Phylogeny and systematics of Lithodora ...

TAXON 57 (1) • February 2008: 79–97

Thomas & al. • Phylogeny and systematics of Lithodora

Phylogeny and systematics of Lithodora (Boraginaceae—Lithospermeae) and its affinities to the monotypic genera Mairetis, Halacsya and Paramoltkia based on ITS1 and trnLUAA-sequence data and morphology Daniel C. Thomas1,2, Maximilian Weigend2 & Hartmut H. Hilger2 1

2

Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh, EH3 5LR, UK. [email protected] (author for correspondence) Freie Universität Berlin, Institut für Biologie – Systematische Botanik und Pflanzengeographie, Altensteinstr. 6, 14195 Berlin, Germany

Lithodora (Boraginaceae tribe Lithospermeae) comprises nine species of often narrowly endemic dwarf shrubs and shrubs. The genus shows a Mediterranean distribution with the centre of diversity in the western Mediterranean region. Lithodora is distinguished from its allies in Lithospermeae by a shrubby habit, the absence of both faucal scales and an annulus, and a very unusual mericarpid morphology, and it has therefore been regarded as a natural group in the past. Phylogenetic relationships of Lithodora were investigated with nuclear ribosomal ITS1 and chloroplast trnLUAA intron sequences using parsimony and likelihood analyses. The results strongly indicate a polyphyly of Lithodora, which falls into two clades: The majority of species forms a monophyletic group (“Lithodora II”) in a well supported clade together with a Lithospermum s.l. clade (including the New World genera Onosmodium and Macromeria) and our sample of Buglossoides species. A smaller number of species (“Lithodora I”) falls into a second well supported clade including the monotypic genera Mairetis, Paramoltkia and Halacsya. A critical reevaluation of carpological characters traditionally used to delimit Lithodora corroborates the results of the molecular analysis and indicates that the cupulate areole and the mericarpid appendage found in the genus are homoplasious. The polyphyly of Lithodora is formally recognised by removing “Lithodora II” as a newly established genus, Glandora. The seven newly required combinations in the genus are provided.

KEYWORDS: Glandora gen. nov., ITS1, Lithodora, Mediterranean, phylogeny, trnLUAA intron

INTRODUCTION Boraginaceae tribe Lithospermeae (DC.) Gürke are an apparently monophyletic group (Hilger & Böhle, 2000; Långström & Chase, 2002) with centres of diversity in the Mediterranean basin, and the United States and Mexico. Fruit anatomy indicates that tribe Lithospermeae is closely allied to Echiochilon Desf., Ogastemma Brummitt, Antiphytum DC. ex Meisn. and Sericostema Stocks ex Wight (Seibert, 1978) and it is now understood to represent the sister group to tribe Echiochileae (Riedl) Långström & M.W. Chase (Långström & Chase, 2002). The systematics of tribe Lithospermeae was last studied in detail by Johnston (1924, 1952, 1953a, b, 1954a, b), who recognised several small segregate genera of Lithospermum L. In the Mediterranean region these include the monotypic genera Mairetis I.M. Johnst., Moltkiopsis I.M. Johnst., and Neatostema I.M. Johnst., and the slightly larger genera Buglossoides Moench (ca. ten species), and Lithodora Griseb. (nine species). Delimitation of Lithospermeae has been anything but straightforward and there has been considerable confusion with regards to the systematics of some taxa placed into Lithospermeae

by Johnston (1924, 1953a). Johnston (1954b) himself removed some of the genera he had originally described as segregates from Lithospermum (Mairetis, Moltkiopsis, Neatostema) to Eritricheae (Benth. & Hook.) Gürke. Riedl (1967, 1968) subsequently moved these genera to a new subtribe Moltkiopsidinae Riedl in his tribe Trigonotideae (Popov) Riedl. However, Riedl (1967, 1968) also included taxa in Trigonotideae which have since been shown to belong to Cynoglosseae DC. s.l. (Trigonotis Stev.) and Echiochileae (Antiphytum, Sericostoma) respectively (Långström & Chase, 2002). Seibert (1978) already expressed serious doubts about the justification of “Trigonotideae” in general and argued for an affinity of Mairetis, Moltkiopsis and Neatostema to Lithospermeae on the basis of a detailed study of fruit morphology and anatomy. This view was, however, not adopted by Riedl (1997), who retained them in “Trigonotideae”, but reluctantly admitted palynological similarity to Lithospermeae. Lithodora was universally placed in Lithospermeae (Johnston, 1953b, 1954a; Seibert, 1978; Bigazzi & Selvi, 1998), but Johnston (1924) and Melchior (1964) emphasized a doubtful affinity to Boragineae Bercht. & J. Presl. Riedl (1968) also emphasized that because of its 79


