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Boraginaceae), and the Phylogeny of Boraginoideae

Boraginaceae), and the Phylogeny of Boraginoideae

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** -)2'/)*) %*()'-) %%*(* 3443 Dissertation for the Degree of Doctor of Philosophy in Systematic Botany presented at Uppsala University in 2002

Abstract

Långström, E. 2002. Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae. Acta Univ. Ups. Comprehensive Summaries og Uppsala Disserta- tions from the Faculty of Science and Technology 693. 34 pp. Uppsala. ISBN 91-554-5257-4.

Echiochilon, Ogastemma and Sericostoma are revised resulting in the recognition of 15 species of Echiochilon and one Ogastemma species. Several species are placed in synonymy and three new species are described, E. baricum, E. callianthum and E. cyananthum. The single species of Sericostoma is shown to be nested within Echiochilon. The plastid atpB gene was sequenced for Echiochilon and Ogastemma from the Old World and Antiphytum from the New World, plus for a selection of 33 other Boraginaceae taxa. They were analysed together with selected outgroup taxa to give a framework of the tribes of Boraginoi- deae. The analysis gave support for establishing the new tribe Echiochileae for Antiphytum, Echio- chilon and Ogastemma, and for merging the traditionally accepted tribe Eritrichieae with Cyno- glosseae. The ITS region was sequenced for all but one species of Echiochilon and for representa- tives of Antiphytum and Ogastemma. Phylogenetic analysis of Echiochilon revealed that the strongly zygomorphic-flowered species form a paraphyletic group. The morphological data gave results fairly congruent with the ITS phylogeny. Biogeographic interpretations of the ITS and atpB phylogenies indicated a trans-Atlantic dispersal of Antiphytum as the most plausible explanation to the Old/New World disjunction. Analyses using DIVA (Dispersal Vicariance Analysis) of the distributions of the Echiochilon species indicated an ancestor to Echiochilon with a wide distribution over northern Africa and Arabia to India.

Key words: Antiphytum, Echiochilon, Ogastemma, Boraginaceae, Echiochileae, phylogeny, ITS, floral morphology, atpB, tribes, biogeography, Africa, America.

Elisabeth Långström, Uppsala University, Evolutionary Biology Centre, Department of System- atic Botany, Norbyvägen 18D, SE – 752 36 Uppsala, Sweden

© Elisabeth Långström 2002

ISSN 1104-232X ISBN 91-554-5257-4

Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2002 To my son Arvid, the light of my life PREFACE

This thesis is based on the following papers, which will be referred to in the text by their respective Roman numerals (I–III)

I Lönn, E. 1999. Revision of the three Boraginaceae genera Echiochilon, Ogastemma and Sericostoma. Botanical Journal of the Linnean Society 130: 185–259.

II Långström, E. & Chase, M. W. 2002. Tribes of Boraginoideae (Boraginaceae) and placement of Echiochilon, Ogastemma and Sericostoma: A phylogenetic analysis based on atpB plastid DNA sequence data. Accepted for publication in Plant Sys- tematics and Evolution.

III Långström, E. & Oxelman, B. Phylogeny of Echiochilon (Boraginaceae) based on ITS sequences. Manuscript.

Published and accepted papers are reproduced with the publishers’ kind permission.

I have published my papers under two names, as Lönn when I was married and as Långström when not. All morphological work and lab work was done by myself except one sequence in paper III. I planned projects II and III and wrote the major parts of paper II and III. TABLE OF CONTENTS

INTRODUCTION ...... 6 TAXONOMY AND MORPHOLOGY OF ECHIOCHILON AND OGASTEMMA ...... 8 PHYLOGENETIC STUDY ...... 8 The atpB gene ...... 9 The ITS region ...... 10 PHYLOGENY OF BORAGINOIDEAE ...... 10 PHYLOGENY OF ECHIOCHILON ...... 12 Phylogeny based on molecular ITS data ...... 12 Phylogeny based on morphological data ...... 12 Discussion of the Echiochilon phylogenies ...... 12 BIOGEOGRAPHY...... 17 TAXONOMIC TREATMENT OF OGASTEMMA AND ECHIOCHILON ...... 19 Ogastemma ...... 19 Echiochilon ...... 20 Key to the species of Echiochilon and Ogastemma ...... 22 SVENSK SAMMANFATTNING – SWEDISH SUMMARY...... 26 ACKNOWLEDGEMENTS ...... 28 REFERENCES ...... 30

E. Långström

INTRODUCTION

Plant systematists describe and name the plants of the world and unravel the phylogenetic relationship among taxa. Morphological work, molecular techniques and comput- erised mathemathical methods are commonly used to reconstruct the phylogenies of the organisms. Systematic knowledge is used also by other disciplines (all biology, e.g. ecology, pharmacognocy and conservation biology), and systematists use information from other disiplines, trying to understand how evolution operates among plants. Infor- mation from plant systematics is used, for example, when considering how to preserve genetic diversity or for getting a lead on where to look for a desired chemical substance known from another plant. Boraginaceae is a family of 2300 species with a cosmopolitan distribution. The members of Boraginaceae have many uses but none of major economic importance, except for some ornamentals, timber and dye plants. Many Boraginaceae plants have me- dicinal properties and they are used in traditional medicine for treating wounds, skin diseases, fever, chest pain etc. (Neuwinger 2000). Symphytum officinale, for example, has in Europe even been used for healing bone. Flora projects in Northeast Africa and the Arabian Peninsula (Flora of Ethiopia and Eritrea, Flora of Somalia and Flora of the Arabian peninsula and Socotra) have revealed the need for revisions of many genera in these areas. A lot of new material has been collected which has made it clear that the old classifications are outdated. One such genus was Echiochilon, and the two related monotypic genera Sericostoma and Ogastemma (paper I). Echiochilon has the bulk of its species in the Horn of Africa and the Arabian Peninsula. The species of Echiochilon were until 1957 (Johnston 1957) divided in two genera, Echiochilon and Sericostoma, based on floral symmetry with the zygomorphic- flowered species referred to Echiochilon and the actinomorphic-flowered species referred to Sericostoma. The first Echiochilon species described was one of the actinomorphic-flowered species (as Heliotropium persicum Burm.f. in 1768, later transferred to Sericostoma) and the name Echiochilon was introduced in 1900 for one of the zygomorphic-flowered species. In 1957 Johnston transferred all Sericostoma species but one to Echiochilon, and in paper II the last species of Sericostoma was transferred to Echiochilon. In paper III the relationships of the Echiochilon species is investigated with morphological and molecular data. The family delimitation of Boraginaceae is controversial. Traditionally Boragina- ceae and Hydrophyllaceae were two separate families considered to be closely related. This was confirmed by broad scale molecular analyses using rbcL (e.g. Olmstead et al. 1992, Chase et al. 1993). Later studies showed that Hydrophyllaceae is nested within Boraginaceae (Olmstead et al. 1993) which was corroborated by studies of other genes (ndhF, Ferguson 1999; atpB, Långström and Chase in press). Some authors have taken the view that Boraginaceae should be further divided, recognising the different subfamilies, Boraginoideae, Ehretioideae, Cordioideae, Heliotropioideae and Wellstedioi- deae, as families (e.g. Hutchinson 1969, Heywood 1993, Gottschling and Hilger 2001,

6 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

Gottschling et al. 2001). There has been much controversy at the tribal level within Boraginoideae as well (e.g. Popov 1953, Riedl 1967, 1968, 1997, Al-Shehbaz 1991, Popova and Zemskova 1995, Retief and Van Wyk 1997) Much work has been done in palynology (e.g. Erdtman 1966, Nowicke and Ridgeway 1973, Sahay 1979, Díez and Valdés 1991, El-Ghazaly 1991, 1995, Perveen at al. 1995, Scheel et al. 1996) and fruit morphology and gynoecial development (e.g. Hil- ger 1985) to improve the knowledge of Boraginaceae and to give a clearer picture of the tribal relationships in Boraginoideae. The biotechnological methodology has given new tools for easing the task of inferring relationships among taxa. Echiochilon and its close relatives have always been difficult to place among the tribes of Boraginoideae (Boragineae, Cynoglosseae, Eritrichieae, Lithospermeae, My- osotideae, Trigonotideae and Trichodesmeae; Riedl 1967, Al-Shehbaz 1991). Echio- chilon has been variously allocated to three different tribes, Echieae (by most authors included in Lithospermeae), Eritrichieae and Lithospermeae. Ogastemma has traditionally been placed in Eritrichieae, but was lately included in Trigonotideae by Riedl (1997). Paper II is a phylogenetic analysis of the atpB chloroplast gene aiming to give a framework of the tribes to be able to place Echiochilon within Boraginoideae. This study is divided in two parts, one part dealing mainly with morphology and taxonomy (I) and another dealing mainly with phylogenetic relationships within Echio- chilon (III) and among genera in Boraginoideae (II) based on molecular data. All authors to plant names used in the summary are listed in Table 1.

