 1992; Struwe & al., 1994; Oxelman & al., 1999), no comprehensive overview had been accomplished. To determine, in more detail, the relationships within Loganiaceae s.str. as well as for the excluded taxa, we sampled genera from other closely related orders. The results are shown in Fig. 4. Loganiaceae s.str. consists of the genera Antonia, Bonyunia, Gardneria, Geniostoma, Labordia, Logania, Mitrasacme, Mitreola, Neuburgia, Norrisia, Spigelia, Strychnos, and Usteria. The previously recognised families Antoniaceae, Geniostomataceae, Spigeliaceae, and Strychnaceae are all reduced into Loganiaceae. The genera Antonia, Bonyunia, and Usteria form a monophyletic group with high sup- port from the sequence data in paper II. These genera also share such char- acters as valvate aestivation, coriaceous leaves, and some anatomical fea- tures (Leeuwenberg & Leenhouts, 1994; Mennega, 1980). Together they are also well supported as the sister group of the remainder of the family. Gard- neria, Neuburgia, Spigelia, and Strychnos form another monophyletic group but with poor support in our analysis. The clade comprising Geniostoma, Labordia, Logania, Mitrasacme, and Mitreola, is strongly supported by mo- lecular characters and by others such as ochreae instead of stipules (Leeu- wenberg & Leenhouts, 1980) and absence of alkaloids (Bisset, 1980). Gen- iostoma and Labordia are well supported as sister taxa.

20 Table 2. Summary of various recent classifications of the Loganiaceae s.lat. by dif- ferent authors, starting with Leeuwenberg and Leenhouts (1980). ––––––––––––––––––––––––––––––––––––––––––––––––––––– Leeuwenberg Cronquist Thorne Struwe Takhtajan Backlund, and and Oxelman, Leenhouts Albert and Bremer (1980) (1981) (1983) (1994) (1997) (1999) ––––––––––––––––––––––––––––––––––––––––––––––––––––– Androya Log-Budd Bud – Bud • Myo • Anthocleista Log-Pota Log Log-Loga Gen Gen-Pota Gen Antonia Log-Anto Log Log-Loga Str Ant Log Bonyunia Log-Anto Log Log-Loga Str Ant Log Buddleja Log-Budd Bud • Bud • Bud • Bud • Desfontainia Log-Desf Log Log-Desf • Des • Col • Emorya Log-Budd Bud Bud – Bud • Bud • Fagraea Log-Pota Log Log-Loga Gen Gen-Pota Gen Gardneria Log-Stry Log Log-Loga Str Str Log Gelsemium Log-Gels Log Log-Loga Gel Gel Gel Geniostoma Log-Loga Log Log-Loga Geo Geo Log Gomphostigma Log-Budd Bud • Bud – Bud • Bud • Labordia Log-Loga Log Log-Loga Geo Geo Log Logania Log-Loga Log Log-Loga Log Log Log Mitrasacme Log-Spig Log Log-Loga Log Spi Log Mitreola Log-Spig Log Log-Loga Log Spi Log Mostuea Log-Gels Log Log-Loga Gel Gel Gel Neuburgia Log-Stry Log Log-Loga Str Str Log Nicodemia Log-Budd Bud • Bud – Bud • Bud • Norrisia Log-Anto Log Log-Loga Str Ant Log Nuxia Log-Budd Bud Bud – Bud • Sti • Peltanthera Log-Budd Bud Bud – Bud • Ges • Plocosperma Log-Ploc Log Log-Ploc • Plo Plo • Polypremum Log-Spig Log Log-Loga • Bud • Tet • Potalia Log-Pota Log Log-Loga Gen Gen-Pota Gen Retzia Log-Retz Ret Log-Retz • Ret • Sti • Sanango Log-Budd Bud Bud – Bud • Ges • Spigelia Log-Spig Log Log-Loga Str Spi Log Strychnos Log-Stry Log Log-Loga Str Str Log Usteria Log-Anto Log Log-Loga Str Ant Log ––––––––––––––––––––––––––––––––––––––––––––––––––––– The following abbreviations are used for families: Ant=Antoniaceae, Bud=Buddlejaceae, Col=Columelliaceae, Des = Desfontainiaceae, Gel = Gelsemiaceae, Geo = Geniostomataceae, Gen = Gentianaceae, Ges = Gesneriaceae, Log = Loganiaceae, Myo = Myoporaceae, Plo = Plocospermataceae, Ret = Retziaceae, Spi = Spigeliaceae, Sti = Stilbaceae, Str = Strychnace- ae, Tet = Tetrachondraceae. For tribes: Anto = Antonieae, Budd = Buddlejeae, Desf = Des- fontainieae / Desfontainioideae, Gels = Gelsemieae, Loga = Loganieae / Loganioideae, Ploc = Plocospermeae / Plocospermatoideae, Pota = Potalieae, Retz = Retzieae / Retzioideae, Spig = Spigelieae, Stry = Strychneae. In addition: – = not included in study, and • = explicitly excluded from the Gentianales.

21 Of the taxa excluded from the Loganiaceae, Gelsemium and Mostuea are placed in the family Gelsemiaceae (Struwe & Albert, in Struwe & al., 1994), and the position of the genera Anthocleista, Fagraea, and Potalia within Gentianaceae (e.g. Struwe & Albert, in Struwe & al., 1994) is confirmed. The taxa excluded from Gentianales are discussed in paper I, and in the chapter on Buddlejaceae.

Apocynaceae This large family comprises about 5000 species of perennial herbs, , , or small trees distributed mainly in the tropics and subtropics. Com- mon names for some of the taxa in Apocynaceae are dogbane (Apocynum), milkweed (Asclepias), frangipani (Plumeria), and periwinkle (Vinca), these and several other taxa are grown as ornamentals (e.g. Hoya and Nerium), or for production of drugs. The most striking features of Apocynaceae is the presence of a milky sap (latex). The corolla aestivation is often contorted, the fruits can be either apocarpous or syncarpous, and the seeds often have long hairs or tufts. All studied taxa contain alkaloids. Jussieu (1789) widely circumscribed the family, including taxa that were later placed in Logani- aceae or Asclepiadaceae. Brown (1810) first described Asclepiadaceae on the basis of its specialised pollination system, but today it is widely accepted that the group is nested within Apocynaceae (e.g. Struwe & al., 1994; Sennblad & Bremer, 1996; paper II).

