Leaf anatomy and foliar trichomes in Heliotropiaceae and their systematic relevance

Leaf anatomy and foliar trichomes in Heliotropiaceae and their systematic relevance

Flora (2003) 198, 468–485 http://www.urbanfischer.de/journals/flora Leaf anatomy and foliar trichomes in Heliotropiaceae and their systematic relevan...

412KB Sizes 0 Downloads 50 Views

Flora (2003) 198, 468–485 http://www.urbanfischer.de/journals/flora

Leaf anatomy and foliar trichomes in Heliotropiaceae and their systematic relevance Nadja Diane*, Claudia Jacob & Hartmut H. Hilger Institut für Biologie, Systematische Botanik und Pflanzengeographie, FU Berlin, Altensteinstraße 6, D-14195 Berlin, Germany Submitted: Mar 20, 2003 · Accepted: Jun 11, 2003

Summary Leaf anatomy and the distribution of foliar trichome types of 65 species of Heliotropiaceae have been investigated. The aim was to evaluate the systematic relevance of their diversity as compared to recent findings of systematic relationships within the family. The results of leaf anatomy patterns, especially venation, vascular system, various foliar trichome types, and localization of crystals are of surprisingly high systematic value and prove the actual proposed subdivision of this family based on molecular results. Each main clade identified in the molecular studies is well characterized on the basis of leaf anatomy. Key words: Boraginaceae, Boraginales, Heliotropiaceae, leaf anatomy, systematics, trichomes.

Introduction The Heliotropiaceae1 are a family of nearly 450 species. They are easily recognized by their partial inflorescences of scorpioid cymes and by the morphology of the highly modified stigmatic head in the flower. The subdivision of the family is mainly based on generative characters, vegetative ones are scarcely considered. Heliotropiaceae are distributed worldwide mainly in tropical and subtropical regions, and in dry, disturbed areas of warm-temperate to semi-arid regions. Four species are halophytes. Although the indumentum is characteristic and namegiving for the Boraginales (“Asperifolieae”, in German : Rauhblattgewächse), little is known on the structural diversity of the trichomes and the leaf ana-

1

Due to the inclusion of Lennoaceae and Hydrophyllaceae into the old Boraginaceae (Yatskievych et al. 1986; Ferguson 1999; Gottschling et al., 2001), we treat this taxon as “Boraginales”, and the former subfamilies of Boraginaceae as families of their own – a taxonomic status that has been discussed for nearly 200 years.

tomy in general. Most descriptions have been published in the frame of general studies of the Boraginales, many of them decades or even more than a century age old (e.g., Schibler 1887; Solereder 1908; Kragge 1911; Metcalfe & Chalk 1950). Actual investigations of leaf anatomy within Boraginaceae s. str. tribe Boraginaea are of Selvi & Bigazzi (2001). Within Heliotropiaceae, only few detailed studies were made by Bider (1935) of two species of Heliotropium, by Frohlich (1978) of leaf morphology and anatomy of 17 Mexican species of Heliotropium section Orthostachys, by Kumar & Rao (1994) of foliar epidermology of 6 species of Heliotropium, and by DiFulvio (1982) of leaf anatomy of the monotypic Argentinian endemic Ixorhea. A phylogenetic analysis of Heliotropiaceae based on molecular data has shown that the old generic limits and characters are no longer valid (Diane et al. 2002; Hilger & Diane 2003). This is summarized in Table 1. While Förther (1998), in his basic revision of the Heliotropiaceae, still recognized 8 genera (Table 1 column left side: Argusia, Ceballosia, Heliotropium, Hilgeria, Ixorhea, Nogalia, Schleidenia, Tournefortia), the most important result of the molecular studies was

* Corresponding author: Nadja Diane, Institut für Biologie, Systematische Botanik und Pflanzengeographie, FU Berlin, Altensteinstraße 6, D-14195 Berlin, Germany, e-mail: [email protected] 468

FLORA (2003) 198

0367-2530/03/198/06-468 $ 15.00/0

that Heliotropium and Tournefortia, in their old circumscription, are para- and polyphyletic, respectively (Table 1 columns right side). Therefore, Heliotropium sect. Orthostachys and Tournefortia sect. Cyphocyema should be recognized as genera of their own. The oldest available names are Euploca (Nuttall 1837) for the former and Myriopus (Small 1933) for the latter taxon. Schleidenia, Hilgeria, Argusia, Ceballosia, and Nogalia fall into synonymy of either Euploca or Heliotropium. Tournefortia sect. Tournefortia is within Heliotropium s.str. in sistergroup relationship to a HELIOTROPIUM

I2 clade, and represents the woody (lianas, small trees) grade within herbaceous to subshrubby HELIOTROPIUM I species of the New World. The informal HELIOTROPIUM II clade represents the Heliotropium species of the Old World. The aim of the present study, covering 65 species, is 2

Combinations of Heliotropium s.str. have not yet been published. To avoid nomenclatural confusion, informal clades are printed in capital letters.

Table 1. Phylogeny of Heliotropiaceae based on molecular data (Diane et al. 2002; Hilger & Diane 2003).

FLORA (2003) 198

469

a) to add more data of leaf anatomy and especially indumentum characters for the family, thus b) to evaluate the systematic relevance and/or adaptive value of the morphological and anatomical diversity.

Materials and methods Representatives of 65 Heliotropiaceae of all genera and sections of Tournefortia and Heliotropium have been investigated, with the exception of the small Heliotropium sections Monimantha and Rutidotheca of the HELIOTROPIUM II clade. They are listed in Table 2, the species are arranged in alphabetical order of the clades identified according Diane et al. (2002) and Hilger & Diane (2003), see also Table 1.

The leaves were fixed with AFE (acetic acid-formalin-ethanol). For light microscopy, the leaves were dehydrated with FDA (formaldehyde-dimethyl-acetal, Gerstberger & Leins 1978) and, in most cases, embedded in paraplast via a graded ethanol-tertiary butanol series and microtome sectioned, or hand sections with a razor were made. Photographs of safranine-astra blue stained serial sections (10 µm), or sections bleached with chloral hydrate (saturated aqueous solution) and astra blue stained hand sections were taken with a Leitz Dialux 20 microscope and a Canon EOS D30 digital camera. For identification of crystal shapes, crossed polarising filters were used. Calcium oxalate and calcium carbonate were distinguished as described by Horner & Wagner (1992). For SEM studies, the material was dehydrated in a graded ethanol series, critical point-dried with CO2, sputter-coated with gold, and photographed with a LEO 430 Scanning Electron Microscope.

Table 2. List of species investigated, with location of voucher specimens, arranged in alphabetical order of the clades/taxa according to Table 1. (BSB) = Institut für Biologie, Systematische Botanik und Pflanzengeographie, Freie Universität Berlin, Germany; (M) = Botanische Staatssammlung München, Germany, (CANB) = Australian National Herbarium, Canberra, Australia Clade – Taxon ARGUSIA – Argusia A. sibirica (L.) Dandy (as Tournefortia sibirica L.) A. sogdiana (as Messerschmidia sogdiana (Bunge) H. Riedl) CEBALLOSIA – Ceballosia C. fruticosa (L.f.) G. Kunkel ex Förther var. angustifolia (Lam.) G. Kunkel ex Förther EUPLOCA Heliotropium sect. Orthostachys H. bursiferum Wr. ex Griseb. H. campestre Griseb. H. chrysanthum Phil. H. convolvulaceum (Nutt.) A. Gray H. humifusum Kunth H. mendocinum Phil. H. ovalifolium Forssk. H. procumbens Mill. H. rariflorum Stocks subsp. heroense (Schinz) Verdc. H. strigosum Willd. H. styotrichum Craven H. tenuifolium R.Br. Hilgeria H. hypogaea (Urban & Eckman) Förther Schleidenia H. antillanum Urb.