______Table 1. Authors to the genera and species names used in the summary. ______Anchusa L. Echiochilon longiflorum Benth. Antiphytum DC. ex Meissn. Echiochilon pauciflorum (Stocks ex Cordia L. Wight) Långström & M.W.Chase Echiochilon Desf. Echiochilon persicum (Burm.f.) Echiochilon arabicum (O.Schwartz) I.M.Johnst. I.M.Johnst. Echiochilon simonneaui Faurel & Du- Echiochilon baricum Lönn buis Echiochilon callianthum Lönn Echium L. Echiochilon chazaliei (H.Boissieu) Ehretia P.Browne I.M.Johnst. Halacsya Dörfl. Echiochilon collenettei I.M.Johnst. Ogastemma Brummitt Echiochilon cyananthum Lönn Ogastemma pusillum (Coss. & Durieu Echiochilon fruticosum Desf. ex Bonnet & Barratte) Brummitt Echiochilon johnstonii Cufod. Sericostoma pauciflorum Stocks ex Echiochilon jugatum I.M.Johnst. Wight Echiochilon kotschyi (Boiss. & Hohen.) Sericostoma Stocks ex Wight I.M.Johnst. Trichodesma R.Br. Echiochilon lithospermoides (S.Moore) Wellstedia Balf.f. I.M.Johnst. ______

7 E. Långström

TAXONOMY AND MORPHOLOGY OF ECHIOCHILON AND OGASTEMMA (I)

Most species of Echiochilon are shrublets or perennial herbs, more or less adapted to dry conditions. The single species of Ogastemma and one form of Echiochilon longiflorum are annuals. The Echiochilon species have more or less incrusted and often sharp-pointed eglandular hairs covering most of the vegetative parts, and sometimes also stalked or unstalked glandular hairs on the vegetative parts and/or corollas. In Ogastemma only eglandular hairs are found. Both alternate and opposite leaves are found in Ogastemma and Echiochilon. Echiochilon jugatum has all of its leaves opposite and several species have the lowermost leaves opposite and the rest alternate. In Boraginaceae the number of calyx lobes most commonly found is five. A few aberrant genera, e.g. Wellstedia, Ehretia and Echiochilon, include species with only four calyx lobes, and Cordia can have two to ten calyx lobes. The calyx lobes of Echiochilon and Ogastemma are almost always divided to the base. In the strongly zygomorphic-flowered Echiochilon species, four-lobed calyces are found. Most Boraginaceae genera have actinomorphic flowers, but Echiochilon has both actinomorphic-flowered and zygomorphic-flowered species and zygomorphic flowers are also found in Echium and some species of Anchusa and Halacsya (Valentine and Chater 1972). In Ogastemma only actinomorphic flowers are found. Echiochilon and Echium have often been grouped together because of their zygomorphic corollas (e.g. de Candolle 1846, Baillon 1888, Gürke 1897). The Echiochilon species with strongly zygomorphic corollas have a distinct upper lip and the stamens are usually inserted at different heights, the species with oblique zygomorphic corollas do not have a differen- tiated upper lip (stamens inserted at slightly different or equal heights). The species with actinomorphic corollas have the stamens inserted at equal heights. Echiochilon have no faucal appendages (often found in Boraginoideae), but Ogastemma has five weak, circular invaginations of the throat that may be homologous to the faucal appendages of other genera. Hairs inside the throat are always present in Echiochilon, but in Ogastemma they are always absent. The gynoecium is superior, and deeply four-lobed in the genera discussed. Ogastemma and all species of Echiochilon, except E. pauciflorum have a pyramidal gynobase. The style has terminal stigmas in Ogastemma and subterminal stigmas in Echiochilon. Most species of Echiochilon have a bifid or only notched sterile tip protruding beyond the stigmas, but E. pauciflorum has a sterile portion of the style deeply sunken between the stigmas (only visible from above). The stigmas of Ogastemma are totally fused at the top. Ogastemma has tuberculate nutlets and Echiochilon has smooth and shiny to tuberculate nutlets. The pollen grains of Echiochilon are 2- or 3-colporate or colpate, square to rectangular in equatorial view and rectangular-rounded to rounded (when 2-aperturate) or rounded triangular in polar view (when 3-aperturate; El-Ghazaly 1991, paper II). The pollen vary in size between species, the 3-colporate pollen grains from E. kotschyi are larger than the 2-colpate pollen grains from E. baricum and E. lithospermoides (paper

8 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

II). The pollen grains of Ogastemma are 3-colporate, somewhat prolate in equatorial view, and rounded or triangular with the apertures forming the truncate corners in polar view (paper II).

PHYLOGENETIC STUDY (II–III)

In order to infer phylogenetic relationships within Boraginoideae and within Echio- chilon I have, beside analysing morphological characters, extracted and sequenced two DNA regions and analysed the sequences with phylogenetic methods. The atpB gene (II). The atpB is a plastid gene that codes for the beta subunit of ATP synthetase, it is situated in the chloroplast genome near rbcL and is 1497 base pairs long (Fig. 1a). It has been used for reconstruction of angiosperm phylogeny in several groups (e.g. Hoot et al. 1995, Hoot et al. 1997, Chase et al. 1999, paper II). The other subunits of ATP synthetase are encoded in either the chloroplast or the nuclear ge- nomes. ATP synthetase couples proton translocation across membranes with the syn- thesis of ATP, and has a conserved rate of evolution similar to that of rbcL (Hoot et al. 1995).

1494R 766R

atpE atpB spacer rbcL

611F 2F

a

P17

P16 ITS 5.8S ITS 18S NUCLEAR rDNA 26S NUCLEAR rDNA 1 rDNA 2

P2 5

ITS4R

26S-82 R

b

Fig. 1. The atpB gene and ITS region with the primer positions shown.

9 E. Långström

The ITS region (III). The internal transcribed spacers (ITS) of the nuclear ribosomal DNA are situated between 18S and 26S nrDNA with 5.8S inserted between ITS 1 and ITS 2 (Fig. 1b). The spacers contain the signals needed to process the rRNA transcript (Baldwin et al. 1995, Page & Holmes 1998, p. 76), and have often been used for inferring phylogeny (e.g. Baldwin 1992, Baldwin et al. 1995, Andreasen et al. 1999, Oxel- man and Lidén 1995, Böhle et al. 1996, Eldenäs et al. 1998, Wagstaff and Garnock- Jones 1998), mostly to solve problems at subfamily level or lower. For higher-level problems ITS is often too variable to get a good alignment. In Boraginaceae the ITS region is ca 650 base pairs long.