“shrubby habit” Lithodora represented “a primitive type” in Lithospermeae. Lithodora comprises nine species (plus three infraspecific taxa variously considered as distinct species), all of which are dwarf shrubs or shrubs (Fernandes, 1972). Lithodora has a Mediterranean distribution with the centre of diversity in the western Mediterranean (Meusel & Kästner, 1990). Several taxa are narrowly endemic (see Fig. 1): L. moroccana I.M. Johnst. is endemic to northeastern Morocco, L. nitida (Ern) R. Fern. to a few mountain ranges in Andalusia (southern Spain), L. oleifolia (Lapeyr.) Griseb. to a small area in the eastern Pyrenees (northeastern Spain), and L. zahnii (Heldr. ex Halácsy) I.M. Johnst. to a small area of the western Peloponnese (Greece). The genus Lithodora was created by Grisebach (1844) to accommodate six shrubby Mediterranean species for-

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merly assigned to Lithospermum. Grisebach (1844) differentiated Lithodora from Lithospermum mainly on the basis of its shrubby (versus herbaceous) growth form and apparent differences in floral morphology. However, most subsequent authors ignored his genus and the name was considered a synonym of Lithospermum or was recognised only at infrageneric level (e.g., Candolle, 1846; Boissier, 1879; Gürke, 1897), until Johnston published a detailed analysis of Lithodora (Johnston, 1953b). He reinstated Lithodora on the basis of its peculiar fruits, which he considered as “unique in the whole Boraginaceae” (Johnston, 1953b: 260). The mericarpids show a circumscissile abscission of the upper, seed-bearing part, while the lower part remains connected with the gynobase forming a cupulate structure (Johnston, 1953b). In addition to the unique fruit morphology, Johnston (1953b) found several other characters distinguishing Lithodora from Lithospermum s.str.

Fig. 1. Distribution map of Halacsya, Lithodora, Mairetis and Paramoltkia. Based on the distribution map in Meusel & al., 1978, vouchers examined in B, BSB, E, FR, and MSB and the following publications: Meusel & Kästner, 1990 (Lithodora); Stevanović & al., 2003 (Halacsya, Paramoltkia); Johnston, 1953b; Sauvage & Vindt, 1954; Silva & Rozeira, 1962; Valdés, 1981; Browicz, 1985; Loidi & al., 1988; Bolòs i Capdevila & al., 2001; Tan & Iatrou, 2001; Valdés & al., 2002; and Bañares Baudet, 2003.

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such as the shrubby growth form and the absence of both corolla invaginations (faucal scales) and basal scales (an annulus) in the corolla tube. Johnston (1953b) also created three sections in Lithodora, mainly based on the occurrence of different stylar polymorphisms (absent, stigmaheight dimorphism, distyly) and the morphology of the detachment scar of the mericarpid ( = cicatrix) and the detachment scar at the receptaculum ( = areole). Section Lithodora (only L. fruticosa (L.) Griseb.) is characterised by a stigma-height dimorphism, i.e., only the length of the styles vary between the morphs, but the level of filament attachment and the filament length are relatively constant between the morphs. The mericarpids have a broad appendage, which does not include a sclerenchymatous, tubular channel. Section Allostema I.M. Johnst. (L. oleifolia, L. moroccana, L. rosmarinifolia (Ten.) I.M. Johnst., L. hispidula (Sibth. & Sm.) Griseb., L. zahnii ) is characterised by distylous flowers and a mericarpid appendage which includes a sclerenchymatous tubular channel. According to Johnston (1953b), section Lasioglottis I.M. Johnst. is characterised by monomorphic flowers and filaments which are affixed at unequal heights on the corolla tube (only L. prostrata (Loisel.) Griseb.), but Fernandes (1970) and Valdés (1981) observed that L. prostrata exhibits true distyly and can thus not be differentiated from section Allostemma. Seibert (1978) doubted Johnston’s theory of a suprabasal abscission of the mericarpids in his very informative carpological overview of the Lithospermeae, thereby weakening this apparent synapomorphy of the Lithodora species to a certain extent. However, he still agreed with Johnston on the basic uniformity of the mericarpids within Lithodora, and he quoted Sprengler (1919: 115), who had emphasized that Lithodora species exhibit “not only great similarities in floral morphology (minor differences of form and size of the corolla), but also in vegetative morphology” (own translation). The flowers of Lithodora show conspicuous characters that differentiate its members from Lithospermum s.str. and/or Buglossoides: the corollas in Lithodora are blue or purple in contrast to the yellow, orange or whitish corollas predominant in Lithospermum s.str. They lack both faucal scales and an annulus (Sprengler, 1919; Johnston, 1953b), while corolla invaginations are present in most species of Lithospermum s.str. (faucal scales) and Buglossoides (vertical pleats), and almost all species in these two genera exhibit an annulus (Johnston, 1954a; Ralston, 1993). However, other genera of Lithospermeae also exhibit a shrubby or subshrubby habit and blue corollas (e.g., Moltkia Lehm.), and an annulus and faucal scales are missing in a range of taxa in the Lithospermeae, for example in Moltkia as well as in Paramoltkia Greuter. In Arnebia Forssk. faucal scales are always missing while an annulus is developed in some species, but missing in others. Overall, none of the morphological characters de-