Collection Skvortsov 29.06.1963 s.n. (M), Russia Nikitin & Iwanow 28.?.1974 s.n. (M), Turkmenistan

Hilger anno 1986 s.n. (BSB, AFE-coll.), Spain (Tenerife)

Hilger Cuba 99/25 (BSB, AFE-coll.), Cuba N. De la Barra s.n. (BSB), Bolivia Hilger et al. Arg 95/92 (BSB, AFE-coll.), Argentina Hilger & Hofmann USA 92/77 (BSB, AFE-coll.), USA (California) Hilger Cuba 99/1 (BSB, AFE-coll.), Cuba Hilger et al. Arg 95/77 (BSB, AFE-coll.), Argentina Hilger Nam 93/5 (BSB, AFE-coll.), Namibia Hilger et al. Arg 95/26 (BSB, AFE-coll.), Argentina Hilger Nam 93/23 (BSB, AFE-coll.), Namibia Hilger Kenya 91/7 (BSB, AFE-coll.), Kenya Craven 9687 (CANB), Australia Craven 9688 (CANB), Australia Briggs 226 (M), Cuba Hilger Cuba 99/26 (BSB, AFE-coll.), Cuba

HELIOTHAMNUS – Heliotropium sect. Heliothamnus H. adenogynum I. M. Johnst. Cano 10058 (M), Peru H. arborescens L. commercial cultivated plant (BSB, AFE-coll.), not indicated H. incanum Ruiz & Pav. Weigend et al. 5818 (BSB, AFE-coll.), Peru H. mandonii I. M. Johnst. Weigend, cult. Botanical Garden of München-Nymphenburg, Germany 01.09.1997 (BSB, AFE-coll.), Ecuador H. rufipilum (Benth.) I. M. Johnst. Förther et al. 10252 (MSB, AFE-coll.), Guatemala H. submolle Klotzsch Weigend & Skrabal 5890 (BSB, AFE-coll.), Peru 470

FLORA (2003) 198

Table 2. (continued) Clade – Taxon

Collection

HELIOTROPIUM I – Heliotropium species of the New World H. amplexicaule Vahl Hilger et al. Arg 95/70 (BSB, AFE-coll.), Argentina H. angiospermum Murray Hilger Cuba 99/44 (BSB, AFE-coll.), Cuba H. curassavicum L. Hilger et al. Arg 95/82 (BSB, AFE-coll.), Argentina H. elongatum (Lehm.) I. M. Johnst. Hilger et al. Arg 95/18 (BSB, AFE-coll.), Argentina H. indicum L. Hilger Cuba 99/22 (BSB, AFE-coll.), Cuba H. krauseanum Fedde Weigend & Förther 97/727 (BSB), Peru H. spec. nov. aff. krauseanum Fedde Weigend et al. 5887 (BSB, AFE-coll.), Peru H. microstachyum Ruiz & Pav. Hilger et al. Arg 95/54 (BSB, AFE-coll.), Argentina H. nicoteanaefolium Poir. Hilger et al. Arg 95/56 (BSB, AFE-coll.), Argentina H. paronychioides A. DC. Hilger et al. Arg 95/90 (BSB, AFE-coll.), Argentina H. patagonicum (Speg.) I. M. Johnst. Weigend et al. 5940 (BSB, AFE-coll.), Argentina H. pinnatisectum R. L. Pérez-Mor. Weigend et al. 5901 (BSB, AFE-coll.), Argentina H. transalpinum Vell. Hilger et al. Arg 95/23 (BSB, AFE-coll.), Argentina H. veronicifolium Griseb. Hilger et al. Arg 95/29 (BSB, AFE-coll.), Argentina HELIOTROPIUM II Heliotropium species of the Old World H. aegyptiacum Lehm. H. arbainense Fresen. H. asperrimum R. Br. H. ciliatum Kaplan H. digynum (Forrsk.) Asch. ex C. Chr. H. erosum Lehm. H. europaeum L. H. giessii Friedr.-Holzh. H. hirsutissimum Grauer H. nelsonii C. H. Wright H. oliverianum Schinz H. suaveolens M. Bieb. H. supinum L. H. zeylanicum (Burm. f.) Lam. Nogalia N. drepanophyllum (Baker) Verdc. IXORHEA – Ixorhea I. tschudiana Fenzl MYRIOPUS – Tournefortia sect. Cyphocyema T. psilostachya Kunth T. salzmannii DC. T. volubilis L. TOURNEFORTIA – Tournefortia sect. Tournefortia T. argentea L. f. T. bicolor Sw. T. glabra L. T. gnaphalodes (L.) Kunth T. hirsutissima L. T. luzonica I. M. Johnst. T. microcalyx (Ruiz & Pav.) I. M. Johnst. T. rollotii Killip T. ternifolia Humb., Bonpl. & Kunth

Schultka 1995/5 (BSB, AFE-coll.), Kenya Hilger 23. 05. 1980 s.n. (BSB, AFE-coll.), Israel Craven 9671 (CANB), Australia Hilger Nam 93/10 (BSB, AFE-coll.), Namibia Hilger Israel 94/23 (BSB, AFE-coll.), Israel Hilger 1986 s.n. (BSB, AFE-coll.), Spain (Tenerife) cult. Botanical Garden Berlin-Dahlem, Germany (BSB, AFE-coll.), not indicated Hilger Nam 93/3 (BSB, AFE-coll.), Namibia Kagiampaki 24.07.2000 s.n. (BSB), Greece (Crete) Hilger Nam 93/6 (BSB, AFE-coll.), Namibia Hilger Nam 93/16 (BSB, AFE-coll.), Namibia Raus & Schiers 19040 (B), Greece Hilger anno 1985 s.n. (BSB, AFE-coll.), Italy: Sicily Hilger Kenya 91/3 (BSB, AFE-coll.), Kenya Kilian 6603 (BSB, AFE-coll.), Jemen Hilger Arg 95/65 (BSB, AFE-coll.), Argentina Weigend & Weigend 2000/339 (M), Peru Franca & Melo 16843 (M), Brasil cult. Botanical Garden Berlin-Dahlem, Germany (BSB, AFE-coll.), not indicated Panahi 28.04.1933 s.n. (B), Hawaii Hilger Cuba 99/12 (BSB, AFE-coll.), Cuba Howard & Howard 8604 (B), Dominican Republic Hilger Cuba 99/34 (BSB, AFE-coll.), Cuba Köhler 12.04.1995 s.n. (BSB, AFE-coll.), Cuba Liede 3302 (BSB), Philippines Weigend & Dostert 97/5 (M), Peru Schnetter 23.02.1983 (BSB, AFE-coll.), Colombia Weigend et al. 5676 (BSB, AFE-coll.), Peru

FLORA (2003) 198

471

Results The distribution of anatomical characters observed is shown in Table 3. The species are listed according to their clades in Table 1.

General leaf morphology Generally, phyllotaxis is alternate, rarely pseudo-opposite, or pseudoternate. The leaves are simple and usual-

ly petiolate, rarely sessile, and estipulate. The lamina is linear, elliptic, or ovate to subcircular, the margin mostly entire, rarely sinuate to pinnatifid, repand, crenate, or dentate, usually flat or sometimes revolute. The leaf apex is acute or acuminate, rarely rounded. The leaf base is cuneate or decurrent, rarely truncate to subcordate. The leaf surface is planar or bullate; a dense indumentum is usually present, and only a few species are glabrous.