PHYLOGENY OF BORAGINOIDEAE (II)

Echiochilon and its close relatives have always been difficult to place within Boragi- noideae. To get a framework to place Echiochilon and its relatives, a phylogenetic analysis based on atpB plastid DNA data was performed. The atpB gene was amplified and sequenced for 38 ingroup and 12 outgroup taxa. The ingroup taxa were selected from each of the seven tribes of Boraginoideae following Riedl (1967) and Al-Shehbaz (1991): Boragineae, Cynoglosseae, Eritrichieae, Lithospermeae, Myosotideae, Trigonotideae and Trichodesmeae, and four tribes of Hy- drophyllaceae (Hydrophylleae, Nameae, Phacelieae and Wigandieae). The outgroup taxa were chosen from Solanales, Gentianales and Lamiales, because these taxa have been inferred to be the taxa most closely related to Boraginaceae, and Garrya Douglas ex Lindl. and Oncotheca Baill. were also selected as outgroup taxa and used to root the tree, according to earlier analyses and classifications (e.g. Chase et al. 1993, Olmstead et al. 1993, APG 1998, Savolainen et al. 2000, Soltis et al. 2000). One of the 779 trees (arbitrarily chosen) from the analysis is shown in Fig. 2 with branches absent from the consensus tree indicated by dotted lines, branch lengths given below the branches and bootstrap frequency (boot) above the branches. The following categories are used to describe the bootstrap and jackknife results: 50–74, weakly supported; 75–84, moderately supported; 85–95, well supported; 95–100, strongly supported. Boraginoideae formed a strongly supported clade. Boragi- neae and Lithospermeae received bootstrap frequencies of 100 and 83 respectively (clade C and F respectively in Fig. 2). The members of Erithrichieae and Cynoglosseae came out nested in a way that made it impossible to separate them, but together forming a moderately supported (if Trichodesma is excluded, only weakly if Trichodesma is included). The name Cynoglosseae is used for the clade since that is the older of the two names. Another genus with an uncertain position is Omphalodes which comes out in an isolated position within Boraginoideae. The genera Antiphytum, Ogastemma, Echiochilon and Sericostoma formed clade E (Fig. 2; boot 84). Since Sericostoma is nested within Echiochilon a recombination was made to form E. pauciflorum. Clade E was given tribal status with the name Echio- chileae.

10 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

Fig. 2. One of the 779 most parsimonious trees (879 steps, CI=0.60, RI=0.76) from analysis of the atpB data from 50 taxa. Node B corresponds to Boraginoideae. Numbers above the branches are bootstrap percentages, the nodes lacking numbers received less that 50% bootstrap support. Numbers below the branches are estimated numbers of substitutions (ACCTRAN optimisation). Groups not present in the strict consensus tree are indicated with dotted lines. Classification according to Table 4 abbreviated as follows.; the tribes of Boraginoideae: Bo=Boragineae, Cy=Cynoglosseae, Er=Eritrichieae, Li=Lithospermeae, My=Myosotideae, Td=Trichodesmeae, Tr=Trigonotideae; the subfamilies of Boraginaceae except Boraginoideae: BC=Cordioideae, BE=Ehretioideae, BH=Heliotropioideae; HH=Hydrophylleae, HN=Nameae, HP=Phacelieae, HW=Wigandieae. Alternate or earlier classifications are written within parenthesis.

11 E. Långström

PHYLOGENY OF ECHIOCHILON (III)

In order to reach a better understanding of the relationships among the species of Echiochilon phylogenetic analyses were performed, both with molecular and morphological data. The ITS region gave good resolution within the genus and was amplified and sequenced for all species but one, which so far is only known from the type. Phylogeny based on molecular ITS data. ITS sequences for 24 taxa were aligned, gap-coded and analysed using PAUP* ver. 4.0b4a (Swofford 1998; 654 characters, 115 parsimony informative). The resulting strict consensus tree is presented in Fig. 3 with jackknife values and bootstrap percentages shown below the branches. The geographical distribution areas are shown and defined in Fig. 4. Phylogeny based on morphological data. A morphological matrix of 31 characters was compiled (Table 2) and used to place E. simonneaui and to find groups that are supported by morphological characters (presented in Table 3). Ca 800 herbarium sheets were thoroughly examined to find the 31 characters. Ogastemma was used as outgroup based on earlier suggestions of relationship (Johnston 1957). The strict consensus tree of the five resulting trees is presented in Fig. 3 with bootstrap and jackknife values above the branches. Discussion of the Echiochilon phylogeny. The results from the ITS data and morphological data are fairly congruent. The main differences between the phylogenies are the respective positions of E. johnstonii and the presence of clade J in the morphological analysis (Fig. 3). Clade J groups the actinomorphic flowered species E. jugatum and E. kotschyi. In a total evidence analysis (not presented) these taxa were placed one branch lower as a response to the morphological characters moving E. johnstonii to- wards an isolated position as sister to clade C and moving E. kotschyi together with E. jugatum. An incongruence length difference test (ILD; Farris et al. 1994) rejects the null hypothesis of incongruence. It seems that the ITS data dominate in the combined data set, and it is therefore better to view the trees separately and compare them visu- ally. The Boraginoideae phylogeny based on atpB plastid sequence data (paper II) and the ITS data set both support the tribes Echiochileae and Cynoglosseae. The positions of Antiphytum, Echiochilon and Ogastemma using ITS are different from paper II where Echiochilon and Ogastemma form the sister group to Antiphytum (but with less than 50% bootstrap support and Bremer support only 1). The new tribe Echiochileae (paper II) is strongly supported by ITS data (clade A in Figs. 3 and 4), and characterised by having more or less spherical pollen (but in Echiochilon square in equatorial outline) with colpi as aperture type. Echiochilon is strongly supported (clade C) and characterised by zygomorphic calyx and corolla (11:1 and 14:1 in Table 3), corolla with hairs inside the throat (18:1) and subterminal stigmas (26:1). All characters are optimised on an arbitrarily chosen most parsimonious tree in Fig. 4.

12 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

______Table 2. The data matrix used in the morphological part of the phylogenetic analysis of Echiochilon and Ogastemma. The letters are coded as following: a=(01), c=(12), e=(012), -=inapplicable. ______Character 00000 00001 11111 11112 22222 22223 3 12345 67890 12345 67890 12345 67890 1 Taxon ______E.arabicum 01001 21000 10010 011a0 1100a 1a112 c E.baricum 01000 21010 00010 00100 11000 1aa1c 2 E.callianthum 01001 c1010 10010 01110 1100a 1111c c E.chazaliei a1010 10010 10011 20100 11000 10200 2 E.collenettei 21a11 21011 0000- 01110 11000 1a110 2 E.cyananthum 0100a 210a0 a0010 0a100 1a000 1a111 2 E.fruticosum 0100a 11010 10111 001a0 11001 11ea0 c E.johnstonii 01aaa 11010 a000- 1a100 11000 11110 2 E.jugatum 210a0 111aa 0100- 10110 00000 11100 2 E.kotschyi a10a0 c11aa 0a00- a0110 0a000 1111a 2 E.lithospermoides 01110 00001 10011 20100 11001 11c1a 2 E.longiflorum eaaaa 0aaa1 10011 2a100 11001 11caa 2 E.pauciflorum 11010 11000 0100- 10100 10010 01011 0 E.persicum 0100a 210a0 a00a0 aa110 1a000 1a11e 2 E.simonneaui 00110 0011a 10011 20100 11000 10210 2 O.pusillum 11000 11000 0110- 200-1 11100 0-111 2 ______

The Echiochilon species with two-lipped corollas divide on two strongly supported clades, D and E, in the molecular analysis (D as sister to E and the rest of Echiochilon), but within the same clade (weakly supported) in the morphological analysis. Characters supporting the North African clade (D) are nutlets with the ventral side obtuse (29:0) and an oblong or boomerang-shaped areola (area where the nutlet was attached to the plant; 28:2). Characters supporting clade E (from the Horn of Africa, Kenya, Ethiopia and the Arabian peninsula), are leaves with hairs only on the lower surface (3:1), ribbed branches (6:0; with a reversal for E. johnstonii), calyx with a conspicuous midrib in fruit (10:1; with a reversal for E. johnstonii) and oblique stigmas (25:1; with a reversal for E. johnstonii). In the morphological analysis E. simonneaui falls within the combined D and E clade, it has all the morphological traits of the species with a two-lipped corolla, and it grows in Morocco (as E. chazaliei). Echiochilon johnstonii does not at all be- long in clade E according to morphology, but is placed there with strong support by ITS data. The position of ‘E. johnstonii’ might be an artefact, it may actually be a hybrid because it grows in a possible hybrid zone between E. johnstonii and E. longiflorum (paper I, III).