fining and delimiting the genus Lithodora sensu Johnston (1953b) is unique in Lithospermeae, with the only exception of the peculiar fruit morphology. On the basis of the apparent overall similarity of the taxa reunited in Lithodora and also on the basis of its apparently unique carpological condition as identified by Johnston (1953b) the genus Lithodora became a widely recognised segregate of Lithospermum (Silva & Rozeira, 1962; Fernandes, 1971, 1972; Valdés, 1981; Meikle, 1983; Greuter & al., 1984; Browicz, 1985; Al-Shehbaz, 1991; Riedl, 1997). The present study initially intended to clarify the systematic position of Lithodora sensu Johnston (1924, 1953b) in the tribe Lithospermeae. However, preliminary molecular data surprisingly associated different species of the genus with different clades in Lithospermeae, indicating a relationship of some Lithodora species with the monotypic Balkan endemics Halacsya Dörfl. and Paramoltkia, which are either considered to be closely allied to Moltkia (Johnston, 1953b; Stevanović & al., 2003) or to Echium L. (Seibert, 1978; Greuter, 1981). As has been indicated above, there are thus several open questions with regards to the systematics of Lithospermeae, which require clarification: Is Lithodora in the circumscription of Johnston (1953b) monophyletic? What are its affinities? Do vegetative and generative characters which were traditionally used to delimit Lithodora, especially the carpological character set, represent autapomorphies of a natural group? Can the shrubby growth habit in Lithodora be interpreted as indicative of “a primitive type” in Lithospermeae? What are the affinities of the obligate serpentine endemics Halacsya and Paramoltkia? Do the monotypic genera Mairetis, Moltkiopsis and Neatostema belong to Lithospermeae? The findings will then be reflected in a—if necessary—modified classification of the genus Lithodora.

MATERIALS AND METHODS Sampling. — Thirty-eight ingroup taxa were incorporated in the molecular matrix including the nine species of Lithodora sensu Johnston (1953b), four species of Buglossoides, and the segregate genera Neatostema and Mairetis. Unfortunately, we did not succeed in obtaining useful sequences of Moltkiopsis. The large genus Lithospermum s.str. is represented by eight taxa representing all growth habits known in the genus (herb, subshrub, dwarf shrub and shrub) and by representatives from both the Old World and the New World in order to provide a preliminary test of its monophyly. The majority of genera associated with Lithospermeae is included in the analysis. We focussed on Old World genera with the exception of Lithospermum s.str. (sensu Johnston, 1954a), which is the only genus of the Lithospermeae present in both 81