Table 3. Leaf anatomy and distribution of foliar trichome types in the Heliotropiaceae. H, habitat (1 = semi-arid with seasonal dry periods, 2 = Andean scrub communities, 3 = humid tropics, 4 = halophytic, coastal and desert sands); Lv, leaf venation (P = hyphodromous venation, F = brochidodromous I, secondaries and tertiaries weakly prominent, S = brochidodromous II without prominent tertiaries, S+ = brochidodromous II, with only a few prominent tertiaries, T = brochidodromous III, prominent tertiaries are opposite percurrent); La, leaf anatomy (B = bifacial, B+ = subbifacial, I = isobilateral, one-layered, I2 = isobilateral, two-layered, Ih = isobilateral, heterogenous, Im = isobilateral, multilayered, S = succulent); Sd, Stomata distribution (H = hypostomatous, A = amphistomatous, H+ = hypostomatous, but with stomata present on the adaxial surface at strongly reduced density); Ko, Kranz-chlorenchyma organization in leaves (+ = present, – = not present); Gt, glandular trichomes (+ = present, – = not present); Tt, foliar trichome types (s = slender, sg = strigose, co = conical, ta = twoarmed, ph = papillose-hispid, – = glabrous); Ct, lithocysts types (ac = multicellular lithocyst complexes, tc = trichome lithocysts, ic = unicellular lithocysts, trichome tip reduced, – = without lithocysts); Cy, idioblasts with crystals (s = crystalsand in mesophyll, d = crystal druses in mesophyll, d+ = crystal tubes in mesophyll, n = crystals needles in epidermis, rarely additionally present in mesophyll or along the bundles, – = crystals lacking). Clade

H

Lv

La

Sd

Ko

Gt

Tt

IXORHEA I. tschudiana

2

F

Ih

A



(+)



MYRIOPUS T. psilostachya T. salzmannii T. volubilis

1 1 1

F F F

B B B

A A H

– – –

– – –

– – –

1 1 1 1 1 1 1 1 1

P S P P P P P P P

I B B I I I B B B+

A A A A A A A A A

– – + + + + + + +

– – – – – – – – –

1 1 1

P P P

B+ B+ I

A A A

+ + +

1

P

B

A

1

P

?

2 2 2 2

T T T T

B B B B

EUPLOCA Heliotropium sect. Orthostachys (subsect.) H. ovalifolium (Ebracteata) H. procumbens (Ebracteata) H. campestre (Ebracteata) H. chrysanthum (Bracteata) H. mendocinum (Bracteata) H. convolvulaceum (Bracteata) H. styotrichum (Bracteata) H. tenuifolium (Bracteata) H. rariflorum subsp. hereroense (Bracteata) H. strigosum (Bracteata) H. bursiferum (Bracteata) H. humifusum (Bracteata) Schleidenia H. antillanum Hilgeria H. hypogaea HELIOTHAMNUS H. mandonii H. arborescens H. rufipilum H. incanum 472

FLORA (2003) 198

Ct

Cy









d

d+

s s s

ac – ac

– – –

– – –

n – –

– – –

– – –

sg sg sg sg sg sg – sg sg

– s – – – – s – –

ac ac – – – – – – ac

– – – – – – – – –

– – – – – – – – –

– – – – – n – – –

– – – – – – – – –

– – – – – – – – –

– – –

sg sg sg

– – –

ac – –

– – –

– – –

– – –

– – –

– – –

+



sg









n





A

+



sg















H+ H+ H+ H+

– – – –

+ + + +

co co co co

– – s s

– – – –

tc tc tc tc

– – – –

– – – –

d d d d

d+ – – d+

Table 3. (continued) Clade

H

Lv

La

Sd

Ko

Gt

Tt

H. submolle H. adenogynum

2 2

T T

B B

H+ H+

– –

+ +

co co

s –

– –

tc tc

– –

– –

d d

d+ –

HELIOTROPIUM I (section) H. krauseanum (Cochranea) H. spec. nov. (Cochranea) H. nicoteanaefolium (Heliotrophytum) H. amplexicaule (Heliotrophytum) H. paronychioides (Plagiomeris) H. pinnatisectum (Plagiomeris) H. transalpinum (Coeloma) H. veronicifolium (Coeloma) H. microstachyum (Hypsogenia) H. elongatum (Tiaridium) H. indicum (Tiaridium) H. angiospermum (Schobera) H. patagonicum (Platygyne) H. curassavicum (Platygyne)

1 1 1 1 1 1 1 1 1 1 1 1 5 5

T S T S S S T T S T T T P P

B B B+ I2 B+ I2 B B+ Im B B+ B S S

H H A A A A A A A A A A A A

– – – – – – – – – – – – – –

+ + + – – – + + – – – + – –

co co co co co co co co co co co co – –

– – – – – – ta ta – – – – – –

– – – – – – – – – – – – – –

tc tc tc tc tc tc tc tc tc tc tc tc – –

– – – – – – – ic ic ic ic ic ic ic

– – – – – – – – – – – – – –

d d d d – – d – – – – – – d

– – – – – – – – – – – – – –

TOURNEFORTIA T. gnaphalodes T. argentea T. rollotii T. glabra T. hirsutissima T. bicolor T. ternifolia T. microcalyx T. luzonica

5 5 3 3 3 3 3 3 3

P T T T T T T T T

S ? B B B B B B B

A A H H H H H H H+

– – – – – – – – –

+ + + + – – + + –

– co co co co co co co co

s – s – – – – – –

– – – – – – – – –

– tc tc tc tc tc tc tc tc

– – ic ic ic ic ic ic ic

– – – – – – – – –

d d – – – – – – –

s – – – – – – – –

CEBALLOSIA C. fruticosa var. angustifolia

1

S

B+

A



+

co



ac

tc

ic



d



ARGUSIA A. sibirica A. sogdiana

1 1

S S

B+ ?

A A

– –

+ –

co ph

– s

– ac

tc –

– –

– –

d d

s d+

1 1 1 1 1 1 1 1 1 1 1 1 1 1

S S S T T S S S S S S+ S+ S+ S

B+ B+ B+ B+ B+ B B+ I I I ? I B+ B

A A A A A A A A A A A A A A

– – – – – – – – – – – – – –

– + + + + – – + – – – + + +

– ph ph ph ph ph ph ph ph ph ph ph ph ph

s s – – – s – – s s – – – –

ac ac ac ac ac ac ac – – – – – – –

– – – – – – – – – – – – – –

– – – – – – – – – – – – – –

– – – – – – – – – – – – – –

d d d d d – d d d d – d – d

d+ d+ s d+ d+ – s d+ d+ – – – – –

1

S

B+

A



+

ph















HELIOTROPIUM II H. erosum (Pterotropium) H. asperrimum (Pterotropium) H. zeylanicum (Zeylanica) H. hirsutissimum (Odontotropium) H. arbainense (Odontotropium) H. supinum (Chamaetropium) H. nelsonii (Pseudocoeloma) H. ciliatum (Pseudocoeloma) H. aegyptiacum (Heliotropium) H. oliverianum (Heliotropium) H. suaveolens (Heliotropium) H. giessii (Heliotropium) H. europaeum (Heliotropium) H. digynum (Pleurolasia) Nogalia N. drepanophyllum

Ct

Cy

FLORA (2003) 198

473

Fig. 1. (a)–(d) Leaf venation; (a) Heliotropium chrysanthemum; (b) Tournefortia volubilis; (c) H. digynum (left side), H. europaeum (right side); (d) T. microcalyx. Scale bars: 1 cm.