______

13 E. Långström

Table 3. Morphological characters used unordered in the phylogenetic analysis of Echiochilon. ______1. Phyllotaxy: alternate (0), opposite at base (1), all opposite (2). 2. Vegetative parts, hair type: bulbous-based hairs (0), disc-based hairs (and discs) (1). 3. Indumentum, leaves: both surfaces covered (0), upper surface glabrous (1). 4. Indumentum, calyces: both surfaces (0), outer surface only (1). 5. Glandular hairs on vegetative parts: absent (0), present (1). 6. Young branches: finely ribbed (0), coarsely ribbed (obvious when dry) (1), not ribbed (2). 7. Leaves, bracts, pedicels: slightly decurrent (0), not (1). 8. Leaf-base: cuneate or attenuate (0), clasping (1). 9. Leaves, midrib: conspicuous, (0) not conspicuous (1). 10. Calyx lobes, midrib: not visible (0), conspicuous in fruit (1). 11. Calyx symmetry: actinomorphic (0), zygomorphic with the abaxial lobe largest (1). 12. Calyx: hairy inside at the base (0), glabrous (1). 13. Hyaline margins of calyx lobes: absent (0), present (1). 14. Corolla symmetry: actinomorphic (0), zygomorphic (1). 15 Zygomorphic corolla: oblique (0), with upper and lower lip (1). 16. Unicellular hairs on outer surface of corolla: on the tube and lobes (0), only on the lobes (1), absent (2). 17. Glandular hairs on corolla: absent (0), present (1). 18. Unicellular hairs inside corolla throat: absent (0), present (1). 19. Unicellular hairs inside corolla throat: white (0), yellow (1), brownish (2). 20. Filaments inserted: above (0), below the constriction/the middle of the corolla tube (1). 21. Inflorescence: flowers axillary (0), flowers in terminal inflorescence (1). 22. Anthers: exserted from corolla throat (0), included in corolla throat (1). 23. Anthers: not compressed (0), dorsiventrally compressed (1). 24. Gynobase1: elevated pyramidal (0), plane or depressed (1). 25. Stigmas: horizontal (0), oblique (1). 26. Stigmas: terminal (0), subterminal (1). 27. Sterile tip of the style: notched/depressed at the apex (0), bilobed (1). 28. Areola2 form: rounded (0), triangular (1), transversely oblong or boomerang-shaped (2). 29. Ventral side of nutlet: obtuse (0), with keel (1). 30. Number of longitudinal ridges on the dorsal side of the nutlets: none (0), one (1), three (2). 31. Nutlet stipe: long vertical (0), short oblique (1), absent (2). 1 Gynobase is the base of the gynoecium where the nutlets are attached, either a flat area or on a conical protrusion. 2 Areola is the area on the nutlet where it has been attached to the gynobase. ______

Clade F includes the rest of Echiochilon, which are actinomorphic-flowered or have oblique zygomorphic corollas. Clade F is supported by, among other characters, an actinomorphic calyx and corolla (11:0 and 14:0) and corolla with hairs on the lobes only (16:1). Echiochilon pauciflorum is separated from clade G by a well supported node and has some autapomorphies not found in the rest of Echiochilon: gynobase plane or depressed (24:1), areola rounded (28:0) and nutlets with a relatively long vertical stipe attaching the nutlet to the gynobase (31:0). The Arabian species Echiochilon arabicum and E. callianthum are the only group within clade G that get any support at all. Morphological characters they have in common are glandular hairs (or unstalked glands) both on corollas (17:1) and on vegetative parts (5:1), a slightly zygomoprhic calyx (11:1) and nutlets with a median dorsal

14 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae ridge from the top to the base, and a shorter ridge on each side on the lower half of the nutlets (30:2).

Fig. 3. Strict consensus tree from the ITS data (left) and morphological data (right). The labels on the branches correspond to groups discussed in the text. The vertical bars to the right marked B, L, C and E mark the tribes Boragineae, Lithospermeae, Cynoglosseae and Lithospermeae respectively. The figures above the branches represent jackknife and bootstrap percentages, respectively. The arrow points to a branch inserted on branch E.

15 E. Långström

Fig. 4. One of the 54 most parsimonious trees from the ITS analysis. Branches K, L and M are not present in the strict consensus tree, here indicated by dotted lines. The labels on the branches correspond to those in Fig. 3 and are discussed in the text, and the labels in the grey box above the tree show the geographical distribution. The characters optimised on the branches have different symbols for synapomorphies (black boxes), parallelisms (parallel lines) and reversals (crosses).

16 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

BIOGEOGRAPHY (II, III)

Echiochileae has an American to northern African/western Asian trans-Atlantic disjunction (geographical distribution shown in Fig. 5). Old World to New World disjunctions have recently been discussed by e.g. Macey et al. (1999), Thulin (1999), Lavin et al. (2000), Sanmartín et al. (2001) and Tiffney and Manchester (2001). The simplest explanation for the Echiochileae disjunction using the atpB and ITS phylogenies, is a dispersal of Antiphytum (or a precursor of Antiphytum and Ogastemma) from Eurasia or Africa to the American continent. The ITS phylogeny supports a later dispersal than the atpB phylogeny since the split between Ogastemma and Antiphytum comes later. There is better support for these branches from the ITS phylogeny than the atpB phylogeny. For Echiochileae there are two possible explanations for the trans-Atlantic distribution: 1) a vicariance of a widespread Echiochileae, 2) a recent dispersal to the Ameri- can continent. The old vicariance pattern between Africa and the American continent (the breakup of Gondwanaland ca 100 Myr ago) is not possible here because Echio- chileae can be assumed to be much younger than 100 Myr. Wikström et al. (2001) dated the split between Borago and Hydrophyllum (corresponds to clade A, Fig. 2) to ca 56–59 Myr. A dispersal via the Thulean Bridge which connected southern Europe to eastern North America through southern Greenland ca 70–45 Myr (Tiffney 1985, San- martín et al. 2001) seems more plausible.

Fig. 5. Distributions of Antiphytum, Ogastemma and Echiochilon labelled with A, O and E in the cladograms from analyses of atpB and ITS respectively that show their relationships and respective pattern.

17 E. Långström

According to Raven and Axelrod (1974) the Boraginaceae are thought to have a mainly Eurasian origin with repeated introductions to the southern land masses. At least for Boraginoideae this is supported by the fact that all tribes have their main distribution in the Old World (Boragineae - almost exclusively Eurasian, Cynoglosseae s. lat. - ca 2/3 Old World, Lithospermeae - more than 2/3 Old World primarily northern hemisphere, Trichodesmeae - all Old World; Al-Shehbaz 1991). A scenario for how the present distribution pattern of Echiochilon has arisen, following the hypothesis of Raven and Ax- elrod (1974), could be that Echiochilon and Ogastemma originated in Eurasia and spread southwards because of the stepwise climatic cooling starting in the Early Eocene (Tiffney and Manchester 2001), which allowed thermophilic plants to migrate to the south. Another scenario is indicated by a DIVA analysis based on the geographical distribution (shown below the taxon names in Fig. 4) of the ITS phylogeny of Echiochilon. In DIVA the cost for vicariance or speciation within one area=0, and the cost for dispersal or extinction=1. The distribution pattern was simplified by letting all species of clade G (Figs. 1–3) represent a distribution area of a combined Horn of Africa, Arabia and In- dia (HAI). Including the North American (Am) distribution of Antiphytum in DIVA analysis of the ITS phylogeny adds Am to the base of the tree, but according to the discussion above this is disregarded and interpreted as a dispersal event. The DIVA analysis based on ITS (Fig. 6a) shows that Echiochilon and Ogastemma had an ancestor widely distributed in northern Africa, over Arabia to India. Several vicariance events and four dispersals have produced the distribution of today. A DIVA analysis of the Boraginoideae atpB phylogeny where Antiphytum is the sister of Echiochilon and Ogastemma gave a result where Echiochileae could have originated in Eurasia (Fig. 6b) and Echiochilon/Ogastemma could have entered the southern land masses via the Mediterranean region when Europe and Africa connected in the Miocene (20–17 Myr), when the Mediterranean Basin was formed (but probably not during the earlier connection during the Palaeocene; 63 Myr). An origin of Echio- chileae in Eurasia would more easily explain the dispersal of Antiphytum to America, possibly via the Thulean Bridge. The optimisation gave three dispersal events. The DIVA optimisations are totally dependent on the geographical range of the taxa chosen. I made a simplification by treating the tribes Boragineae and Lithospermeae as being Eurasian as most of their species are Eurasian, and Cynoglosseae as African/Eurasian.