the Old and the New World, and the closely allied North American genera Macromeria D. Don and Onosmodium Michx. Representatives of the tribes Echiochileae and Boragineae were chosen as outgroup taxa on the basis of the study by Långström & Chase (2002) and the observations of Seibert (1978). Voucher information, collection and herbarium details, and GenBank accession numbers of the taxa used for the molecular analysis are presented in the Appendix. DNA extraction, amplification and sequencing. — DNA was extracted from fresh or silica-gel dried leaves or leaves of herbarium material, as described by Weigend & al. (2004). The primers P1 and P2 (Baldwin, 1992) were used for the PCR-fragment amplification of the ITS1 region, and the primers C and D (Taberlet & al., 1991) for the amplification of the trnLUAA intron. Amplified products were purified using QIAquick™ columns according to the manufacturer’s protocol (Qiagen Inc.). The purified amplification products served as templates for cycle sequencing with a Quick Start Kit (Beckman Coulter) according to manufacturer’s instructions. Sequencing of the ITS1 region and the trnLUAA intron was done using the same primers as in the amplification. A CEQ 8000 (Beckman Coulter) automated sequencer was used to generate the data. Sequences of Echium vulgare L. and E. wildpretii H. Pearson were taken from the study by Böhle & al. (1996), sequences of Anchusa officinalis L. and Borago officinalis L. were taken from the study by Hilger & al. (2004). Editing, alignment and gap coding. — The initial sequence data were edited using Chromas Pro V. 1.33 (Technelysium Pty. Ltd. 2003–2005). The alignment was performed manually in Multicolor Sequence Alignment Editor (Hepperle, 2001). A secondary structure model of the ITS1 transcript has been proposed for Boraginales (Gottschling & al., 2001). This model consists of an open multibranch loop with four pairing regions (helices I to IV). The alignment of the ITS1 sequences proved difficult in the part corresponding to helix I of the transcript. Therefore, a sequence of 39 characters was excluded from the ITS1 matrix. The rest of the alignment was aided by the investigation of the framework dictated by the secondary structure of the ITS1 transcript, with separate aligning of homologous regions that were identified by mutual sequence comparison. Gaps were coded as missing data, but eleven indels in the ITS1 region and three indels in the trnLUAA intron were coded as additional parsimonyinformative binary characters (present/absent). The alignment is available on TreeBASE. Phylogenetic analysis. — Phylogenetic relationships were analysed using winPAUP 4.0b10 (Swofford, 2002). To assess character congruence between the ITS1 and the trnLUAA datasets, a partition homogeneity test (Farris & al., 1994) as implemented in winPAUP 4.0b10 was performed 82

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with 1,000 replicates with no more than 1,000 trees saved per replicate. Heuristic parsimony searches of combined nrDNA and cpDNA datasets were performed with 1,000 random sequence additions, all characters unweighted and unordered, and the following options in effect: tree bisection-reconnection (TBR) branch swapping, collapse zero length branches, and MULTREES. Confidence limits for trees were assessed by performing 1,000 replicates of bootstrapping (Felsenstein, 1985) with 100 addition sequence replicates per bootstrap replicate using equal weighting, TBR branch swapping and MULTREES on. Further support was estimated using jackknife analysis. We ran 1,000 replicates with 37% deletion, using random addition sequence (10 replicates) and TBR branch swapping. Maximum likelihood calculations were executed assuming a GTR + G + I model and a rate variation among sites following a gamma distribution. GTR + G + I was chosen as the best-fit substitution model using the hierarchical likelihood ratio test (hLRT) in Modeltest 3.7 (Posada & Crandall, 1998). The settings proposed by Modeltest were: Basefreq = (0.3589 0.1687 0.1798), Nst = 6, Rmat = (1.0000 1.3194 1.0000 1.0000 2.7946), Shape = 0.6277, Pinvar = 0.3470. Likelihood bootstrap searches were performed with 1,000 replicates using the “fast bootstrap” option (bootstrapping without branch-swapping). “Fast bootstrapping” was shown to provide estimates of support similar to, though generally less than, bootstrapping with branch-swapping within a reasonable computing time (Mort & al., 2000). Scanning electron microscopy. — Herbarium material for scanning electron microscopy (SEM) was directly mounted on double-sided adhesive tape and sputter coated with gold. Material from the FAA collection was first dehydrated in a graded ethanol series, critical point dried following the standard procedure, and than mounted and sputter coated as above. The material was subsequently viewed and photographed with a LEO VP 430 scanning electron microscope.

RESULTS Molecular phylogeny. — The partition homogeneity test indicated that our data from two distinct marker regions were not significantly different from random partitions of the combined dataset (P = 0.46; Cunningham, 1997), thereby justifying the combined analysis of both loci. The combined matrix of ITS1 and trnLUAA intron (group I) data contained 42 taxa and 722 characters (including 14 indel codes). Of these 252 (11 indels) were derived from the ITS1 dataset and 470 (3 indel codes) from the trnLUAA intron dataset. Analyses of the combined dataset yield trees with similar overall topologies in both parsimony and maxi-

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mum likelihood searches. The heuristic search produced 5,520 shortest trees of 536 steps with a consistency index (CI) of 0.63 and a retention index (RI) of 0.73. Figure 2 shows the strict consensus tree of the 5,520 most parsimonious trees with bootstrap support values above the branches and jackknife values below. Figure 3 shows the single optimal tree obtained by maximum likelihood


analysis of the combined data with bootstrap support values above the branches or to the right of nodes. Bootstrap (bs) and jackknife support values ( jk) given in brackets in the following text are derived from the parsimony analysis. The ingroup, i.e., all analysed Lithospermeae taxa, is strongly supported as monophyletic (bs: 97, jk: 99).