Gelsemiaceae The smallest family in Gentianales is Gelsemiaceae with two genera (Gel- semium and Mostuea) and eleven species. They are tropical and subtropical shrubs or vines. Gelsemium sempervirens, the Carolina jessamine, is culti- vated as an ornamental and is also used for medicinal purposes. The plants of Gelsemiaceae lack latex as well as stipules. The flowers are heterostylous, white or yellow and have twice-dichotomously divided stigmas, latrorse anthers, and imbricate corolla aestivation. Gelsemiaceae was described by Struwe & al. (1994), and is closely related to Apocynaceae. The taxa have earlier been placed in Loganiaceae (Leeuwenberg & Leenhouts, 1980), and Gelsemium has been placed in both Apocynaceae (Jussieu, 1789) and Gen- tianaceae (Bartling, 1830). Although the hypothesized sister group relationship between Gel- semiaceae and Apocynaceae is not very strongly supported by our analyses (paper II), it has further support from anther anatomy (Johri & al., 1992), cytotaxonomy (Moore, 1947), and phytochemistry (e.g. Jensen, 1992). The alternative hypothesis would be that Gelsemiaceae is sister to Lo- ganiaceae, which is supported by corolla aestivation, anatomy (Leeu- wenberg & Leenhouts, 1980), and late sympetaly (Erbar, 1991).

22 Gentianaceae The family Gentianaceae differs from the rest of the order by a generally herbaceous habit (a few taxa are woody), and the plants are found mainly in temperate regions. It comprises approximately 87 genera and 1600 species with a worldwide distribution. Gentianaceae are recognised by their lack of stipules, imbricate aestivation, and campanulate or salverform corolla. The consists of two carpels, often with a glandular disk at the base. The fruits are often dehiscent capsules, sometimes berries. Gentianaceae was first described by Jussieu (1789), and the circumscription of the family has not changed much since then. The most striking change was the inclusion of the woody tribe Potalieae (Struwe & al., 1994; Struwe & Albert, 1997), for- merly a member of Loganiaceae (Leeuwenberg & Leenhouts, 1980). Place- ment of Potalieae in Gentianaceae is also clearly shown in paper II. These results are based on molecular characters, but morphological features sup- porting this hypothesis are monadelphous filaments (not found in Fagraea), dextrally contorted , and septate parenchyma. The presence of several chemical substances such as e.g. gentianine also support a close relationship. The Potalieae is a strongly supported group in our study.

Rubiaceae By far the largest family in the order Gentianales is Rubiaceae, comprising ca. 630 genera and about 10 200 species. The majority of taxa are found in the tropics and subtropics and have a woody habit. The best known members of the family are Coffea (coffee), and Cinchona, from which the anti- plasmodial alkaloid quinine is extracted. Many of the flowers are colourful, and there are several genera such as Ixora and Gardenia that are grown as ornamentals. There are several characters that separate Rubiaceae from the other families in the order, such as inferior ovary, obturator, Casparian thickenings, and lack of internal phloem. Also this family was described by Jussieu in 1789. In paper II, the position of Rubiaceae as sister to the rest of Gentianales is very strongly supported. The systematics of the family will be further dis- cussed below.

23 Rubiaceae systematics

There has never been much doubt about the monophyly of the Rubiaceae, but the systematic placement of the family has been discussed, both within the order Gentianales (Verdcourt, 1958; Bremekamp, 1966; Robbrecht, 1988; Bremer, 1996) and segregated as the order Rubiales (Bentham & Hooker, 1873; Cronquist, 1981, 1988). Although the Rubiaceae is a very clear-cut group, the intrafamilial classi- fication has been controversial and there have been disagreements about which characters are most suitable for delimiting tribes and subfamilies. Schumann (1891) recognised two subfamilies, characterised by one or many per . The eight subfamilies delimited by Bremekamp (1966) were supported by seed structures, raphides, and secondary pollen presenta- tion, and Verdcourt (1958) accepted three of those. Robbrecht (1988) di- vided Rubiaceae into four subfamilies. Currently three monophyletic sub- families are recognised; Cinchonoideae, , and Rubioideae as out- lined by Bremer and co-workers (Bremer & al., 1995; Bremer, 1996). The tribal delimitations within Rubiaceae have varied considerably be- tween different studies, and some paraphyletic groups and taxa with uncer- tain positions remain at tribal level.

Figure 5. Plocama calabrica (as Putoria calabrica in Schumann, 1891).

24 The tribes Paederieae & Putorieae – paper III

One of the tribes in Rubiaceae that has been considered to be paraphyletic for some time is Paederieae (Rubioideae), but there has been no comprehen- sive phylogenetic study of that tribe until now. In paper III we have ana- lysed three sets of nucleotide sequences from all genera previously placed in Paederieae, with the exception of Pseudopyxis, from which we failed to am- plify the DNA regions used (Table 3).

Historical outline Paederieae was first described by de Candolle (1830) who recognised three genera; Lygodysodea, Lecontea, and Paederia. Schumann (1891) added the genera Aitchisonia, Leptodermis, Pseudopyxis, and Spermadictyon, and his classification of the tribe lasted until Puff (1982) and Robbrecht (1988) ex- panded the tribe with 11 genera. Putorieae was also first recognised by de Candolle (1830), but as a sub- tribe of in which he placed Plocama, Putoria, Serissa, and some taxa not anymore associated with either Paederieae or Putorieae. Some years later Sweet (1839) raised Putorieae to tribal rank to comprise the gen- era Plocama, Putoria, Serissa, and Ernodea. Schumann (1891) moved these taxa to , and Putorieae was taken out of use. In the classifica- tions of Puff (1982) and Robbrecht (1988, 1993), the taxa of Putorieae (ex- cept Ernodea) were included in Paederieae.

25 Identifying the problems Although results from some previous molecular studies of Rubiaceae have suggested that the tribe Paederieae (sensu Puff and Robbrecht) is para- phyletic (Andersson & Rova, 1999; Bremer & Manen, 2000), these studies were focused on a higher taxonomic level, and the sampling of taxa from Paederieae was too limited to make any firm conclusions. Another problem has been the "Gaillonia complex", in which the species have been treated as belonging to a single genus Gaillonia s.lat., or alternatively placed in several different genera Choulettia, Crocyllis, Gaillonia, Jaubertia, Pseudogail- lonia, and Pterogaillonia (Linczevski, 1973; Qarar, 1973; Léonard, 1984). A possible close connection between this complex and the genera Putoria and Plocama has also been suggested (Thulin, 1998). A study to investigate these questions was thus initiated (paper III), including all genera previously included in Paederieae.