18 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

Fig. 6a. The resulting optimisation from the DIVA analysis excluding North America. The upper- case letters at the base of each node represent the ancestral areas for the nodes. Circles around the areas at the base of the node represent vicariance areas. The lower-case letters indicate dispersals to the areas. Areas are defined as following: N=northern Africa, A=Arabia, K=Kenya, H=the Horn of Africa, I=Iran, Pakistan and India. Fig. 6b. One of the resulting optimisations from the DIVA analysis of a simplification of the atpB analysis. Areas are defined as following: Afr=Africa, Am=America, Eur=Eurasia. Clade C corresponds to Boragineae which is almost exclusively Eura- sian, Clade D corresponds to Lithospermeae which has more than 2/3 in Eurasia, and clade G corresponds to Cynoglosseae s. lat. which has ca 2/3 of its species in the Old World.

TAXONOMIC TREATMENT OF OGASTEMMA AND ECHIOCHILON (I)

Ogastemma. A genus of richly branched annual herbs up to ca 25 cm high, densely covered with appressed hairs with a discoid base. Lowest branches opposite. Leaves simple, the lowest pairs opposite, then alternate. Flowers produced abundantly along almost the whole length of the branches, the first flowers of the young shoots develop in the axil of the bifurcating stems. Bracts leaflike. Calyx 5-lobed, reaching beyond the corolla, with the lobes somewhat to distinctly unequal in size, somewhat enlarged in fruit, the base becoming subglobose with the tips of the lobes leaning together; lobes with hyaline margins. Corolla usually 5-lobed, actinomorphic, subcylindrical. Pollen 3- aperturate (colpate or colporate), round or subprolate in equatorial view, round or rounded triangular in polar view with the apertures situated in the corners when

19 E. Långström rounded triangular. Stigmas 2, terminal, subglobose. Nutlets beige, ovoid with a conoidal to somewhat rostrate acute apex, densely verrucose, usually 4 developing. Fig. 7. A genus of one species occurring on the Canary Islands, in northern Africa, and Arabia.

Fig. 7. Ogastemma pusillum reprinted from Cosson and Durieu in Bonnet and Barratte (1892–1895). This illustration is the one that was selected as lectotype in paper I.

Echiochilon. A genus of moderately to richly branched perennial (rarely annual) herbs and shrublets, up to ca 60 (–100) cm high, ± glabrous to densely covered with disc- based or bulbous-based eglandular hairs, sometimes also with glandular hairs on some or all parts of the plants. Leaves simple, alternate, opposite or alternate with the lowest pairs opposite. Flowers either in clearly defined many-flowered cymes terminating the branches or interspersed among the leaves all the way along the branches. Bracts leaflike. Calyx 5-lobed, less than half as long to equalling the corolla tube, with the lobes equal to very unequal in size; lobes in one species with hyaline margins. Corolla

20 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

5-lobed, actinomorphic, slightly zygomorphic with the adaxial side somewhat prolonged, to strongly zygomorphic with the adaxial side distinctly prolonged and the 2 adaxial lobes forming an erect upper lip different in shape from the 3-lobed lower lip, ± funnel-shaped, usually with a waist. Pollen 2–3-colporate or colpate, square to rectangular in equatorial view and rectangular-rounded to rounded (when 2-aperturate) or rounded triangular in polar view (when 3-aperturate). Stigmas 2, subterminal, with a bifid or only notched sterile tip protruding beyond or sunken between the stigmas. Nutlets white, beige, reddish or brown, ovoid to cordate, ± smooth and shiny to verrucose, with conoidal apex. Fig. 8. A genus with 15 species with a distribution in Mauritania, Morocco, Algeria, Tuni- sia, Libya, Egypt, Sudan, Djibouti, Somalia, Ethiopia, Kenya, Israel, Jordan, Iran, Pakistan, India, Saudi Arabia, Bahrain, United Arab Emirates, Oman, and Yemen.

Fig. 8. Echiochilon pauciflorum reprinted from Stocks ex Wight (1848). This illustration (here with many details removed) is the one that was selected as lectotype for Sericostoma pauciflorum in paper I.

21 E. Långström

Key to the species of Ogastemma and Echiochilon (1+15 species)

1. Calyx lobes with margins hyaline at the base; style with the stigmas terminal; corolla actinomorphic with a weak invagination below each lobe, reminding of small faucal appendages, annual ...... O. pusillum 1. Calyx lobes with green or blue margins (if hyaline then corolla strongly zygomorphic and style with a conspicuous sterile tip); style with a sterile tip protruding beyond or sunken between the stigmas; corolla lacking any sign of faucal appendages, perennial (rarely annual) ...... 2 2. Outer surface of corolla glabrous or with glandular hairs only ...... 3 2. Outer surface of corolla with eglandular hairs, sometimes also with glandular hairs ...... 7 3. Glandular hairs on vegetative parts and corolla absent; corolla zygomorphic with a two- lobed upper lip ...... 4 3. Glandular hairs abundant on vegetative parts and outside of the corolla; corolla zygomorphic, obliquely funnel-shaped, with a ± heart-shaped upper lip ...... E. longiflorum 4. Stigmas ± horizontal ; sterile tip of the style ± clavate, with a small notch or depression at the tip; leaves triangular and leathery, or obovate to oblong and fleshy and covered with appressed eglandular hairs below ...... 5 4. Stigmas oblique to strongly oblique; sterile tip of the style conspicuously bifid; leaves (narrowly to broadly) lanceolate, oblanceolate to oblong and of normal or somewhat leathery texture, or ovate and somewhat fleshy (with eglandular hair discs below), or obovate and fleshy (thickly covered with bulbous-based eglandular hairs) ...... 6 5. Leaves triangular, leathery, with rigid white cone-shaped hooked bulbous-based eglandular hairs on margins and midrib ...... E. simonneaui 5. Leaves obovate or oblong, fleshy, with the lower surface fairly densely covered with appressed disc-based eglandular hairs and the upper surface with scattered appressed disc- based eglandular hairs ...... E. chazaliei 6. Corolla with a ± distinctly heart-shaped upper lip; nutlets reddish and heart-shaped in dorsal outline or beige to whitish and ovoid in dorsal outline ...... E. longiflorum 6. Corolla with the upper lip not distinctly heart-shaped; adaxial lobes of the same shape and size as the abaxial lobes; nutlets brownish, ovoid in dorsal outline ... E. lithospermoides 7. Most leaves opposite ...... 8 7. Most leaves alternate ...... 9 8. Corolla large (tube ca. 7.5–9 mm long), narrowly funnel-shaped, with glandular hairs (usually abundant) on the vegetative parts and corollas ...... E. collenettei 8. Corollas small (tube ca. 3.2–3.7 mm long), broadly funnel-shaped, without glandular hairs on the vegetative parts and corollas ...... E. jugatum 9. Flowers interspersed among the leaves along the whole of the branches, not in well defined cymes terminating the branches ...... E. kotschyi 9. Flowers in cymes terminating the leafy branches ...... 10 10. Flowers zygomorphic, blue, pink or white and pink ...... 11 10. Flowers actinomorphic or only slightly zygomorphic, mostly white or yellowish ...... 15