Fig. 2. (a)–(c) Leaf anatomy; (a) H. antillanum: bifacial leaf and Kranz-type leaf anatomy; (b) H. europaeum: predominantly bifacial leaf; (c) H. giessii: isobilateral leaf. Scale bars: 50 µm.

Venation (Table 3 column 2 Lv ; Figs. 1a – d) In general, leaf venation is brochidodromous, differing in the prominence of secondary and tertiary veins on the abaxial surface, the orientation of the tertiary veins, or in the shape of the intercostal areas delimited by the secondary and tertiary veins. 474

FLORA (2003) 198

Four types of venation are found. The description follows the terminology of Hickey (1973) and Ash et al. (1999). 1 – hyphodromous (abbrevation in Table 3 and 4: P, Fig. 1a). Only one prominent midvein is visible on the abaxial leaf surface. All higher order veins are weakly developed and immer-

sed in the mesophyll. This condition is found generally in EUPLOCA [with the only exception of H. procumbens (see type 3)], and in the succulent leaves of the halophytic H. curassavicum, and H. patagonicum of HELIOTROPIUM I, and T. gnaphalodes of the TOURNEFORTIA clade. 2 – brochidodromous I (F, Fig. 1b). Several very fine and barely prominent secondary veins curve near the margin and join in a series of arches. The tertiaries are very fine, not prominent, randomly reticulate to weakly alternate percurrent, enclosing irregular four- to five-sided intercostal areas. This type of venation is found in MYRIOPUS and IXORHEA. 3 – brochidodromous II (S, Fig. 1c). Several prominent secondary veins curve near the margin and join in a series of arches. Near the margin the secondaries are often immersed in the mesophyll (Fig. 1c left, S). Most or all tertiaries are not prominent. If elevated tertiaries are present they are opposite to additional alternate percurrent (Fig. 1c right, S+) and enclosing regular intercostal areas. This venation type is found in most species of HELIOTROPIUM II and several species of HELIOTROPIUM I, as well as in ARGUSIA and CEBALLOSIA, in EUPLOCA only in H. procumbens. 4 – brochidodromous III (T, Fig. 1d). Like as type 3, with several prominent secondary veins curve near the margin and join in a series of arches. All tertiary veins are prominent and opposite percurrent, i.e., the tertiaries link neighboring secondaries in parallel rows without branching, in addition some tertiaries may alternate percurrent. Characteristic are the regular intercostal areas delimited by the tertiaries. Veins of higher order are also mostly elevated, leading to a characteristic rugose surface pattern of the lamina. This venation type is found in all species of HELIOTHAMNUS, many species of HELIOTROPIUM I, and all species of TOURNEFORTIA, except the succulent T. gnaphalodes (see type 1). Within HELIOTROPIUM II this venation type is found in H. hirsutissimum and H. arbainense.

Leaf anatomy (Table 3 column 3 La ; Figs. 2a – c) In general, the leaves of Heliotropiaceae are bifacial, subbifacial or isobilateral with a one-layered palisade tissue. Four additional types are restricted to a few species. 1 – bifacial (B, Fig. 2a). This is the most common type, with a single adaxial layer of palisade parenchyma. Bifacial leaves are found in many species of EUPLOCA, HELIOTROPIUM I and II (species see Table 3). In MYRIOPUS, HELIOTHAMNUS, and TOURNEFORTIA it is the only leaf type with the exception of the halophytic T. gnaphalodes. 2 – subbifacial (B+, Figs. 2b, 4a, g). This differs from type 1 only by an additional, often discontinuous, one-layered palisade or palisade-like tissue on the abaxial side. The abaxial palisade cells are shorter than adaxial ones. This leaf type is found in EUPLOCA, HELIOTROPIUM I and II (species see Table 3), CEBALLOSIA and Argusia sibirica. 3 – isobilateral, one-layered (I, Fig. 2c). With a well developed and more or less uniform, one-layered palisade tissue on both sides. This type is found in EUPLOCA and in HELIOTROPIUM II (species see Table 3).

4 – isobilateral, two-layered (I2). An adaxial, two-layered palisade tissue, and a two-layered palisade-like tissue of shorter cells on the abaxial side is present in HELIOTROPIUM I (H. pinnatisectum, H. amplexicaule). 5 – isobilateral, heterogenous (Ih, Fig. 4f). Restricted to IXORHEA and characterized by a two-layered adaxial and a one-layered abaxial palisade parenchyma.

In types 1–5 the spongy mesophyll is well developed and consists of isodiametric cells. 6 – isobilateral, multilayered (Im, Fig. 4c). Characterized by a 6-layered palisade parenchyma, lacking spongy mesophyll, and found only in H. microstachyum (HELIOTROPIUM I). 7 – succulent (S, Fig. 4b). The succulent leaves are isobilateral without a differentiation in chlorenchyma and water-storage tissue (all mesophyll cells are able to store water). They are found in the halophytic species of HELIOTROPIUM I (H. curassavicum, H. patagonicum). A differentiation in a two-layered palisade tissue each side and a water-storage tissue in the center parts of the leaves found is only in halophytic TOURNEFORTIA (T. gnaphalodes).

Epidermis and stomata (Table 3 column 4 Sd) In all taxa investigated the leaf epidermis consists of a single cell layer. The cells are polygonal, and the adaxial epidermal cells are usually slightly larger than those on the abaxial side. The anticlinal cell walls are straight, irregular to sinuous, and of varying shape on both surfaces. The cuticle is thin (5 to 10 µm), in some EUPLOCA species thickened up to 20 or 25 µm. Ornamentations are lacking. Most of the species have amphistomatous leaves (A) with nearly homogenous stoma distribution on either side (species see Table 3). Hypostomatous leaves (H) are found in MYRIOPUS (T. volubilis), HELIOTROPIUM I (sect. Cochranea), and the non-halophytic TOURNEFORTIA. In HELIOTHAMNUS and T. luzonica (TOURNEFORTIA) there are some stomata present on the adaxial side (H+). The stomata are always anomocytic (Ranunculaceous type), randomly orientated, oval in shape and variable in size, with (3–) 5 (–6) accessory cells (Fig. 4d). The stomata are on the same level of the epidermis except in IXORHEA and HELIOTROPIUM I (sect. Cochranea) where they are elevated (Figs. 4e, f) in a chimney-like form, and the accessory cells form a ring.