Table 3. Summery of previous classifications of genera sometimes placed in Paed- erieae s.lat. Ant=Anthospermeae, Cof=Coffeeae, Gue=Guettardeae, Mit or Mor- MI=Mitchella-group, Mor=Morindeae, Pae=Paederieae, Psy=Psychotrieae, Put=Putorieae, SSpe= subtribe Spermacocinae of Spermacoceae, SPut= subtribe Putorineae of Spermacoceae, Spe=Spermacoceae. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Genera DC. Hook. Schu. Puff Robb. Robb. Bremer Paper 18301 18732 18913 19824 19885 19936 20007 II –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Aitchisonia – – Pae Pae Pae Pae – Put Choulettia – – – Pae Pae Pae Pae Put Crocyllis – Ant Ant Pae Pae Pae Pae Put Gaillonia SSpe Spe Spe Pae Pae Pae Pae Put Jaubertia – – – Pae Pae Pae Pae Put – Ant Ant Pae Pae Pae Pae – Leptodermis Gue Pae Pae Pae Pae Pae Pae Pae Mitchella Gue Ant Ant Pae inc. sed. Mit Mor-MI Mor Paederia Pae Pae Pae Pae Pae Pae Pae Pae Plocama SPut Ant Ant Pae Pae Pae Pae Put Pseudogaillonia – – – Pae Pae Pae Pae Put Pseudopyxis – Pae Pae Pae Pae Pae Pae Pae Pterogaillonia – – – Pae Pae Pae Pae Put Putoria SPut Ant Ant Pae Pae Pae Pae Put Cof Psy Psy – Psy Pae – – Serissa SPut Ant Ant Pae Pae Pae Pae Pae Spermadictyon Gue Pae Pae Pae Pae Pae Pae Pae –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 1 (de Candolle, 1830), 2 (Hooker, 1873), 3 (Schumann, 1891), 4 (Puff, 1982), 5 (Robbrecht, 1988), 6 (Robbrecht, 1993), 7 (Bremer & Manen, 2000).

26 Phylogenetics of Paederieae & Putorieae Phylogenetic analyses based on nucleotide sequence data from the chloro- plast DNA regions rbcL, rps16, and ndhF were performed. The results in paper III show, with strong support, that Paederieae s.lat. is paraphyletic and separable into two large , Paederieae s.str. and Putorieae (Fig. 7). The tribe Paederieae s.str. comprises the genera Leptodermis, Paederia, Serissa, and Spermadictyon. The Japanese genus Pseudopyxis, from which we were not able to obtain PCR products, most probably belongs in Paed- erieae as well. With the exception of Serissa, these genera have dry fruits that have two to five, one-seeded pyrenes (Puff, 1982). When ripening the exocarp splits into valves. The fleshy fruits of Serissa have probably evolved secondarily from the dry ones. The genus Paederia is monophyletic and is further divided into two monophyletic sister groups, one comprising the Asian, and the other the African species. The endemic species of Paederia from also form a monophyletic group. Paederieae s.str. is sister to a moderately supported clade comprising Putorieae, , Theligoneae, Kelloggia, and Saprosma. Putorieae is resurrected as a tribe, and all the taxa included in this circum- scription have earlier been placed in Paederieae s.lat. The members of the Putorieae are diagnosed by fruits splitting into two indehiscent, one-seeded mericarps, with the exception of the fleshy fruits of . In our study Putorieae has strong support, and so has its sister group relationship to the clade that includes Rubieae, Kelloggia, and Theligoneae. The tribe Ru- bieae is well supported with Kelloggia, a genus comprising two species, placed as its sister (as previously shown by Nie & al., 2005). If Kelloggia is to be included in Rubieae or placed in a monogeneric tribe is left undecided in our study, because a more thorough investigation has to be performed. The placement of the genus Saprosma is also a matter of debate. Tradition- ally it has been included in Psychotrieae (e.g. Schumann, 1891; Robbrecht, 1988), but later Robbrecht (1993) placed it in Paederieae. The position of Saprosma in our study as sister to Putorieae, Rubieae, Kelloggia, and Theli- goneae is only moderately supported. Further studies of Saprosma, including more data as well as more species, are needed before making a decision on its placement. The taxa within Putorieae form several well-supported clades based on molecular data, but we have not been able to find morphological characters that support these clades. One example is the clade to which the former ge- nus Putoria belongs, and this will be discussed in more detail below. The solution proposed is to accept a single genus, using the oldest name Plo- cama. The previously recognised genera Aitchisonia, Choulettia, Crocyllis, Gaillonia, Jaubertia, Pseudogaillonia, Pterogaillonia, and Putoria are here reduced to Plocama.

27 We thus accept 34 species of Plocama (see realignments and synopsis be- low), which previously was a monotypic genus with the endemic species P. pendula from the . Plocama pendula is strongly supported as sister to a clade with the southern African P. crocyllis (=Crocyllis anthos- permoides) and, on a strongly supported internal node, P. yemenensis ( and Oman) and P. tinctoria (Socotra and ), thus indicating a biogeographical link between and these distant regions in Af- rica and southern Arabia. P. pendula is the only species in Putorieae with fleshy fruits that do not split into mericarps. The fruits serve as food for the lizards Gallotia galloti and G. stehlini, also endemics of the Canary Islands (Barquin Diez & Wildpret, 1975; Molina Borja, 1986; Mendoza-Heuer, 1987). It seems likely that the fleshy of P. pendula is an adaptation to saurochory.

Figure 6. Plocama tinctoria drawn by Margaret Tebbs.

28 Luculia Ophiorrhiza 51 Gaertnera 100 100 Morinda 70 Mitchella • 85 Danais 89 Anthospermum 64 Mycetia 100

100 Spermadictyon suaveolens • 100 Leptodermis potaninii • Serissa foetida • 100 P 83 • A Paederia pilifera • E 100 Paederia pospischilii • D E 95 Paederia sambiranensis • R 87 Paederia majungensis • I Paederia lanata • E 92 86 Paederia taolagnaroensis • A E Paederia farinosa • 71 87 Paederia bojeriana • Paederia mandrarensis • 100 Saprosma foetens • Saprosma fruticosum • Theligonum cynocrambe THE 92 • 75 100 100 R Rubia U 100 B 100 I E 100 100 Asperula A E 100 Putoria calabrica • Putoria brevifolia • 100 Pterogaillonia calycoptera • 99 P 100 Pseudogaillonia hymenostephana • U Gaillonia olivieri • 98 T 97 Aitchisonia rosea • O 100 Gaillonia eriantha • R I 100 Jaubertia aucheri • E Choulettia reboudiana • 67 A Plocama pendula • E 99 84 Crocyllis anthospermoides • 95 Gaillonia yemenensis • Gaillonia tinctoria •

Figure 7. The single most parsimonious tree from combined data of rbcL, rps16, and trnT-F. Bootstrap values are shown above the branches. THE=Theligoneae. Results from paper III (Backlund & al., manuscript). Dots indicate taxa that have been in- cluded in Paederieae. Taxa in the tribe Putorieae are labelled according to pre- revision classification. Taxa of the tribe Rubieae are composed of sequence data from several species, thus only generic names are given.