22 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

11. Vegetative parts covered with loosely appressed to spreading sharp pointed eglandular hairs, and the whole plant covered with glandular hairs; corollas with an expanded throat with patent lobes ...... E. callianthum 11. Vegetative parts covered with appressed eglandular hairs (only spreading on the margins of the leaves and calyces); glandular hairs mostly in the inflorescence when present; corollas obliquely trumpet- or funnel-shaped ...... 12 12. Adaxial corolla lobes larger than the abaxial, pink or white with pink mottlings in the throat, outer part of the corolla densely covered with curly to straight appressed hairs ...... E. baricum 12. All corolla lobes ± equal in size, blue (rarely white), sparsely or densely covered outside with hairs on throat, waist and lobes ...... 13 13. Margins of the calyx lobes and bracts normally blue, red or hyaline; nutlets ovoid or narrowly ovoid in dorsal outline, often bent, saddle-like, in side view, not conspicuously verrucose, not ridged ...... E. fruticosum 13. Margins of the calyx lobes or bracts green; nutlets ovoid in dorsal outline, ± straight back, verrucose and with at least one longitudinal ridge on the dorsal side ...... 14 14. Corolla 8.5–9 mm long, with spreading lobes; nutlets with two extra ridges on the sides of the longitudinal dorsal ridge; leaves rather closely appressed to the stem ...... E. arabicum 14. Corolla 5–5.5 mm long, with spreading to patent lobes; nutlets with only one longitudinal dorsal ridge; leaves either closely appressed to the stem or recurving ...... E. cyananthum 15. Hairs inside corolla throat white; nutlets ovoid to broadly ovoid, not ridged ...... 16 15. Hairs inside corolla throat yellow; nutlets ovoid, often with one to three longitudinal ridges on the dorsal side ...... E. persicum 16. Sterile tip of style at least 1.5–2 times longer than the height of the stigmas, conspicuously bifid ...... E. johnstonii 16. Sterile tip of style shorter than the height of the stigmas, only with a small depression at the tip; nutlets with a vertical short stipe and the attachment scar basal ...... E. pauciflorum

Ogastemma pusillum (Coss. & Durieu ex Bonnet & Barratte) Brummitt is an annual herb with white actinomorphic flowers in inflorescences terminating the leafy branches. Leaves alternate, with the basal pairs opposite, lanceolate. Calyx lobes with hyaline margins at the base. Nutlets beige, ovoid with a conoidal or somewhat rostrate apex, verrucose. O. pusillum grows on sandy or rocky ground from sea level up to 1100 m on Fuerteventura (Canary Islands), Mauritania, Morocco, Algeria, Tunisia, Libya, Egypt, Kuwait, Bahrain, Qatar, United Arab Emirates, Saudi Arabia and Oman.

Echiochilon jugatum I.M.Johnst. is a perennial shrublet with white to yellow actinomorphic flowers interspersed among the recurved connate opposite leaves. Nutlets white to beige, narrowly ovoid to ovoid, smooth. Echiochilon jugatum grows on sand from sea level to 200 m above sea level in Bahrain, Qatar, United Arab Emirates and Oman.

23 E. Långström

Echiochilon kotschyi (Boiss. & Hohen.) I.M.Johnst. is a perennial shrublet with white to yellow actinomorphic flowers interspersed among the ± recurved alternate leaves; the basal pairs of leaves sometimes opposite. Nutlets white to beige, ovoid to broadly ovoid, smooth. Echiochilon kotschyi grows on sand from sea level to 30 (–150) m above sea level in Iran, United Arab Emirates, Oman and Yemen. Sometimes cushion- forming and locally dominating the landscape. Synonym (not recorded in paper I): Sericostoma pauciflorum var. qeshmensis A.Ghahreman & F.Attar (Ghahreman and Attar 1996) from Qeshm island in Iran.

Echiochilon johnstonii Cufod. is a perennial herb or shrublet (sometimes forming dense cushions) with white or pink actinomorphic flowers in inflorescences terminating the leafy branches. Leaves alternate, oblanceolate to linear. Nutlets white, pinkish or beige, ovoid to broadly ovoid, obscurely verrucose. Echiochilon johnstonii grows on sand, rock or coral from 10–50 (–150) m above sea level in southern Somalia.

Echiochilon fruticosum Desf. is a perennial shrublet with blue zygomorphic flowers with an upper and lower lip in unilateral inflorescences terminating the leafy branches. Calyx with blue or hyaline margins. Leaves alternate, lanceolate to oblanceolate. Nut- lets beige or whitish, narrowly ovoid to ovoid (sometimes bent on the middle), somewhat verrucose. Echiochilon fruticosum grows on sandy or rocky ground from sea level up to 1500 m in Morocco, Algeria, Tunisia, Libya, Egypt, Syria, Israel, Jordan and Saudi Arabia. The most widespread and common Echiochilon species.

Echiochilon chazaliei (H.Boissieu) I.M.Johnst. is a perennial shrublet with white, pink or blue zygomorphic flowers with an upper and lower lip in few-flowered inflorescences terminating the leafy branches. Leaves alternate, with the basal pairs often opposite, spathulate to oblong. Nutlets beige or whitish, ovoid, verrucose or smooth. Echiochilon chazaliei grows on sandy or stony ground near the sea between 2–50 m above sea level in Morocco and Mauritania.

Echiochilon simonneaui Faurel & Dubuis is a perennial shrublet with pink zygomorphic flowers with an upper and lower lip in unilateral inflorescences terminating the leafy branches. Leaves alternate, narrowly triangular. Nutlets beige-brown, ovoid, verrucose. Echiochilon simonneaui is only known from one collection from the inland of Morocco where it locally dominates the vegetation on the slopes of the valley Seguiet el Hamra (Faurel and Dubuis 1959).

Echiochilon lithospermoides (S.Moore) I.M.Johnst. is a perennial herb or shrublet with reddish to bluish zygomorphic flowers with an upper and lower lip in unilateral inflorescences terminating the leafy branches. Leaves alternate, lanceolate to oblong. Nutlets reddish brown to dark brown, ovoid to broadly ovoid, verrucose. Echiochilon lithospermoides grows on sandy soil or loam in grassland or open bushland on 850–2100 m above sea level in southern Ethiopia and Kenya.

24 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

Echiochilon longiflorum Benth. is an annual to perennial herb or shrublet with reddish, bluish, yellowish or whitish zygomorphic flowers with an upper and lower lip with different coloured markings on the corolla, sometimes with glandular hairs on the vegetative parts and corollas. Flowers in unilateral inflorescences terminating the leafy branches. Leaves alternate (rarely with the basal pairs opposite), narrowly lanceolate to broadly ovate or obovate. Nutlets reddish, beige or grey, conspicuously cordate to ovoid. Echiochilon longiflorum grows on sandy or rocky ground in open habitats or in grassland or bushland on an altitude ranging from sea level to 950 m above sea level in Ethiopia, Somalia, Yemen and Oman. It is a variable species growing on volcanic ground, limestone and gypsum. Several partly geographically correlated forms can be recognised.

Echiochilon baricum Lönn is a perennial herb or shrublet with pink or white zygomorphic flowers with an oblique corolla with reddish or yellow markings in the corolla throat. Flowers in unilateral inflorescences terminating the leafy branches. Leaves alternate, narrowly oblanceolate. Nutlets beige to reddish beige, ovoid, verrucose. Echio- chilon baricum grows on stony ground on 10–250 m above sea level in the Bari region in Somalia. It has only been collected twice.

Echiochilon cyananthum Lönn is a perennial shrublet with blue zygomorphic flowers with an oblique corolla in inflorescences terminating the leafy branches. Leaves alternate, oblanceolate. Nutlets orange-beige, ovoid, verrucose. Echiochilon cyananthum grows on sandy or rocky ground on altitudes ranging from sea level up to 1800 m. It was treated as a part of E. arabicum by Johnston (1957) but has been separated out as a species (paper I).