Fig. 2. Parsimony analysis of combined ITS1 and trnLUAA data. Strict consensus tree of the 5,520 most parsimonious trees (length = 536, CI = 0.63, RI = 0.73) of the heuristic search. Bootstrap support values > 50% are presented above the branches, jackknife values > 50% below. BOR, Boragineae; ECH, Echiochileae; LIRA I, “Lithodora I”; LIRA II, “Lithodora II”.

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Lithodora falls into two monophyletic assemblages: “Lithodora I” (bs: 84, jk: 90) comprises L. hispidula, L. zahnii, and L. fruticosa. Within this clade L. fruticosa is the sister taxon to a strongly supported L. zahnii/L. hispidula clade (bs: 100, jk: 100). “Lithodora I” is a subclade of a moderately supported clade A (bs: 87, jk: 91), which also contains Paramoltkia doerfleri (Wettst.) Greuter & Burdet, Halacsya sendtneri (Boiss.) Dörfl. and Mairetis mi-

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crosperma (Boiss.) I.M. Johnst. The relationships among “Lithodora I” and the other taxa in clade A are only poorly resolved, but there is weak support for a sister group relationship of this clade to Neatostema apulum (L.) I.M. Johnst. (bs: 77, jk: 84). Moltkiopsis ciliata (which we failed to sequence successfully) will almost certainly be retrieved in this clade. Its presence or absence does not modify the morphological or phylogenetic interpretations, since it is

Fig. 3. Likelihood analysis of combined ITS1 and trnLUAA data. Single optimal likelihood tree obtained by a heuristic search using the GTR + G + I substitution rate matrix, plus a gamma distribution of rate variation among sites and a proportion of invariant sites (settings: Basefreq = [0.3589 0.1687 0.1798], Nst = 6, Rmat = [1.0000 1.3194 1.0000 1.0000 2.7946], Shape = 0.6277, Pinvar = 0.3470). Bootstrap support values generated by “fast bootstrapping” are shown above the branches or to the right of nodes (percentages < 50% not shown). Broken lines indicate branches with < 50% bootstrap support.

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morphologically similar and evidently closely related to Neatostema and Mairetis (Johnston, 1953a, 1954b; Riedl, 1967, 1968). “Lithodora II” forms a strongly supported clade (bs: 95, jk: 97), comprising L. nitida, L. oleifolia, L. moroccana, L. rosmarinifolia, L. prostrata and L. diffusa (Lag.) I.M. Johnst. Within this clade L. nitida and L. oleifolia form a weakly supported sister group to the other species in this clade (not resolved in the strict consensus tree of the MP analysis). The relationships among these species are well resolved: Lithodora moroccana forms a well supported clade with L. rosmarinifolia (bs: 91, jk: 94), and L. prostrata forms a moderately supported clade with L. diffusa (bs: 88, jk: 87). “Lithodora II” is retrieved as monophyletic group within a well supported clade B (bs: 93, jk: 96) together with a Lithospermum s.l. clade

(bs: 91, jk: 96, including Macromeria and Onosmodium) and our sample of Buglossoides species. Buglossoides section Buglossoides is strongly supported as monophyletic (bs: 100, jk: 100), however, the monophyly of the genus Buglossoides is not supported. A sister group relationship between Buglossoides /Lithospermum on the one hand and “Lithodora II” on the other is only weakly supported (bs: 74, jk: 79). Morphology. — Selected vegetative and generative characters of twelve species and subspecies of Lithodora and of Neatostema, Mairetis, Moltkiopsis, Halacsya and Paramoltkia are summarized in Table 1. Selected vegetative characters and floral characters are illustrated in Fig. 4. Figures 5 and 6 illustrate systematically important morphological characters of the mericarpids

Clade

Taxon

Growth form

Habit

Special underground organs

Leaf (lamina form)

Leaf (indumentum)

Inflorescence

Pedicellus

Style polymorphisms

Table 1. Selected morphological characters of Lithodora and allied taxa (continued on next page).