29 The Mediterranean Plocama – paper IV

Paper IV has a close connection with paper III, and is a taxonomic revision of a part of Plocama, based on herbarium specimens and literature.

Historical outline According to the molecular results presented in paper III, the Mediterranean species of Plocama form a well supported clade. These taxa were earlier recognised as Putoria, separated mainly by their fleshy fruits that have sometimes been called drupes or berries. In fact, the fleshy fruit wall dries up when mature, and the fruit splits into two one-seeded mericarps, just as it does in the species previously placed in the genus Gaillonia. Other examples of shared features between Plocama calabrica, P. brevifolia, and species previously placed in Gaillonia are reddish, exserted and inrolled margins.

Identifying the problems The of the Mediterranean taxa of Plocama has been quite com- plex. The species circumscriptions are in need of clarification, and several species and varieties have been described, many of which have not been typified. The need of a revision was the starting point for paper IV.

Nomenclatural considerations Literature dealing with the different taxa were studied and herbarium speci- mens examined. This resulted in the acceptance of two species of Mediterra- nean Plocama, the widespread P. calabrica and P. brevifolia in Morocco and Algeria. Due to the wide morphological variation within these species, there have been several earlier suggestions to recognise a number of species and infraspecific taxa (e.g. Boissier & Reuter, 1856; Lange, 1868; Pomel, 1874). We found that there is a continuum of variation within each of the species, but that the two proposed species can be easily separated from each other.

30 Key to the species of Plocama in the Mediterranean area 1. Flowers 4-15 (rarely more) in terminal cymes; calyx lobes usually 4, subequal 1. P. calabrica 1. Flowers solitary, terminal; calyx lobes usually 2, sometimes with small teeth in the sinuses 2. P. brevifolia

Figure 8. Plocama calabrica, modified after Cirillo's foetidissima (1784).

31 Conclusions and future prospects

During the last decades phylogenetic analysis has become one of the most powerful tools in the life sciences (e.g. Hestmark, 2000). Many of the early studies addressed higher level systematics, e.g. the separation of Archeae (Sogin & al., 1991) or the relationship between plants, animals, and fungi (Wainright & al., 1993). As in completing a jigsaw puzzle, once the frame and corners are constructed, a huge amount of work remains to figure out how all the other pieces fit. This shift in trends has become evident for me during my nine years as a PhD student. When I started with this PhD project in 1996 I was dealing with systemat- ics at ordinal and family level, with focus on molecular data in papers I and II. My last study, which I reached via a tribal delimitation in paper III, is a taxonomic revision of a group of taxa within a genus, based on morphology, presented in paper IV. Although it might seem like a very broad project, with taxonomically distant substudies, there are many similarities in the ways of approaching the problems and how to solve them. The uniting trait is that the four different studies have included questions on taxa with uncer- tain taxonomical positions and obscure delimitations. To solve these prob- lems different sets of tools, in each specific case represented by different types of data, have been used. The ultimate goal has been to formulate phy- logenetic hypothesis by recognising monophyletic groups, with the addi- tional objective that these also should be recognisable in the field. The large jigsaw of plant systematics is far from laid. Some issues rele- vant to the narrow field of this thesis that merit further study include: • the position of the genus Saprosma • the tribal classification of the genus Kelloggia • further generic delimitations in the Loganiaceae

The systematic conclusions of my studies are summarised in the following realignment and synopsis.

32 Realignment and synopsis of the Gentianales

Suggested realignments according to results in paper I and II:

Families of the Gentianales: Apocynaceae Gelsemiaceae Gentianaceae Loganiaceae Rubiaceae

Taxa excluded from Gentianales: Buddleja, Emorya, Gomphostigma, and Nicodemia to Buddlejaceae, Lamiales (Oxelman & al., 1999) Nuxia and Retzia to Stilbaceae, Lamiales (Bremer & al., 1994; Oxelman & al., 1999) Peltanthera and Sanango to Gesneriaceae, Lamiales (Oxelman & al., 1999) Androya to Myoporaceae, Lamiales (Oxelman & al., 1999) Plocosperma to Plocospermataceae, euasterids I (Oxelman & al., 1999) Polypremum to Tetrachondraceae, Lamiales (Oxelman & al., 1999) Desfontainia to Columelliaceae, Dipsacales (Backlund & Bremer, 1997)

Synopsis of Paederieae and Putorieae (Rubiaceae), according to results in paper III and IV:

Genera in the Paederieae: Five genera, Leptodermis, Paederia, Pseudopyxis, Serissa, and Spermadictyon

Genus in the Putorieae: A single genus, Plocama, including as synonyms the previously recognised genera Aitchi- sonia, Choulettia, Crocyllis, Gaillonia, Jaubertia, Pseudogaillonia, Pterogaillonia, and Puto- ria – 34 species,

P. afghanica, P. asperuliformis, P. aucheri, P. botschantzevii, P. brevifolia, P. bruguieri, P. bucharica, P. calabrica, P. calcicola, P. calycoptera, P. crocyllis, P. crucianelloides, P. dubia, P. eriantha, P. hymenostephana, P. iljinii, P. inopinata, P. jolana, P. kandaharensis, P. macrantha, P. mestscherjakovii, P. olivieri, P. pendula, P. puberula, P. putorioides, P. reboudiana, P. rosea, P. somaliensis, P. szovitsii, P. thymoides, P. tinctoria, P. trichophylla, P. vassilczenkoi, P. yemenensis

Excluded species: Putoria pulchella { Asperula pulchella (Ehrendorfer & Schönbeck-Temesy, 2005) Putoria indica { Neanotis indica (Lewis, 1966)