Echiochilon persicum (Burm.f.) I.M.Johnst. is a perennial herb or shrublet with white or yellowish (rarely reddish or bluish) actinomorphic flowers (or slightly zygomorphic flowers with an oblique corolla) in inflorescences terminating the leafy branches, sometimes with glands on the vegetative parts. Leaves alternate, narrowly oblong to oblanceolate. Nutlets whitish, beige or reddish beige, ovoid, verrucose with small or small and large verrucae. Echiochilon persicum grows on soil, sandy or rocky ground on altitudes ranging from sea level up to 2400 m in Bahrain, Qatar, United Arab Emir- ates, Iran, Pakistan, Sudan, Saudi Arabia, Oman, Yemen, Djibouti and Somalia. It is a variable species growing on granite, limestone and gypsum. Several partly geographically correlated forms can be recognised.

Echiochilon collenettei I.M.Johnst. is a perennial shrublet with yellow and/or white actinomorphic flowers in inflorescences terminating the leafy branches, with glands on the vegetative parts and corollas. Leaves opposite, oblanceolate to lanceolate. Nutlets reddish beige, ovoid, verrucose. Echiochilon collenettei grows on gravelly ground on

25 E. Långström

(60–) 500–1100 m above sea level in northern Somalia and Oman. It is a uniform species despite the disjunct distribution.

Echiochilon arabicum (O.Schwartz) I.M.Johnst. is a perennial herb or shrublet with blue zygomorphic flowers with an oblique corolla with purple veins in inflorescences terminating the leafy branches, sometimes with glands and glandular hairs on the vegetative parts and corollas. Leaves alternate, oblanceolate, often closely appressed to the stem. Nutlets beige to reddish beige, oviod to broadly ovoid, verrucose. Echiochilon arabicum grows on stony ground on altitudes between 50–200 m above sea level in SE Yemen and SW Oman.

Echiochilon callianthum Lönn is a perennial herb or shrublet with blue and white zygomorphic flowers with an oblique corolla with purple veins in inflorescences terminating the leafy branches, vegetative parts and corollas covered with glandular hairs. Leaves alternate, oblanceolate. Nutlets beige to reddish beige, ovoid, verrucose to almost smooth. Echiochilon callianthum grows on sandy or rocky ground on altitudes between 30–900 m above sea level in SE Yemen and Oman.

Echiochilon pauciflorum (Stocks ex Wight) Långström & M.W.Chase is a perennial herb or shrublet with white actinomorphic flowers in few-flowered inflorescences terminating the leafy branches. Leaves alternate, with the basal pairs opposite, lanceolate. Nutlets brownish, ovoid with a vertical prolonged basal stipe, smooth to verrucose. Echiochilon pauciflorum grows on sandy and rocky ground in Pakistan and India on sea level up to ca 500 m above sea level. It has also been reported as a weed in fields.

SVENSK SAMMANFATTNING

Systematisk botanik är ett intressant och högst aktuellt forskningsområde. Somliga tror att det är ett avsomnat ämne som tog slut i och med Linnés död, men i tidningsartiklar från år 1999 och 2000 kunde man läsa att nu är Linnés gamla läror falsifierade och forskningen sker på ett helt annorlunda sätt och ger en revolutionerande ny klassifikation. Systematisk botanik är definitivt inget avsomnat ämne, men det har heller inte skett ett totalt falsifierande av Linnés forskning. Somligt från Linnés tid lever fortfarande kvar. Vi använder fortfarande hans system för namnsättning på latin, med ett släktnamn och ett artepitet. Varje växt har alltså två namn, ett som visar på släktestillhörighet och ett som specificerar arten. Exempelvis åkerförgätmigej, som kan hittas i hela Sverige, heter Myosotis arvensis där Myosotis betecknar hela förgätmigejsläktet och arvensis är artepitetet som talar om vilken art det är. Delar av den klassifikation som Linné skapade håller också fortfarande men det mesta har förändrats allt eftersom nya kunskaper har tillkommit. Med de moderna molekylära metoder man nu använder kan man få svar på frågor som forskarna länge har tvistat om och det är det som tidningarna har skrivit om,

26 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae men man får inte glömma att det skett en kontinuerlig utveckling mellan Linnés tid och nutid. Min forskning har varit koncentrerad på strävbladsväxterna (familjen Boraginace- ae) där man återfinner förgätmigej (Myosotis scorpioides), blåeld (Echium vulgare), hundtunga (Cynoglossum officinale), oxtunga (Anchusa officinalis), uppländsk vallört (Symphytum x uplandicum), lungört (Pulmonaria officinalis), gurkört (Borago officinalis), paddfot (Asperugo procumbens) och sminkrot (Lithospermum arvensis) m. fl. Strävbladsväxterna är ingen stor familj i Sverige men man finner fler av dem längre sö- derut i Europa, de tycker om värme och många av dem klarar av torka bra. Många av namnen ovan slutar med officinalis eller officinale, vilket visar att de har haft betydelse som medicinalväxter, t ex äkta vallört (Symphytum officinale) har använts till att läka benbrott och lungört (Pulmonaria officinalis) till att bota lungsjukdomar. Det finns även andra användningsområden än de rent medicinska. Gurkörtsblad kan användas i sallad (dock inte nyttigt i större mängder) och blommorna kan användas som dekoration i sallader mm. Sminkroten innehåller ett rött färgämne som använts som smink. Linné noterade under sina resor att bondflickorna i Hälsingland använde de färska rötterna av sminkroten (som på den tiden hade det föga smickrande namnet horlätta; lätta ≈ färga) som smink för att få röda läppar och kinder (Linné 1755). Namnen med olika djurs tungor associerar till de mer eller mindre strävhåriga bladen som faktiskt känns som dju- rens tungor. De växter jag har koncentrerat mig mest på är två släkten (Echiochilon med 15 arter och Ogastemma med endast en) i norra Afrika, på Afrikas horn och i Arabien, Iran, Pakistan och Indien. De är små halvökenbuskar och örter med tidigare osäker placering inom den underfamilj av strävbladsfamiljen som våra svenska strävbladsväxter represen- terar. Mycket nytt material hade samlats och en ny revision (genomgång av artavgräns- ningar) behövdes. Revisionen genomfördes och publicerades (bidrag I) och för att kunna placera släktena inom strävbladsfamiljen användes moderna molekylära (DNA) metoder (bidrag II och III). De molekylära metoder jag använt innebär att man renar fram (extraherar) DNA ur färskt eller torkat växtmaterial (t ex blad eller blommor), väljer ut en ca region av DNA man vill arbeta med, ofta mellan 500–2000 baser lång (baser = byggstenarna i DNA), och mångfaldigar den med hjälp av enzymer som bygger upp många nya likadana DNA kedjor som den man valt ut (PCR-amplifiering). Sedan gör man om mångfaldigandepro- ceduren men denna gång med ett inslag av färgade baser som har en ‘stoppkloss’ och därmed inte bygger vidare på kedjan. Detta leder till olika långa kedjor med en färgad stoppklossbas i änden på varje. Man gör så många kedjor att man får alla möjliga läng- der på kedjorna (från en till t ex 800 baser). Därefter kan man ordna kedjorna i storleks- följd och läsa av färgkoderna för baserna som har namnen A (adenin), T (thymin), G (gu- anin) och C (cytosin). Denna procedur kallas DNA-sekvensering. Sekvenser läggs sedan ihop till en datamatris. För vissa regioner, t ex ITS (bidrag III) måste man arbeta mycket med matrisen för att få underregioner av vald region att passa ihop (samman-