A, L I

Lithodora fruticosa

DS

E

–

L, Obl, E

S-H

C

S

S

A, L I

Lithodora hispidula subsp. hispidula

DS

E

–

Obov

S-H

S, (C)

S

D

LI

Lithodora hispidula subsp. cyrenaica

DS, S

E

–

Obov

S-H

dT, C

S

D

LI

Lithodora hispidula subsp. versicolor

DS, S

E

–

Obov

S-H

dT, C

S

D

A, L I

Lithodora zahnii

DS

E

–

L, Obl

S

dT, C

S

D

A

Paramoltkia doerfleri

HS

E

R

Obl, E

S

C

SP

–

A

Halacsya sendtneri

HS

A, E

–

Obov, Obl, Ov

S

C

SP

–

A

Mairetis microsperma

A

A

–

Obov, Obl

S

C

S, SP

–

A

Neatostema apulum

A

E

–

Obov, L, Ov

S

C, T

S, SP

–

–

Moltkiopsis ciliata

HS

A, E

–

Ov, Obl

S-H

C

S

–

B, L II

Lithodora nitida

DS

A, D

S

Obov, E

S

C

S

D

B, L II

Lithodora oleifolia

DS

A, D

S

Obov, E

S-T

C

P

D

B, L II

Lithodora moroccana

DS

A, D

–

Obov, Obl

S-T

C

S

D

B, L II

Lithodora rosmarinifolia

DS

A, E

–

L, Obl, E

S

C

S

D

B, L II

Lithodora prostrata subsp. prostrata

DS

A, D

–

L, Obl, E

H, S

C

S

D

L II

Lithodora prostrata subsp. lusitanica

DS

A, E

–

L, O, E

H, S

C

S

D

B, L II

Lithodora diffusa

DS

A, D

–

L, O, E

H, S

C

S

D

Clade: A, clade A; B, clade B (L I, “Lithodora I”; L II, “Lithodora II”; see Fig. 2). ― Growth form: A, annual; DS, dwarf shrub; HS, subshrub; S, shrub. ― Habit: A, ascending; D, decumbent; E, erect. ― Special underground organs: –, no special underground organs present; R, rhizome; S, perennial underground stolons. ― Leaf (lamina form): E, narrowly elliptic; L, linear; Obl, narrowly oblong; Obov, obovate or narrowly obovate; Ov, narrowly ovate. ― Leaf (indumentum): H, hispid; S, strigose; S-H, strigose-hispid; S-T, strigose-tomentose. ― Inflorescence: C, cymoid; dT, depauperate thyrsoids; S, flowers solitary (depauperate cymoids); T, thyrsoids. ― Pedicellus: S, flowers sessile or subsessile; P, distinctly pedicellate; SP, shortly pedicellate. ― Style polymorphisms: –, absent; D, distyly; S, stigma-height dimorphism. 85

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and the receptaculum: the cicatrix ( = detachment scar at the mericarpid), the cicatrix appendage, the tubular funicular channel, and the areole ( = detachment scar at the receptaculum). Fruit morphology. — The terminology of this section follows Seibert (1978) and Hilger (1985). The mericarpids in Lithodora are ovoid, and straight, or slightly (L. oleifolia) to strongly (L. hispidula, L. zahnii) incurved towards the style. They are gradually narrowed to the base or suprabasally constricted and then exhibiting a thicker collar about the base (L. prostrata, L. fruticosa) or a short, stout neck (L. hispidula, L. zahnii). The ventral keel is protruding, at least in the distal portion. The mericarpids are dorsally convex, a dorsal keel is missing

or weakly developed in the distal part (L. fruticosa, L. zahnii, L. prostrata subspp.). The mericarpids are whitish, grey, brownish, or black. The surface is smooth, or slightly tumulose (Lithodora nitida), minutely tuberculate (L. prostrata subspp.), muriculate (L. hispidula subspp.), or minutely rugulose (L. fruticosa, L. zahnii). The cicatrix is horizontal to slightly oblique. All species except L. nitida show a distinct peg-like or broadly pyramidal to asymmetrical cicatrix appendage in central to slightly ventral position. Around this appendage, the cicatrix is often distinctly hollowed-out by the collapse of the abscission tissue (L. diffusa, L. moroccana, L. oleifolia, L. prostrata subspp.) or the cicatrix around the appendage is only weakly sunken (L. rosmarinifolia, L. hispidula,

Clade

Taxon

Petal colour

Annulus

Glandular hairs (throat)

Indument (corolla tube)

Stamina insertion

Cicatrix appendage

Areole

Mericarpid (surface)

CaCO3 (pericarp)

Table 1. Continued.