Vascular system and Kranz-type leaf anatomy (Table 3 column 5 Ko) All species investigated exhibit collateral bundles with adaxial xylem. Collenchymatic tissue is frequently associated to the main veins. Veins of higher order are surrounded by parenchymateous single-layered bundle FLORA (2003) 198

475

Fig. 3. (a)–(l) key of all trichome types found in Heliotropiaceae. Scale bars: 50 µm.

sheaths and their cells are mostly with, rarely without chloroplasts. Kranz-type leaf anatomy is present in all species of EUPLOCA except H. ovalifolium and H. procumbens. The bundle sheath cells contain a large number of chloroplasts centripetally appressed to the cell wall (Figs. 2 a, 4 g). There are no additional radially arranged mesophyll cells around the veins (Figs. 2a, 4g). 476

FLORA (2003) 198

Foliar trichome types and lithocysts (Figs. 3a – l; Table 3 columns 6 – 8, Gt, Tt, Ct) In most taxa, both leaf surfaces are more or less densely covered with different types of uniseriate trichomes forming an indumentum of variable texture and density. Only the halophytic and succulent leaves of H. patagonicum and H. curassavicum (HELIOTROPIUM I)

Fig. 4. FLORA (2003) 198

477

1 – glandular, multicellular (Fig. 3a). Trichomes consist of two or three stalk cells, and a terminal more or less spherical gland cell (Fig. 4h). Glandular trichomes occur only in combination with other trichome types, and are thus always part of a heterotrichous indumentum. An exception is IXORHEA where it is the only trichome type. Usually they are much smaller than the eglandular, unicellular trichomes. They are found in HELIOTHAMNUS, some species of HELIOTROPIUM I and II, and TOURNEFORTIA, CEBALLOSIA, and Argusia sibirica. They are lacking in MYRIOPUS and EUPLOCA. The isotrichous IXORHEA is characterized by having sessile, one-celled secretory glands (Figs. 4e, f). 2 – unicellular, unbranched trichomes of slender shape (s, Fig. 3f). The trichomes form a villous to lanate indumentum. They are usually long and slender, with unswollen base, smooth or sculptured with small papillae (Fig. 5a), and are not mineralized. The surrounding epidermis cells show no modification. This trichome type predominantly occurs as a part of a heterotrichous, rarely part of isotrichous indumentum in MYRIOPUS, EUPLOCA, HELIOTHAMNUS, TOURNEFORTIA, HELIOTROPIUM I, and Argusia sogdiana. 3 – unicellular, unbranched trichomes on a pedestal of distinctly enlarged epidermis cells (sg, Fig. 3e). The trichomes are strigose, uniform in length, thick-walled, with a slightly swollen base, and covered with blunt, conical papillae. The surrounding epidermis cells are enlarged and elevate the trichome base above the epidermis. These bases are strongly

curved so that the trichomes are appressed to the epidermis. All trichome tips are pointing to the leaf apex (Figs. 5b, c). This type is only found in EUPLOCA. 4 – unicellular, unbranched, cystolith bearing trichomes (co, tc, Figs. 3i–k). Trichomes are strongly conical (co), born on a bulbous base, and smooth or covered with papillae of different shape and density. The indumentum is anisotrichous, rarely uniform in length on either side of the lamina surface (Fig. 5d). Often the trichomes are reduced to small tips with strongly swollen bases (Figs. 3k, 5e). The surrounding epidermis cells are partly enlarged, but they never elevate the trichome base above the epidermis. Their swollen bases are only slightly curved so that the shafts are not appressed to the epidermis (Figs. 5d, e). The basal part of the enlarged bulbous base is deeply embedded in the mesophyll. In most cases the trichomes of this type bear a cystolith of calcium carbonate of usually mussel- (Figs. 3k, 5h) or rarely bunch-shape (Figs. 5i, 6e) in their bulbose bases. The term trichome lithocyst (tc) is then applied. The cystolith is indistinctly stalked when the trichome is well developed (Figs. 3k, 4c, 5h), and distinctly stalked when the trichome apex is reduced to a tip (Fig. 3k, 5i). This trichome type is found in species of HELIOTHAMNUS, HELIOTROPIUM I, TOURNEFORTIA, CEBALLOSIA, and Argusia sibirica. In HELIOTHAMNUS the trichomes are predominantly uniform in length and rarely contain cystoliths in the bases. In HELIOTROPIUM II, sporadically type 4-like trichomes occur on veins or leaf margins in addition to trichome of type 5. They are uniform in length and never bear a cystolith in their bases. 5 – unicellular, unbranched trichomes with strongly swollen base (ph, Figs. 3g, h). The spear-like trichomes form a papillose-hispid indumentum of uniform length. They are born on strongly swollen bases and have a smooth surface (Figs. 5f, g). The trichome tips and enlarged bases are sometimes mineralized. The deposits of calcium carbonate in the bulbose bases are sometimes cup-shaped (Figs. 3h, 5j), but never form a compact cystolith as described in type 4. The trichomes may be erect (Figs. 3h, 5g) to sharply curved above their bases, but they are not appressed to the epidermis (Figs. 3g, 5f). The surrounding epidermis cells are not modified.

Fig. 4. (a) Heliotropium zeylanicum: predominantly bifacial leaf and spear-like trichome of type 5 (ph) without cystolith; (b) H. patagonicum: isobilateral succulent leaf; (c) H. microstachyum : isobilateral leaf, 6-layered palisade parenchyma, no spongy parenchyma, trichome of type 4 (tc) with a well developed basal unstalked cystolith; (d) H. transalpinum: abaxial anomocytic stoma; (e) Ixorhea tschudiana: abaxial leaf surface with elevated stomata and one-celled secretory glands; (f) I. tschudiana: isobilateral leaf with twolayered adaxial palisade parenchyma, elevated stomata, and secretory gland; (g) H. rariflorum: predominantly bifacial leaf with Kranz-type leaf anatomy; (h) H. angiospermum: glandular, multicellular trichome of type 1. Method of observation: (a–c, f, g) LM, (d, e, h) SEM. Scale bars: (d, h) 10 µm; (e) 50 µm; (a–c, f, g) 100 µm.

Fig. 5. (a) Heliotropium incanum: long and slender trichomes of type 2 (s); (b) Hilgeria hypogaea: trichome of type 3 (sg) on pedestal of distinct, enlarged cells; (c) Heliotropium rariflorum: trichome of type 3 (sg); (d) H. mandonii: trichome of type 4 (co, tc) of different lengths; (e) H. amplexicaule: trichome of type 4 (co, tc) with strongly reduced trichome tip; (f) H. europaeum: trichome of type 5 (ph) with sharply curved trichome tip; (g) H. suaveolens: trichome of type 5 (ph) with erect trichome tip; (h) H. paronychioides: trichome of type 4 (tc) with well developed trichome tip and indistinctly stalked cystolith; (i) H. amplexicaule: trichome of type 4 (tc) with reduced trichome tip and distinctly stalked cystolith; (j) H. giessii: trichome of type 5 (ph) with cup shaped mineralization at the bulbose base. Method of observation: (a–g) SEM; (h–j) LM. Scale bars: (h–j) 10 µm; (d, f) 50 µm; (a–c, e, g) 100 µm.

have glabrous leaves. Multiseriate trichomes lack completely. Eight types of trichomes and lithocysts were identified. All are found on either side of the lamina, but with different density. Sometimes there are gradual transitions between trichome types especially on veins, leaf margins, and petioles. On veins the trichomes are often enlarged and the trichome type is indistinct. For an unambiguous determination of the trichome type we present a key for all types found in the family (Figs. 3a – l). The terminology follows Theobald et al. (1979) and Harris & Harris (1994).