27 E. Långström jämka) så att de delar man jämför är jämförbara (homologa). Andra mer konserverade regioner, t ex atpB (bidrag II), är lättare att sammanjämka. Datamatrisen analyseras med speciella datorprogram som genom ‘fylogenetisk rekonstruktion’ producerar släktträd vilka i bästa fall visar hur evolutionen gett upphov till de arter man analyserat. Denna fylogenetiska rekonstruktion går till så att organismer som har gemensamma karaktärer grupperas tillsammans. Om man till exempel ska göra en fylogenetisk analys av vilka som är närmast släkt av en människa, en elefant och en husfluga kan man använda karaktären ryggrad som finns både hos människan och elefanten men saknas hos flugan och därmed grupperar ihop människan med elefanten. Avsaknad av karaktärer ger däremot ingen fylogenetisk information och alltså inget stöd för någon gruppering. Man kan till exempel inte använda karaktären avsaknad av snabel för att gruppera ihop människan med flugan. I verkligheten används många fler karaktärer för att bestämma släktskap. Olika karaktärer ger stöd för olika grupperingar och antalet möjliga hypoteser blir väldigt stort, men med hjälp av de datoriserade meto- derna försöker man leta rätt på det eller de träd som är kortast (därmed minst komplice- rat) och får ett antal grupperingar som resultat. I bästa fall får man fram ett välförgrenat (helt upplöst) släktträd. Den större analysen för att placera de två släktena Echiochilon och Ogastemma (bidrag II) visade att Echiochilon och Ogastemma (från Afrika och Arabien) placerade sig tillsammans med Antiphytum (från Amerika) på en egen gren. Detta släktskap hade tidigare föreslagits av Johnston (1957) och blev nu bekräftat med molekylära metoder. De tre släktena har alltid varit svårplacerade bland de befintliga tribusarna (grupper av släkten) och med de resultat Boraginaceae-analysen gav kunde en ny tribus, Echiochi- leae, upprättas. Analyserna av släktskap inom Echiochilon (bidrag III) gav en grund för att disku- tera biogeografin inom utbredningsområdet för Echiochilon. Man tror att Boraginaceae har sitt ursprung i Europa och det verkar som en förfader eller en tidig Echiochilon kom till Afrikas mediterranområde från Europa för att sedan sprida sig vidare österut ända till Indien. Det visade sig också att den ovanliga karaktären bilateralsymmetrisk blomkrona är ursprunglig inom släktet men det finns också radiärsymmetriska blommor högre upp i släktträdet.

ACKNOWLEDGEMENTS

I have had several supervisors during my time as PhD-student and they have helped in different ways and are greatly acknowledged for their help. Mats Thulin (paper I) has an unequalled knowledge of the plants of the world, Kåre Bremer (papers I–III) is fair, efficient and good at giving feed-back on manuscripts, Mark Chase (papers II, III) really took a genuine interest in my work and is a generous person creating a relaxed and welcoming atmosphere in his lab (group), and Bengt Oxelman (papers II, III) also has the talent to create a good group feeling, a great sense of humour and interesting music preferences.

28 Systematics of Echiochilon and Ogastemma (Boraginaceae), and the phylogeny of Boraginoideae

I want to thank all past and present friends and colleagues at the Department of systematic botany, the Herbarium and the Department of systematic zoology in Uppsala. My friends from the old times in ‘Alg & moss’: Mats Gustafsson, Mariette Manktelow, Lars-Gunnar Reinhammar and (†) Christine Mmari used to share my time off work as well as coffee and lunch breaks. Eva Andersson and Anna-Lena Fritz, with whom I shared my room, were appreciated discussion partners, and so were Bengt Sennblad, Ulf Swenson, Anders Backlund and Mats Hjertson (my nomenclature guru). My good friends from more recent times in EBC: Johanne Maad and Maria Backlund go way back and we have seen each other after working hours as well as shared lunch and coffee breaks. Dick Anderson is always up to something fun (party mate) and I have con- sulted him, Johannes Lundberg and Jesper Kårehed in all kinds of matters, scientific as well as non scientific. Kristina Articus is a most generous and inspiring person, Annika is my darling work-out partner, Henrik Lantz shares my gardening interest, Björn-Axel Beijer and I use to discuss our friends in common from Kew, Magnus Popp and Per Kornhall makes me laugh all the time, and Hanne Hegre Grundt came here from Tøyen, Norway, with sunshine and laughter in November/December 2001 when things were a bit heavy. My lunch break friends from systematic botany: Thomas Persson, Nahid Hei- dari, Edit Barkhordarian, Ulla Hedenquist (great sense of humour), Sylvain Razafi- mandimbison, Agneta Brandtberg-Falkman, Reija Dufva, Anders Nordin, Starri Heid- marsson, Per Erixon, Katarina Andreasen, Frida Eggens; and from Zoology: Afsaneh Ahmadzadeh, Thomas Jaenson, Isabel Sanmartín (biogeography guru), Hege Vårdal, Johan Nylander, Yonas Tekele, Per Ahlström, Katinka Pålsson, Lars Vilhelmsen, Johan Liljeblad and Christine Dahl, plus Mats Block and Stefan Ås, all contribute to the nice atmosphere here at Systematic Botany and Zoology in EBC. My friends and colleagues at Jodrell Laboratory in Kew, Surrey, made my stay in England absolutely wonderful. In particular I want to thank: Faridah Qamaruz Zaman, Martyn Powell and his Nicola, Jeffrey Joseph, Robyn Cowan, Mary Clare Sheahan (who helped me a lot by copying articles for me when I could not get to work), Liz Caddick, Conny Asmussen, Jonathan Steele, Zuoij, Mark Chase and Mike Fay. It was a great comfort having my Swedish friends Agneta Hörnell and Mats Wedin in England, who could give advice on how everything worked. We had many nice din- ners and excursions together, and their children Tove and Björn made friends with Arvid. I am happy they have moved back to Sweden again so I can see them more often and have a reason to visit Umeå. Bengt and Kristina Sennblad joined the ‘Swedish community’ in London after a while. Abdul Nasser Al-Gifri & Omar made my field trip to Yemen unforgettable. We got a good insight into peoples lives in Yemen, met kind and helpful people that let us spend the night and eat with them, and we had the most luxurious accommodations possible in the Oil camps of Canadian Oxy with air conditioning, shower, packed lunch boxes, din- ner served in the evening, a pool table and ice cream 24 hours a day. I want to thank my childhood friends Ylva and Katarina who always want to give me a good time. Ylva Ocklind and her husband Thomas Söderlund give parties as often

29 E. Långström as possible ;-), in Stockholm or in Grovstanäs by the Baltic sea. I am always invited and can always stay over. Katarina Stenbom, whom I have known since I was one year old, tries to inspire me to get fit, but her husband Robert counteract Katarina’s good inten- tions by feeding me with too much alcohol every time they give a party. An early friend from my Uppsala time is Anna Granholm who I enjoy seeing (with family and dog etc). I am happy to have found my friend from the Sollefteå time, Karin Bölje Svärd, who now lives in Uppsala. Sara Maad finally succeeded in dragging me back to my old choir Sine Nomine, and I really enjoy singing with you again. Gisela Krupke and Kent Wennman have really helped me and Arvid to a good life in Håga Village by being the best possible friends, and they help me by taking Arvid to the sports club every Monday. Mikael Lönn is a good father to Arvid and a good ex husband who has taken care of Arvid more than usual during the last weeks when I have had to work extra long hours. The families Ocklind, Risberg and Jönsson are thanked for being like extra family to me, always caring. Last but not least I want to thank my family. My mother Judith, my father Valter, my brother Lennart and his Caroline, and my son Arvid cannot be thanked enough. My father interested me in plants already at the age of three by teaching me three plants a day in Swedish and Latin, and he has always enjoyed discussing science with me. My mother gives me endless love and support through anything. My brother is a tease, and we have a lot of fun when we meet or talk on the phone, e-mail or SMS. He is the best possible brother, and he and Caroline care so much for Arvid. And my dear Arvid, what would I do without you? You give my life extra value, and I work hard during the days to get more time free to spend with you. I Love you all! Thank you.

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