A, L I

Lithodora fruticosa

P, B

–

–

–

E

F

CT

R

–

A, L I

Lithodora hispidula subsp. hispidula

B, P

–

–

–

E

F

CT

M

–

LI

Lithodora hispidula subsp. cyrenaica

B

–

–

–

E

F

CT

M

?

LI

Lithodora hispidula subsp. versicolor

B, P, R

–

–

–

E

F

CT

M

?

A, L I

Lithodora zahnii

B, (W)

–

–

–

E

F

CT

R

–

A

Paramoltkia doerfleri

P

–

–

–

E

–

F

S

–

A

Halacsya sendtneri

Y

–

–

–

E

–

F

T, T-R

–

A

Mairetis microsperma

B+Y

+

–

–

E

–

F

T

+

A

Neatostema apulum

Y

+

–

+

E

–

F

T

+

B, P

+

–

+

D

–

F

S, T

+

B

–

+

+

E

–

VD

STu

?

B

–

+

+

E

P

D

S

+

B, (W)

–

+

+

E

P

D

S

?

–

Moltkiopsis ciliata

B, L II

Lithodora nitida

B, L II

Lithodora oleifolia

B, L II

Lithodora moroccana

B, L II

Lithodora rosmarinifolia

P, B, (W)

–

+

+

E

P

D

S

+

B, L II

Lithodora prostrata subsp. prostrata

B, (P)

–

+

+

D

P

D

MT

–

L II

Lithodora prostrata subsp. lusitanica

B, (P)

–

+

+

D

P

D

MT

?

B, L II

Lithodora diffusa

B

–

+

+

E

P

D

S

?

Clade: A, clade A; B, clade B (L I, “Lithodora I”; L II, “Lithodora II”; see Fig. 2). ― Petal colour: B, blue; P, purplish; R, pinkish; W, white; Y, yellow. ― Annulus (basal scales): – , absent; +, present. ― Glandular hairs (adaxial corolla tube, corolla throat): – , absent; +, present. ― Indument (abaxial corolla tube): – , absent, +, present. ― Stamen insertion: D, insertion on different levels (for details, see Johnston, 1953a, b); E, stamen insertion on one level. ― Cicatrix appendage: – , absent or minutely protruding vascular strands or funicular channels; F, Lithodora fruticosa-type; P, Lithodora prostrata-type. ― Areole: CT, cupulate, thinwalled; D, cupulate, thick-walled depression in the receptacle; F, flat, slightly concave or with minutely elevated margin; VD, oblique with ventral depression. ― Mericarpid surface: M, muriculate; MT, minutely tuberculate; R, rugulose; S, smooth; STu, slightly tumulose, T, tuberculate; T-R, tuberculate-rugose. ― CaCO3 incrusts in pericarp: data from Seibert (1978), – , absent; +, present; ?, unknown. ― Abbreviations in brackets indicate rare character states. 86

TAXON 57 (1) • February 2008: 79–97


Fig. 4. Growth habit and floral morphology in Lithodora. A, L. hispidula subsp. hispidula (A. Kagiampaki 9–00), habit; B, L. zahnii (D. Thomas 2–05), habit. C–E, L. oleifolia (D. Thomas 3–05). C, habit; D, habit, note perennating underground runners; scale bar = 10 cm; E, flower. F, L. prostrata (D. Thomas 10–05), flowers; G, L. hispidula subsp. hispidula (A. Kagiampaki 9–00), flower; H, L. fruticosa (D. Thomas 7–06), flower. I, L. zahnii (D. Thomas 2–05), flowers.