478

FLORA (2003) 198

Fig. 5. FLORA (2003) 198

479

Fig. 6. 480

FLORA (2003) 198

This trichome type is restricted to HELIOTROPIUM II (with the single exception of H. erosum showing only trichomes of type 2), and it is intermediately developed, covered with papillae on surface, like trichomes of type 4, in Argusia sogdiana (Fig. 6a). 6 – unicellular, two-armed (ta, Fig. 3d). Trichomes are regularly to irregularly two-armed, with a bulbous base and covered with conical papillae (Fig. 6b). The surrounding epidermis cells are not modified. This trichome type is only found in the heterotrichous indumentum of H. amplexicaule and H. veronicifolium of HELIOTROPIUM I together with trichomes of type 4 (Figs. 3i–k). 7 – unicellular lithocysts, with reduced trichome tip (ic, Fig. 3l). These idioblasts can be recognized in surface view as cells with a small diameter in the center of radially arranged and enlarged epidermis cells (Fig. 6c). The main extension of the lithocyst lies deeply embedded in the mesophyll. The cystolith enclosed consists of calcium carbonate, is distinctly stalked, and mussel- (Figs. 3k, 6d) or bunch-shaped (Fig. 6e). Such lithocysts are restricted to HELIOTROPIUM I, TOURNEFORTIA, and CEBALLOSIA (species see Table 3). 8 – multicellular lithocyst complex (ac, Figs. 3b, c). Trichome lithocysts of usually elongated shape, uniform in length, sculptured with small papillae, with one well developed, unstalked, mussel-shaped cystolith in its bulbous base. They are surrounded by radially arranged subsidiary lithocysts, each containing an unstalked mussel-shaped calcium carbonate cystoliths attached to the centripetal cell walls (Figs. 3b, c, 6f–h). This lithocyst complex occurs only sporadically and sparsely as a part of a heterotrichous indumentum in some species of MYRIOPUS, EUPLOCA, many species of HELIOTROPIUM II (species see Table 3), CEBALLOSIA, and Argusia sogdiana. In the last species it is the dominant trichome type.

Fig. 6. (a) Argusia sogdiana: intermediary developed trichome of type 5 (ph), spear-like appearance but ornamented on surface; (b) Heliotropium transalpinum: trichome of type 6 (ta), two-armed, in the background trichome of type 4 with ornamented surface and reduced trichome tip; (c) H. angiospermum : idioblast if of type 7 (ic), on epidermis surface recognizable as cells with small diameter, radially sourrounded by enlarged cells; (d) Heliotropium curassavicum: type 7 (ic), distinctly stalked and mussel-shaped lithocyst, deeply embedded in the mesophyll ; (e) Tournefortia hirsutissima: type 7 (ic); distinctly stalked and bunch-shaped lithocyst, deeply embedded in the mesophyll; (f) Argusia sogdiana: type 8 (ac), multicellular lithocyst complex surrounded by subsidiary lithocysts; (g) Ceballosia fruticosa: type 8 (ac), multicellular lithocyst complex, cystoliths attached to centripetal cell walls; (h) Tournefortia psilostachya: type 8 (ac), multicellular lithocyst complex, under polarization filter; (i) Heliotropium ciliatum: crystal druses in the form of crystal tubes (type 2, d+) in the mesophyll; (j) H. convolvulaceum: epidermis with a large complex of crystal needles (type 3, n); (k) H. convolvulaceum: sector of Fig. 6j with crystal needles (type 3, n). Method of observation: (a–c, f) SEM; (d, e, g–k) LM. Scale bars: (c–e, k) 10 µm; (b, g–i) 50 µm; (a, f, j) 100 µm.

Crystals and idioblasts (Table 3 column 9, Cy ; Figs. 6i – k) Calcium oxalate crystals of various shape are found in different leaf tissues. Three types of crystal idioblasts were identified. 1 – crystal druses or crystalsand (d, s, Fig. 6i). Crystal druses are situated in different densities only in the mesophyll, but never in the epidermis or along bundles. Only sporadically the crystal druses are accompanied by crystalsand. They are abundant in all HELIOTHAMNUS species, IXORHEA, and in most species of HELIOTROPIUM II, CEBALLOSIA, and ARGUSIA. Calcium oxalate crystals occur rarely in species of HELIOTROPIUM I (species see Table 3), while they are completely absent in the non-halophytic TOURNEFORTIA. 2 – crystal tubes (d+, Fig. 6i). The idioblasts form tubes in the palisade parenchyma and contain many crystal druses arranged in one line. They are present in IXORHEA, HELIOTHAMNUS, HELIOTROPIUM II (species see Table 3), and Argusia sogdiana. They are absent in HELIOTROPIUM I and TOURNEFORTIA. 3 – crystal needles (n, Figs. 6j, k). Bundles of calcium oxalate needles are found in low density in the epidermis, rarely additionally in the mesophyll or surround the vascular bundles. Frequently many adjacent epidermal cells containing these crystal needles form large complexes of dendritic shape. They are found in some species of MYRIOPUS and EUPLOCA (species see Table 3).

Discussion The results of leaf anatomy patterns, especially venation, vascular system, various foliar trichome types, and localization of crystals are of surprisingly high systematic value and prove the formerly proposed subdivision of this family based on molecular results (Diane et al. 2002; Hilger & Diane 2003).

General leaf morphology Size and shape of the leaves are of high variability within Heliotropiaceae and are in general not appropriate for systematic studies at higher taxonomic level (Förther 1998). These characters may be interpreted under ecological points of view. Semi-arid habitats promote small narrow- to linearlanceolate leaves, sometimes with revolute leaf margins, and often a dense indumentum. This has evolved several times within most clades of Heliotropiaceae, especially within EUPLOCA, HELIOTROPIUM I, and also MYRIOPUS. On the other hand, humid tropical conditions or permanent water availability promote a broad-leaved shape. Species of HELIOTHAMNUS form a part of Andean scrub communities in rainless habitats. Their leaves are very often characterFLORA (2003) 198

481

ized by a dense indumentum, thus enabling fog condensation, which is the only reliable source of water in this regions.

Venation While leaf size and shape may vary enormously within one group, leaf venation is usually constant. In general, the leaves of Heliotropiaceae show a brochidodromous venation. They differ in prominence of the secondary and tertiary veins, which may be immersed in the mesophyll, the orientation of the tertiaries, or in the shape of the intercostal areas. A brochidodromous venation with fine and barely developed veins of higher order and randomly reticulate to weakly alternate percurrent tertiaries enclosing irregularly four- to five-sided intercostal areas (type 2, F) is characteristic for MYRIOPUS and IXORHEA. Starting from this venation type a reduction to a hyphodromous venation (type 1, P) characterizes the leaves of EUPLOCA species. In addition to EUPLOCA only the halophytic species (H. curassavicum, H. patagonicum, T. gnaphalodes) show that venation (type 1, P), which probably depends on the halophytic habitat conditions which promotes succulence with reduction of the venation. Venation types 3 (S) and 4 (T) are variations, differing in the prominence of the tertiary veins, and depend strongly on leaf size. The secondaries are always elevated. When elevated tertiaries are present, they are opposite percurrent, delimiting regularly intercostal areas. These mixtures of venation types characterize the leaves of the remaining clades of Heliotropiaceae. Only the generally broad leaved HELIOTHAMNUS and TOURNEFORTIA species are characterized by venation type 4 (T) exclusively, that means the larger the leaves are the stronger the veins of higher order may develop. Particularly the regular opposite position of the tertiaries may contribute to stabilizing the lamina and leads in addition to veins of higher order, to a characteristic rugose surface pattern.