33 Sammanfattning (Swedish summary)

Inom växtsystematik studerar man växter, bl.a. hur de ser ut och hur de är släkt med varandra. Den grundläggande biologiska enheten är arten, t.ex. blåsippa, vitsippa och gulsippa. Arter inordnas i släkten, släkten i familjer, familjer i ordningar osv. i den s.k. linneanska hierarkin. Om en familj är stor kan den delas upp i underfamiljer och tribusar. Metoderna man använder i detta arbete varierar, men de flesta växtsystematiker vill idag ordna växterna i grupper som avspeglar deras ursprung, dvs. hur de är släkt med varandra. Man talar i detta sammanhang å ena sidan om monofyletiska grupper som består en gemensam förfader och alla dess ättlingar. Parafyletiska grupper består å andra sidan endast av vissa av ättlingarna och polyfyletiska grupper har olika förfäder. Två monofyletiska grupper som är närmast släkt med varandra kallas för systergrupper. Denna avhandling handlar om fylogenetiska (släktskaps-) studier inom ordningen Gentianales som innehåller ca. 17 200 arter fördelade på de fem familjerna Apocynaceae, Gelsemiaceae, Gentianaceae, Loganiaceae och Rubiaceae. Av dessa är Rubiaceae, kaffefamiljen, den största med ca. 10 200 arter (Mabberley, 1997). De flesta arterna i Gentianales växer i tropiska och subtropiska områden, men några grupper finns i kallare klimat också. Ett exempel är släktet Galium, måror som det finns flera arter av här i Sverige. För att kunna göra dessa fylogenetiska studier behövs information av nå- got slag om växterna. I denna avhandling har jag till största delen använt mig av data i form av DNA-sekvenser från kloroplastgenomet. Sekvenserna sam- las ihop i matriser, ungefär som ett pussel och analyseras genom parsimoni- analys med hjälp av datorprogram. Resultaten från dessa analyser har jag valt att visa i form av träddiagram som representerar den enklaste lösningen för de data som analyserats. Jag gör sedan tolkningen att träddiagrammet avspeglar den undersökta gruppens släktskapsförhållanden. I avhandlingens artikel II bekräftar vi att Gentianales är en monofyletisk ordning som består av familjerna Apocynaceae, Gelsemiaceae, Gentianace- ae, Loganiaceae och Rubiaceae. Vidare visar vi hur de olika familjerna i ordningen är släkt med varandra (Fig. 2), och även i viss mån deras inbördes släktskapsstrukturer. Av särskilt intresse i denna studie är familjen Logania- ceae, som i tidigare system bestått av 29 släkten. Flera av dessa har i olika tidigare studier uteslutits från Loganiaceae, och vi har dels velat bekräfta detta, dels försökt utröna var dessa släkten hör hemma. Tolv släkten hamnar utanför Gentianales (Fig. 2 och 3, se artikel I), tre hamnar inne i Gentianace-

34 ae och två bildar familjen Gelsemiaceae. Kvar i familjen Loganiaceae blir 13 släkten: Antonia, Bonyunia, Norrisia, Usteria, Gardneria, Neuburgia, Spige- lia, Strychnos, Geniostoma, Labordia, Logania, Mitrasacme och Mitreola. En grupp som uteslutits från Loganiaceae är tribusen Buddlejeae. I artikel I studeras placeringen av de taxa som tidigare ingått i Loganiaceae och som uteslutits inte bara från familjen, utan även från Gentianales. I denna studie är urvalet av släkten från f.a. ordningen Lamiales (i artikel I kallad Scrophu- lariales) större än i artikel II för att ge en bättre bild av var dessa släkten hör hemma. De släkten som i Loganiaceae bildat tribusen Buddlejeae är Buddle- ja, Emorya, Gomphostigma och Nicodemia. De bildar en monofyletisk grupp som i vår studie hamnar i Lamiales, men som idag bildar tribusen Buddleje- ae i familjen Scrophulariaceae. En grupp som i en del molekylära arbeten utpekats som troligen varande parafyletisk är tribusen Paederieae i Rubiaceae. Analyser av alla de taxa som någon gång placerats i Paederieae visar i artikel III att de flesta av släktena i själva verket fördelar sig i två monofyletiska grupper. Den ena utgör den nyomskrivna Paederieae som består av släktena Leptodermis, Paederia, Se- rissa och Spermadictyon. Troligtvis hör även Pseudopyxis hit, men vi lycka- des inte få fram sekvenser av den. Den andra gruppen kallar vi Putorieae, eftersom det är ett namn som en gång i tiden använts för att sammanföra en del av de taxa som ingår nu. Det enda släktet i Putorieae blir Plocama som tidigare omfattade endast en art på Kanarieöarna, men som nu består av 34 arter. Släkten som nu inkluderats i Plocama är Aitchisonia, Choulettia, Cro- cyllis, Gaillonia, Jaubertia, Pseudogaillonia, Pterogaillonia och Putoria. I artikel IV görs en revision, dvs. en genomgång av namn, herbariemate- rial och litteraturuppgifter av en grupp taxa från Plocama. Dessa arter, som har en Medelhavsutbredning, var tidigare kända som släktet Putoria och utgör en grupp som får ett starkt stöd av molekylära data. Det har beskrivits över 20 synonyma namn, flera av dessa under artnivå, för något som visar sig vara två separata arter, Plocama calabrica och P. brevifolia, båda mycket variabla i sitt utseende. Mina systematiska studier har berört några olika taxonomiska nivåer inom ordningen Gentianales och jag har lyckats lösa en del problem. Mycket finns dock kvar att göra, både inom den här ordningen, och på många av andra ställen i växternas släktskapsträd.

Att leva är inte nog: solsken, frihet och en liten blomma måste man ha.

H. C. Andersen

35 Acknowledgements

Table 4. A non-comprehensive list of taxa greatly acknowledged for their input, contributions, comradeship, or other influences to the author during the work with this thesis. Accession numbers can be obtained from the author on request. ______Aagaardh, sunniva c Eklund, helena e Antonelli, alexander g Ekman, karl m Ahlström, per i Eriksson, torsten g Ahmadzadeh, afsaneh i, k Erixson, per c Albert, victor g Fahlander, mikael h, k Alsmark, björn l, m ({ Andersson) Farris, steve g g Alsmark, cessie g, l, m ({ cecilia) Fay, mike Alsmark, hans m Felsenstein, joe g c, i Alsmark, ulla m Forshage, mattias Anderberg, arne g Frajman, bozo f, g g Anderson, cajsa c ({ cajsalisa) Friis, elsemarie e, j Andersson eva e, j Fritz, anna-lena c, i Andersson, dick e Garboui, samira i, m Andreasen, katarina b, e, m Gunnarsson, urban e Apelgren, karin e, j, m Gustafsson, mats b Articus, kristina e, m Hedberg, inga b Backlund, anders d, e, g, j, l, m, n Hedberg, olof h, k Backlund, elsa l, n Hedenquist, ulla h, k Backlund, laila l Heidari, nahid e Backlund, nina l ({ kristina) Heidmarsson, starri e, h, m Backlund, per l Hjertson, mats ({ Eriksson) m Backlund, sven l, n Hjertson, malin b Barkhordarian, edit h Holm, kerstin b, j Beier, björn-axel d, e Holm, lennart m Bergström, elisabeth h, k Hoppe, magnus m Björk, lars f Hoppe, tobi ({ Sturesdotter, torborg) Block, mats h, k Hultgårdh, ulla-maj g, j l de Boer, hugo f, i Ivarsson, ulla Brandtberg-falkman, agnetha h, k Jaenson, thomas i, k Bremer, birgitta a, b, j, g Jansson, anne m ({ Gustafsson) Bremer, kåre a, b, j Jondelius, ulf i Carlsson, nicholas m Jonsell, lena g, j Carnelius, anna m Karis, peo g ({ p.-o.) Carpenter, jim g Karlsson, torbjörn m Chase, mark g Klackenberg, jens g Clapham, lena i, k Klum, mattias l, m Constantinescu, ovidiu g Klum, monika l, m({ Germundsson) Dagås, anna-maria m Kool, anneleen c Dahl, christine i, k Kornhall, per e Dahlman, eva l Kårehed, jesper e Delvéus, elin m ({ Bengtsson) Källersjö, marie g Dryselius, elisabeth m ({ Lindberg) Königsson, kristian l, m Dryselius, staffan m Königsson, tetta l, m ({ Nilsson) Eggens, frida c Lantz, henrik d, e Ejdeholm, ellem m ({ lars-magnus) Larsson, arne l Ekenäs, catarina c Larsson, barbro l