Vascular system and Kranz-type leaf anatomy Most important with regard to the vascular system is the arrangement of bundle sheaths in EUPLOCA. The sheath cells contain a large amount of chloroplasts centripetally appressed against the cell wall. This corresponds to the Kranz-chlorenchyma organization of Haberlandt (1896). δ 13C-measurements (H. scabrum Retz., H. zeylanicum Lam., Das & Raghavendra 1973 ; H. marifolium Retz., H. paniculatum R. Br., Sankhla et al. 1975) revealed that the C4-dicarboxylic acid pathway is indeed present. Comparable obser482

FLORA (2003) 198

vations were made by Frohlich (1978) for species of Heliotropium sect. Orthostachys of Mexico. Furthermore, he described the mesophyll cells as arranged more or less radially around the bundle sheath. We cannot confirm this observation. We did not find mesophyll cells arranged that way. Aditionally, Napp-Zinn (1984) pointed out that in dicotyledons the Kranz-type leaf anatomy is not developed with radially arranged mesophyll. C4-species are restricted to the EUPLOCA clade. Revision of H. zeylanicum (HELIOTROPIUM II) could not confirm the observations of C4-anatomy by Das & Raghavendra (1973). The C4 photosynthetic pathway developed only in the EUPLOCA lineage, and thus the genus “Heliotropium” must be substituted by Euploca in the list of genera in which both C3 and C4 photosynthesis evolved. All species of subsect. Bracteata investigated, and some species of subsect. Ebracteata are C4-plants, and thus Kranz-type leaf anatomy is not appropriate for the characterization of the two subsections of Orthostachys, as was suggested by Förther (1998). The presence of Kranz-anatomy in “Hilgeria” and “Schleidenia” corresponds to our molecular studies (Diane et al. 2002; Hilger & Diane 2003) which showed that these two taxa are nested within EUPLOCA.

Character combinations (Table 4) and systematic implications A summary of the results is shown in Table 4.

EUPLOCA The EUPLOCA clade includes species of the current taxonomic circumscription of Heliotropium sect. Orthostachys, Schleidenia [which Johnston (1928) already treated as subsection Axillaria of section Orthostachys], and Hilgeria. The leaves are characterized by a hyphodromous venation (type 1, P – except H. procumbens), bilateral to isobilateral anatomy (B, B+, I), amphistomatous leaves (A), trichomes of type 3 (sg) and, sporadically, additional lithocyst complexes of type 8 (ac) in Old World species. In two species crystal needles (n) are present in the epidermis and surrounding the bundles. Kranz-type leaf anatomy is restricted to species of this clade. Glandular trichomes, trichomes of the types 4 (co, tc), 5 (ph), 7 (ic), and calcium oxalate druses (d) in the mesophyll are lacking. Summarizing, strongly supported by leaf anatomical and molecular results (Diane et al. 2002, Hilger & Diane 2003), we plead for a nomenclatural recombination within this clade to the genus Euploca Nutt. (Nuttall 1837).

HELIOTHAMNUS The species belonging to Heliotropium sect. Heliothamnus are characterized in the overall occurrence of brochidodromous venation type 4 (T), bilateral leaf anatomy (B), hypostomatous

Table 4. Summary of the results on leaf anatomy and distribution of foliar trichome types of the Heliotropiaceae. Abbrevations as in Table 3. Highlighted are significant characters. Clade

Taxon

H

Lv

La

Sd

Ko

Gt

IXORHEA

Ixorhea

2

F

Ih

A

-

(+)

-

-

-

-

-

-

d

d+

MYRIOPUS

Tournefortia sect Cyphocyema

1

F

B

AH

-

-

-

s

ac

-

-

n

-

-

Heliotropium sect. Orthostachys

1

P

B B+ I

A

+

-

sg

s

ac

-

-

n

-

-

Schleidenia

1

P

B

A

+

-

sg

-

-

-

-

n

-

-

Hilgeria

1

P

?

A

+

-

sg

-

-

-

-

-

-

-

HELIOTHAMNUS

Heliotropium sect. Heliothamnus

2

T

B

H+

-

+

co

s

-

tc

-

-

d

d+

sect. Cochranea

1

ST

B

H

-

+

co

-

-

tc

-

-

d

-

HELIOTROPIUM I

Heliotropium species of the New World

halophytes

5

P

S

A

-

-

-

-

-

-

ic

-

d

-

remaining species

1

ST

B B+ I2 Im

A

-

+

co

ta

-

tc

ic

-

d

-

halophytes

5

PT

S

A

-

+

co

s

-

tc

-

-

d

s

remaining species

3

T

B

H

-

+

co

s

-

tc

ic

-

-

-

1

S

B+

A

-

+

co

-

ac

tc

ic

-

d

-

A. sibirica

1

S

B+

A

-

+

co

-

-

tc

-

-

d

s

A. sogdiana

1

S

?

A

-

-

ph

s

ac

-

-

-

d

d+

Heliotropium species of the Old World

1

ST

B B+ I

A

-

+

ph

-

ac

-

-

-

d

d+ s

Nogalia

1

S

B+

A

-

+

ph

-

-

-

-

-

-

-

EUPLOCA

TOURNEFORTIA

Tournefortia sect. Tournefortia

CEBALLOSIA

Ceballosia

ARGUSIA

Argusia

HELIOTROPIUM II

leaves with strongly reduced density of stomata on the adaxial leaf surface (H+), glandular trichomes and trichomes of type 4 (co, tc), and the occurrence of calcium oxalate druses (d) usually additional in the form of crystal tubes (d+) but lack of unicellular lithocysts (type 7, ic). The occurrence of crystal tubes and the lacking of lithocysts separates these Andean species from the HELIOTROPIUM I clade.

HELIOTROPIUM I and TOURNEFORTIA Molecular results (Diane et al. 2002; Hilger & Diane 2003 show a close relationship between HELIOTROPIUM I and TOURNEFORTIA, which is also reflected in leaf anatomy. Both lineages are characterized by the presence of glandular trichomes, trichomes of type 4 (co, tc), and unicellular lithocysts of type 7 (ic). They lack calcium oxalate crystals

Tt

Ct

Cy

in the form of crystal tubes (d+), multicellular lithocyst complexes (type 8, ac), trichomes of types 3 (sg) and 5 (ph), and calcium oxalate needles (n) in the epidermis. The exclusive occurrence of venation type 4 (T), bilateral (B) and hypostomatous leaves (H), and the lack of calcium oxalate druses (d) – except the halophytes – separates TOURNEFORTIA from HELIOTROPIUM I. On the other hand, in HELIOTROPIUM I – except sect. Cochranea – , additional venation type 3 (S), and subbifacial to isobilateral (B+, I2, Im), always amphistomatous (A) leaves, and the occurrence of calcium oxalate druses (d) are characteristic.

HELIOTROPIUM II This clade comprises Heliotropium species of the Old World including Nogalia (based on Heliotropium drepanophyllum Baker). With HELIOTROPIUM I they share brochidodromous FLORA (2003) 198

483

venation types 3 (S) and 4 (T), bi- to isobilateral (B, B+, I) and amphistomatous (A) leaf anatomy, glandular trichomes and calcium oxalate druses in the mesophyll (d). They differ from HELIOTROPIUM I and TOURNEFORTIA by the exclusive presence of trichomes of type 5 (ph), lithocyst complexes of type 8 (ac), and crystal tubes (d+). Unicellular lithocysts (type 7, ic) have not been found. With HELIOTHAMNUS they share crystal tubes and the lack of unicellular lithocysts.

Staatssammlung München, the Botanical Garden and Botanical Museum (BGBM) of Berlin-Dahlem and administration of the Botanical Garden of München-Nymphenburg (all Germany) for herbarium material. We thank Horst Lünser (Berlin) for drawings and Maximilian Weigend (Berlin) for helpful comments on the text.