36 Larsson, josefin g, l Ronne, marta l, m Larsson, karolina c, i Ronquist, fredrik i Larsson, sonny g, m Rova, johan f, g Laurent, nadina e Ryding, olof g Lepage, olivier m Ryman, svengunnar h, j Lidén, magnus g Rättzén, henrik m Lidfeldt-rahm, barbro h Röcklinsberg, cristoph m ({ Röckle) Lindholm, petra g, m ({ Rosenblad) Röcklinsberg, helena m ({ Nilsson) Lundberg, johannes e Samuelsson-Ekman, annemarie m Långström, elisabeth e ,f ,k , m ({ Lönn) Sanmartin, isabell i Lönn, kristina e Savic, sanja c, d Maad, johanne e, m Schriber, ulla m ({ Nilsson) Magnusson, jessica h, m ({ Rönnholm) Schriber, svante m Magnusson, jonas m Sennblad, bengt e, g, m Manen, jean-françois f, g Sennblad, kristina m Manktelow, mariette f, m ({ Steiner) Sernhed, anette m ({ Axelsson) Marcussen, thomas f Sernhed, björn m Martinsson, karin e, g, j, m Sidall, mark g Mayer, veronika g, m Smedmark, jenny g McDowell, tim f, g Sorelius, joakim m Medhani, gebrehiwet e Sorelius, martha m el Mhalli, fawzeia c, i Stone, douglas f Moberg, roland g, h, j Strand, maria l Monroy, carlota i Strand, mats l ({ Larsson) Nilsson, harald m Stridh, arne g Nilsson, l. anders g, j Struwe, lena e, g Nordin, anders e Sundquist, agneta h Nylander, johan g, i Swenson, ulf e, g, j, m l Nyström, britta Söderström, bosse m ({ bo) Oh, il-chan c, d Tapper, anna-lena h l Olausson, monika Tekle, yonas c, i Oxelman, bengt b Thulin, mats a, b, j g Persson, eva Tibell, leif b Persson, nisse h ({ nilserik) Ulväng, göran m Persson, thomas i, k, m Wallberg, andreas c, i Philipsson, conny j Wedén, christina e Popp, magnus e Wedin mats e Puff, christian g Wheeler, ward g Pålsson, katinka i Wikström, niklas b Raikova, olga i Vinnersten, annika e, m Rautenberg, anja c Vinterbäck, lena m Razafimandimbison, sylvain f, g Vrede, katarina m c, m Reese-naesborg, rikke Vrede, tobias m ({ Lundgren) e Reinhammar, lars-gunnar Vårdahl, hege i Ringius, åsa m ({ Sjöberg) Ås, stefan h, k Ronne, erik l, m ______a supervisors, b senior researcher at Dept. of Syst. Bot., c present graduate student colleagues, d roommates, e previous graduate student colleagues, f associated researchers, g external researchers, h technical and administrative personel, i zoologists and other fellow biologists, j inspiring teachers, k lunchmates, l relatives by blood or law, m friends and pseudo- relatives, n love of my life. Also acknowledged are following non-personfied entities: "Da- gis"-families, Whiskymates of SMS, Curators and personel at herbaria consulted, Librairians at Uppsala University library, Cousins, aunts and onkels, Tetrapods (Fåksi, Adoxa & Coffea), Tetrawheels (Gösta, Svea, Adrian & Victoria).

37 References

Andersson, L. & Rova, J. H. E. 1999. The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Pl. Syst. Evol. 214: 161-186. Angiosperm Phylogeny Group 2003. An update of the angiosperm phylogeny group classi- fication for the orders and families of flowering plants: APG II. Bot. J. Linn. Soc. 141: 399-436. Backlund, A. & Bremer, B. 1997. Phylogeny of the Asteridae s.str. based on rbcL sequences, with particular reference to the Dipsacales. Pl. Syst. Evol. 207: 225-254. Backlund, M., Oxelman, B. & Bremer, B. 2000. Phylogenetic relationships within the Gentianales based on ndhF and rbcL sequences, with particular reference to the Loganiaceae. Amer. J. Bot. 87: 1029-1043. Barquin Diez, E. & Wildpret, W. de la Torre. 1975. Diseminación de plantas canarias. Datos iniciales. 5: 38-60. Bartling, F. G. 1830. Ordines naturales plantarum eorumque characteres et affinititates. Göttingen, Germany: Dieterichianus. Bentham, G. 1856. Notes on Loganiaceae. J. Indian Bot. Soc. 1: 52-114. Bentham, G. & Hooker, J. D. 1862-1883. Genera Plantarum, vols. 1-3. London, UK: Lovell Reeve & co. Bentham, G. & Hooker, J. D. 1873. Genera Plantarum, vol. 2. London, UK: Lovell Reeve & co. Bisset, N. G. 1980. Phytochemistry. In: A. J. M. Leeuwenberg (ed.), Engler & Prantl's Die Natürlichen Pflanzenfamilien, Fam. Loganiaceae, vol. 28b: 211-237. Berlin, Germany: Duncker & Humblot. Boissier, P. E. & Reuter, G. F. 1856. Diagnoses Plantarum Orientalium Novarum, ser.2, part 2. Lipsiae. Bremekamp, C. E. B. 1966. Remarks on the position, the delimitation and the subdivision of the Rubiaceae. Acta Bot. Neerl. 15: 1-33. Bremer, B. 1996. Phylogenetic studies within the Rubiaceae and relationships to other families based on molecular data. Opera Bot. Belg. 7: 33-50. Bremer, B., Andreasen, K. & Olsson, D. 1995. Subfamilial and tribal relationships in the Rubiaceae based on rbcL sequence data. Ann. Missouri Bot. Gard. 82: 383-397. Bremer, B., Bremer, K., Heidari, N., Erixon, P., Olmstead, R. G., Anderberg, A. A., Källersjö, M. & Barkhordarian, E. 2002. Phylogenetics of based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels. Molec. Phylo. Evol. 24: 274-301.