References MYRIOPUS MYRIOPUS and TOURNEFORTIA have no close relationships. While the species of TOURNEFORTIA generally resemble HELIOTROPIUM I species and show nearly identical leaf anatomical features, the character set of MYRIOPUS species differs markedly. It is characterized by venation type 2 (F), bilateral hypo- and amphistomatous leaves, simple trichomes (type 2, s), lithocyst complexes (type 8, ac), and crystal needles (n) in the epidermis, while trichomes of type 4 (co, tc), unicellular lithocysts of type 7 (ic), and crystal druses (d) are lacking. Leaf anatomy thus strongly supports the molecular results of Diane et al. (2002) and Hilger & Diane (2003). As in EUPLOCA, a re-establishment of the genus Myriopus Small (Small, 1933) is inevitable.

ARGUSIA, CEBALLOSIA, IXORHEA It is also possible to obtain systematic indications for CEBALLOSIA and ARGUSIA. Apparently the two species of Argusia do not constitute a natural group. Leaf anatomy of both Argusia species differs strongly, and A. sibirica seems to be more closely related to HELIOTROPIUM I species by sharing the same trichomes of type 4 (co, tc). A. sogdiana on the other hand seems to be connected with HELIOTROPIUM II by the presence of lithocyst complexes (type 8, ac), crystal tubes (d+), and the trichomes of type 5 (ph). Ceballosia differs from HELIOTROPIUM I species only by having multicellular lithocyst complexes (type 8, ac) and seems to belong to this lineage. Ixorhea is morphologically very aberrant and leaf anatomy does not at all contribute to clarify its systematic position.

Combining all data, leaf anatomy of Heliotropiaceae is of unexpectedly high value with regard to their relationships and strongly supports the actual subdivision within this family based on molecular data (Table 1, Diane et al 2002, Hilger & Diane 2003).

Acknowledgement The authors wish to thank the following collectors who provided us with plant material: Lyn Craven (Canberra, Australia), Harald Förther (München), Anna Kagiampaki (Iraklion, Greece), Norbert Kilian (Berlin), Egon Köhler (Berlin), Sigrid Liede (Bayreuth), Monika Löschner (Berlin), Wolfgang Schultka (Gießen), Jürgen Skrabal (Berlin), Maximilian Weigend (Berlin). We thank the Botanische 484

FLORA (2003) 198

Ash, A.; Ellis, B.; Hickey, L. J.; Johnson, K.; Wilf, P. & Wing, S. (eds.) (1999): Manual of leaf architecture – morphological description and categorization of dicotyledonous and net-veined monocotyledonous angiosperms by Leaf Architecture Working Group. – Smithsonian Institution. Washington DC. Bider, J. (1935): Beiträge zur Pharmakognosie der Boraginaceen und Verbenaceen. Vergleichende Anatomie des Laubblattes. – Inaugural-Diss. Univ. Basel. Das, V. S. R. & Raghavendra, A. S. (1973): A screening of the dicotyledonous weed flora for the occurrence of C4 dicarboxylic acid pathway of photosynthesis. – Proc. Indian Acad. Sci. 77: 93–100. Diane, N.; Förther, H. & Hilger, H. H. (2002): A systematic analysis of Heliotropium, Tournefortia, and allied taxa of the Heliotropiaceae (Boraginales) based on ITS1 sequences and morphological data. – Amer. J. Bot. 89: 287–295. DiFulvio, T. E. (1982): Sobre la anatomia foliar y estomatogenesis de Ixorhea tschudiana (Heliotropiaceae). – Kurtziana 15: 9–18. Ferguson, D. M. (1999): Phylogenetic analysis and relationships in Hydrophyllaceae based on ndhF sequence data. – Syst. Bot. 23: 253–268. Förther, H. (1998): Die infragenerische Gliederung der Gattung Heliotropium L. und ihre Stellung innerhalb der Subfam. Heliotropioideae (Schrad.) Arn. (Boraginaceae). – Sendtnera 5: 35–241. Frohlich, M. W. (1978): Systematics of Heliotropium section Orthostachys in Mexico. – Cambridge (Mass.). Ph.D. Thesis (unpubl.). Gerstberger, P. & Leins, P. (1978): Rasterelektronenmikroskopische Untersuchungen an Blütenknospen von Physalis philadelphica (Solanaceae). Anwendung einer neuen Präparationsmethode. – Ber. Deutsch. Bot. Ges. 91: 381–387. Gottschling, M.; Hilger, H. H.; Wolf, M. & Diane, N. (2001): Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny of Boraginales. – Plant Biol. 3: 629–636. Haberlandt, G. (1896): Physiologische Pflanzenanatomie. – 2. ed., W. Engelmann. Leipzig. Harris, J. G. & Harris, M. W. (1994): Plant identification terminology: an illustrated glossary. – Spring Lake Publishing, Spring Lake, Utah. Hickey, L. J. (1973): Classification of the architecture of dicotyledonous leaves. – Amer. J. Bot. 60: 17–33. Hilger, H. H. & Diane, N. (2003): A systematic analysis of Heliotropiaceae (Boraginales) based on trnL and ITS1 sequence data. – Bot. Jahrb. Syst. 125: 1–33.

Horner, H. T. & Wagner, B. L. (1992): Association of four different calcium crystals in the anther connective tissue and hypodermal stomium of Capsicum annuum (Solanaceae) during microsporogenesis. – Amer. J. Bot. 79: 531–541. Johnston, I. M. (1928): Studies in the Boraginaceae. VII. 1. The South American species of Heliotropium. – Contr. Gray Herb. 81: 3–83. Kragge, H. (1911): Über die Festigkeit der Blätter der Boraginaceae und verwandter Familien. – Inaugural-Diss. Univ. Kiel, Petschmann, Hamburg. Kumar, K. V. & Rao, B. H. (1994): A contribution to the foliar epidermology of Heliotropium. – J. Swamy Bot. Cl. 11: 12–19. Metcalfe, C. R. & Chalk, L. (1950): Anatomy of the dicotyledons: 195. Boraginaceae. 2: 945–954. Clarendon Press, Oxford. Napp-Zinn, K. (1984): Anatomie des Blattes, 2. Blattanatomie der Angiospermen. – Handbuch der Anatomie, Gebrüder Borntraeger, Berlin–Stuttgart. Nuttall, T. (1837): Collections towards a flora of the territory of Arkansas. – Trans. Amer. Philos. Soc., n.s. 5: 189.

Sankhla, N.; Ziegler, H.; Vyas, O. P.; Stichler, W. & Trimborn P. (1975): Eco-physiological studies on Indian arid zone plants. V. A screening of some species for the C4-pathway of photosynthetic CO2-fixation. – Oecologia. 21: 123–129. Schibler, W. (1887): Beiträge zu einer vergleichend-systematischen Anatomie des Blattes und Stengels der Boragineen. – Diss. Univ. Bern. Selvi, F. & Bigazzi, M. (2001): Leaf surface and anatomy in Boraginaceae tribe Boragineae with respect to ecology and taxonomy. – Flora 196: 269–285. Small, J. K. (1933): Manual of the southeastern flora. – University of North Carolina Press, New York, USA. Solereder, H. (1908): Systematische Anatomie der Dikotyledonen. – Enke, Stuttgart. Theobald, W. L., Krahulik, J. L. & Rollins R. C. (1979): Trichome description and classification. In: Metcalfe, C. R. & Chalk, L. [eds.], Anatomy of the dicotyledons, 2nd ed., Vol. 1: 41–96. – Clarendon Press, Oxford. Yatskievych G., Maxon J. & Charles T. (1986): A revision of the Lennoaceae. – Syst. Bot. 11: 531–548.

FLORA (2003) 198

485