38 Bremer, B., Jansen, R. K., Backlund, M., Lantz, H. & Kim, K.-J. 1999. More characters or more taxa for a robust phylogeny-case study from the coffee family (Rubiaceae). Syst. Biol. 48(3): 413-435. Bremer, B. & Manen, J.-F. 2000. Phylogeny and classification of the subfamily Rubioideae (Rubiaceae). Pl. Syst. Evol. 225: 43-72. Bremer, B., Olmstead, R. G., Struwe, L. & Sweere, J. A. 1994. rbcL sequences support exclusion of Retzia, Desfontainia and Nicodemia (Buddlejaceae) from the Gentianales. Pl. Syst. Evol. 190: 213-230. Bremer, B. & Struwe L. 1992. Phylogeny of the Rubiaceae and the Loganiaceae: congruence or conflict between morphological and molecular data? Amer. J. Bot. 79: 1171-1184. Brown, R. 1810. Prodromus florae Novae Hollandieae. London, UK: Taylor. Candolle, A. P. de 1830. Prodromus Systematis Naturalis Regni Vegetabilis, vol. 4. Paris: Truettel and Würtz. Carlquist, S. 1992. Wood anatomy of sympetalous dicotelydon families: a summary, with comments on systematic relationships and evolution of the woody habit. Ann. Missouri Bot. Gard. 79: 303-332. Cirillo, D. 1784. De Essentialibus Nonnullarum Plantarum Characteribus Commentarius. Naples, Italy: Botan. Florent. Soc. Cronquist, A. 1981. An integrated system of classification of flowering plants. London, UK: Columbia University Press. Cronquist A. 1983. Some realignments in the . Nord. J. Bot. 3: 75-83. Cronquist, A. 1988. The evolution and classification of flowering plants. Bronx, New York, USA: New York Botanical Garden. Dahlgren, R. 1983. General aspects of angiosperm evolution and macro-systematics. Nord. J. Bot. 3: 119-149. Downie, S. R. & J. D. Palmer. 1992. Restriction site mapping of the chloroplast DNA inverted repeat: a molecular phylogeny of the Asteridae. Ann. Missouri Bot. Gard. 79: 266-283. Edwards, A. W. F. & Cavalli-Sforza, L. L. 1964. Reconstruction of evolutionary trees. In: V. H. Heywood & J. McNeill (eds.), Phenetic and Phylogenetic Classification. London, UK: Systematics Association Publ. No. 6. Ehrendorfer, F. & Schönbeck-Temesy, E. 1980. Rubiaceae. In: C. C. Townsend & E. Guest (eds.), Flora of Iraq, vol. 4, part 1: 564-628. Baghdad: Ministry of Agriculture and Agrarian Reform. Endress, M. E., Sennblad, B., Nilsson, S., Civeyrel, L., Chase, M. W., Huysmans, S., Grafström, E. & Bremer, B. 1996. A phylogenetic analysis of Apocynaceae s.str. and some related taxa in Gentianales: a multidisciplinary approach. Opera Bot. Belg. 7: 59-102. Erbar, C. 1991. Sympetaly - a systematic character. Bot. Jahrb. 112: 417-451. Felsenstein, J. 1973. Maximum likelihood and minimum-steps methods for estimating evolutionary trees from data on discrete characters. Syst. Zool. 22: 240-249. Hennig, W. 1950. Grundzüge einer Theorie der phylogenetischen Systematik. Berlin, Germany: Deutscher Zentralverlag. Hennig, W. 1966. Phylogenetic systematics. Urbana, USA: University of Illinois Press. Hestmark, G. 2000. Temptations of the tree. Nature 408: 911.

39 Hooker, J. D. 1873. Rubiaceae. In: G. Bentham & J. D. Hooker (eds.), Genera Plantarum, vol. 2: 7-151. London: Lovell Reeve. Huelsenbeck, J. P. & Ronquist, F. 2001. MrBayes: Bayesian inference of phylogeny. Biometrics 17: 754-755. Jensen, S. R., Nielsen, B. J. & Dahlgren, R. 1975. Iridoid compounds, their occurrence and systematic importants in the angiosperms. Bot. Not. 128: 140-180. Jensen, S. R. 1991. Plant iridoids, their biosynthesis and distribution in angiosperms. In: B. Harborne & F. A. Tomas-Barberan (eds.), Ecological chemistry and biochemistry of plant terpenoids. Oxford, UK: Clarendon Press. Jensen, S. R. 1992. Systematic implications of the distribution of iridoids and other chemical compounds in the Loganiaceae and other families of the asteridae. Ann. Missouri Bot. Gard. 79: 284-302. Johri, B. M., Ambegaokar, K. B. & Srivastava, P. S. 1992. Comparative embryology of angiosperms, vol.2. Berlin, Germany: Springer. Jussieu, A.-L. 1789. Genera Plantarum secundum naturales disposita. Paris, France: Vidaum Herissant. Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge, UK: Cambridge University Press. Lange, J. 1868. Rubiaceae. In: M. Willkomm & J. Lange (eds.), Prodromus florae Hispanicae seu synopsis methodica omnium plantarum in Hispania, vol. 2. Stuttgart, Germany: Sumtibus E. Schweizerbart (E. Koch). Leeuwenberg, A. J. M. & Leenhouts, P. W. 1980. Taxonomy. In: A. J. M. Leeuwenberg (ed.), Engler and Prantl's Die natürlichen Pflanzenfamilien, Fam. Loganiaceae, vol. 28b1: 8-96. Berlin, Germany: Duncker & Humblot. Léonard, J. 1984. Contribution à la connaissance de la flore de l'Iran. – VI. Le complexe Gaillonia A. Rich. ex DC. (Rubiaceae). Bull. Jard. Bot. Belg. 54: 493-497. Lewis, W. H. 1966. The Asian genus Neanotis nomen novum (Anotis) and allied taxa in the Americas (Rubiaceae). Ann. Missouri Bot. Gard. 53: 32-46. Li, S., Pearl, D. K. & Doss, H. 1996. construction using Marcov chain Monte Carlo. Technical Report 583, Dept. of Statistics, Ohio State University. Linczevski, I. 1973. De genere Gaillonia A. Rich. ex DC. (Rubiaceae). Novosti Sist. Vyssh. Rast. 10: 224-236. Lindley, J. 1833. Nixus Plantarum. London, UK: Ridgway & Sons. Mabberley, D. J. 1997. The plant-book. Cambridge, UK: Cambridge University Press. Martin, W. & Embley, T. M. 2004. Early evolution comes full circle. Nature 431: 134-136. Martius, C. F. P. 1827. Nova genera et quas in itinere per Brasiliam, vol. 2. Munich, Germany: Wolf. Mau, B. & Newton, M. A. 1997. Phylogenetic inference for binary data on dendograms using Markow chain Monte Carlo. Journ. Computational and Graphical Statistics 6: 122-131. Mendoza-Heuer, I. 1987. Makaronesische Endemiten: zur Blütenbiologie von Plocama pendula Ait. (Rubiaceae). Bauhinia 8: 235-241. Mennega, A. M. W. 1980. Anatomy of the secondary xylem. In: A. J. M. Leeuwenberg (ed.), Engler and Prantl's Die natürlichen Pflanzenfamilien, Fam. Loganiaceae, vol. 28b1: 112-161. Berlin, Germany: Duncker & Humblot.

40 Molina Borja, M. 1986. Notes on the diet of Gallotia stehlini (Fam. Lacertidae) as obtained from behaviour observations. Vieraea 16: 23-26. Moore, R. 1947. Cytotaxonomic studies in the Loganiaceae. 1. Chromosome numbers and phylogeny in the Loganiaceae. Amer. J. Bot. 35: 404-410. Nei, M. 1987. Molecular evolutionary genetics. New York, USA: Columbia University Press. Nie, Z.-L., Wen, J., Sun, H. & Bartholomew, B. 2005. Monophyly of Kelloggia Torrey ex Benth. (Rubiaceae) and evolution of its intercontinental disjunction between western North America and eastern Asia. Amer. J. Bot. 92: 642-652. Olmstead, R. G., Bremer, B., Scott, K. M. & Palmer, D. J. 1993. A parsimony analysis of the Asteridae sensu lato based on rbcL sequences. Ann. Missouri Bot. Gard. 80: 700-722. Olmstead, R. G. & Reeves, P. A. 1995. Evidence for the of the Scrophulariaceae based on chloroplast rbcL and ndhF sequences. Ann. Missouri Bot. Gard. 82: 176-193. Oxelman, B., Backlund, M. & Bremer, B. 1999. Relationships of the Buddlejaceae s.l. investigated using parsimony jackknife and branch support analysis of chloroplast ndhF and rbcL sequence data. Syst. Bot. 24: 164-182. Oxelman, B., Kornhall, P., Olmstead, R. G. & Bremer, B. 2005. Further disintegration of Scrophulariaceae. Taxon 54: 411-425. Oxelman, B., Lidén, M. & Berglund, D. 1997. Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Pl. Syst. Evol. 206: 393-410. Pomel, A. 1874. Nouveaux Matériaux pour la Flore Atlantique. Paris, France: Savy. Puff, C. 1982. The delimitation of the tribe Anthospermeae and its affinities to the tribe Paederieae (Rubiaceae). Bot. J. Linn. Soc. 84: 355-377. Punt, W. 1980. Pollen Morphology. In: A. J. M. Leeuwenberg (ed.), Engler & Prantl's Die Natürlichen Pflanzenfamilien, Fam. Loganiaceae, vol. 28b: 162-191. Berlin, Germany: Duncker & Humblot. Qarar, M. 1973. Die Gattung Gaillonia A. Rich. (Rubiaceae-Anthospermeae) und ihre Diferenzierung im Saharo-Sindischen und Irano-Turanischen Raum. Wien, Austria: Universität Wien (unpublished thesis). Robbrecht, E. 1988. Tropical woody Rubiaceae. Opera Bot. Belg. 1: 1-271. Robbrecht, E. 1993. Supplement to the 1988 outline of the classification of the Rubiaceae. Index to genera. Opera Bot. Belg. 6: 173-196. Schumann, K. 1891. Rubiaceae. In: A. Engler & K. Prantl (eds.), Die natürlichen Pflanzenfamilien, vol. 4: 1-156. Leipzig, Germany: Wilhelm Engelmann Verlag. Scogin, R. 1992. The distribution of acetoside among angiosperms. Biochem. Syst. Ecol. 20: 477-480. Sennblad, B. & Bremer, B. 1996. The familial and subfamilial relationships of Apocynaceae and Asclepiadaceae evaluated with rbcL data. Pl. Syst. Evol. 202: 153-175. Sogin, M. L. 1991. Early evolution and the origin of eukaryotes. Curr. Opinion Gen. Dev. 1: 457-463. Struwe, L. & Albert, V. A. 1997. Floristics, cladistics, and classification: three case studies in Gentianales. In: J. Dransfield, M. J. E. Coode, & D. A. Simpson, (eds.), Plant diversity in Malesia III. Kew, UK: Royal Botanic Gardens. Struwe, L., Albert, V. A. & Bremer, B. 1994. Cladistics and family level classification of the Gentianales. Cladistics 10: 175-205.

41 Stuessy, Tod F. 1990. . NY, USA: Columbia Univ. Press. Sugiura, M. 1992. The chloroplast genome. Pl. Molec. Biol. 19: 149-168. Sweet, R. 1839. Hortus Britannicus, ed. 3. London: J. Ridgway. Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Molec. Biol. 17: 1105 1109. Takhtajan, A. 1987. Systema Magnoliophytorum. Leningrad, Russia: Nauka. Takhtajan, A. 1997. Diversity and classification of flowering plants. New York, USA: Columbia University Press. Thorne, R. F. 1983. Proposed new realignments in the angiosperms. Nord. J. Bot. 3: 85-117. Thorne, R. F. 1992. An updated phylogenetic classification of the flowering plants. Aliso 13: 365-389. Thulin, M. 1998. Gaillonia (Rubiaceae-Paederieae) in and Arabia. Nordic J. Bot. 18: 31-38. Tomas-Barberan, F. A., Grayer-Barkmeijer, R. J., Gil, M. I. & Harborne, J. B. 1988. Distribution of 6-hydroxy-, 6-methoxy- and 8-hydroxyflavone glycosides in the Labiatae, the Scrophulariaceae and related families. Phytochemistry 27: 2631-2645. Verdcourt, B. 1958. Remarks on the classification of the Rubiaceae. Bull. Jard. Bot. État 28: 209-281. Wagenitz, S. J. 1959. Die systematische Stellung der Rubiaceae–ein Beitrag zum System der Sympetalen. Bot. Jahrb. 79: 17-35. Wainright, P. O., Hinkle, G., Sogin, M. L., & Stickel, S. K. 1993. The monophyletic origins of the Metazoa: An unexpected evolutionary link with fungi. Science 260: 340-342. Yang, Z. & Rannala, B. 1997. Bayesian phylogenetic inference using DNA sequences: A Markow chain Monte Carlo method. Mol. Biol. Evol. 14: 717-724.

42

 ( !  (                      

3 1      4         

        4         5 (  ( ! 5          2    6   7            8   9  7     2 5 7      2                 &   !     (       4         6 :   ; 5 -<<+5     7 2        =&   !     (       4         >6?

      2 1 2  66    1 2 1 11!,*-.