87


L. zahnii ) or flat (L. nitida, L. fruticosa). The areoles are distinctly cupulate in all species except L. nitida, which shows only a weakly developed depression in ventral position of the oblique areole. In spite of many shared characters in the morphology of the mericarpids, two distinct types can be differentiated on the basis of the morphology of the cicatrix and the areole:

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1. Lithodora fruticosa-type: The mericarpids show a centrally to slightly ventrally positioned, broadly pyramidal to asymmetrical appendage (Fig. 5A–C). In contrast to the cicatrix appendages of the L. prostrata-type the appendage of the L. fruticosa-type does not include a sclerenchymatous, tubular channel. However, a sclerenchymatous tubular channel leaves the mericarpid in ventral position directly beneath the keel. The areoles are

Fig. 5. Scanning electron micrographs of mericarpids and areoles: Lithodora fruticosa-type. A, L. fruticosa (D. Thomas 7–06), mericarpid; B, L. hispidula subsp. hispidula (A. Kagiampaki 9–00), mericarpid; C, L. zahnii (D. Thomas 2–05), mericarpid. D–E, L. fruticosa (D. Thomas 7–06). D, mericarpid, attached; E, cupulate areole. F–G, L. hispidula subsp. hispidula (A. Kagiampaki 9–00). F, mericarpid, attached; G, cupulate areole. Abbreviations: am, abortive mericarpid; ca, cupulate areole; cx, cicatrix appendage; mc, mericarpid; s, style; vs, vascular strand. Scale bars = 1 mm.

88

TAXON 57 (1) • February 2008: 79–97

characterised by a hard, thin-walled, cupulate structure. In contrast to the depression of the receptaculum characteristic of the L. prostrata-type, the walls of the cupulate areole reach a height of up to ca. 1 mm above the attachment level of the abortive mericarpids (Fig. 5D–G). A sclerenchymatous tubular channel filled by a vascular strand runs


through the ventral wall of the areole and is recognisable as a ridge on the inner wall. It slopes down the wall and inwards at the bottom of the cupulate areole (Fig. 5E, G). This channel is a continuation of the channel which leaves the mericarpid in ventral position directly below the keel (L. fruticosa, L. hispidula subspp., L. zahnii ).

Fig. 6. Scanning electron micrographs of mericarpids and areoles: Lithodora prostrata-type. A, L. prostrata (T. Raus 20775), mericarpid; B, L. diffusa (D. Thomas 5–05), mericarpid; C, L. rosmarinifolia (E. di Gristina 15.02.03), mericarpid; D, L. oleifolia (H. Hilger 25.5.04), mericarpid; E, L. nitida (D. Thomas 1–05), mericarpid; F, L. diffusa (D. Thomas 5–05), areole; G, L. prostrata (T. Raus 20775), mericarpid, still attached; areole; H, L. diffusa (D. Thomas 5–05), mericarpid, still attached; areole; I, L. nitida (F. Garcia & al. 2378/79), areole. Abbreviations: am, abortive mericarpid; ca, cupulate areole; cx, cicatrix appendage; mc, mericarpid; oa, oblique areole; s, style; vs, vascular strand. Scale bar = 1 mm.

89


2. Lithodora prostrata-type: The mericarpids show a ventrally to centrally positioned, peg-like cicatrix appendage mainly consisting of a sclerenchymatous, tubular channel (Fig. 6A–D). After detachment this channel is recognisable as a hole at the proximal end of the appendage. In L. nitida the “appendage” is only very weakly developed and the sclerenchymatous funicular channel is only slightly protruding in ventral position (Fig. 6E). The areoles are cup-shaped, thick-walled depressions in the receptaculum in all species in this group except of L. nitida (Fig. 6F–H). In that latter species the oblique areole exhibits only a weakly developed depression in ventral position (Fig. 6I). The remains of the vascular strand, which occupied the tubular channel in the cicatrix appendage before the mericarpid detachment, arise bristle-like in ventral to central position from the bottom of the cupulate areole (L. diffusa, L. moroccana, L. nitida, L. oleifolia, L. prostrata subspp, L. rosmarinifolia) (Fig. 6F–I).

DISCUSSION Johnston’s interpretation of the carpological conditions in Lithodora as unique in the whole Boraginaceae and as crucial differential characters of the genus (Johnston, 1953b) is recognised as erroneous on the basis of the data presented here. Lithodora is polyphyletic and falls into two clades: “Lithodora I”, which comprises the western Mediterranean L. fruticosa, and the eastern Mediterranean species L. hispidula and L. zahnii. This clade is closely allied with the three monotypic, Mediterranean genera Mairetis, Paramoltkia and Halacsya. “Lithodora II”, which comprises the western Mediterranean species L. nitida, L. oleifolia, L. moroccana, L. rosmarinifolia, L. prostrata and L. diffusa, and is closely allied to Lithospermum s.str. and Buglossoides. The two clades here retrieved do not coincide with the infrageneric classifications of Lithodora by Johnston (1953b). Section Allostema is polyphyletic as L. hispidula and L. zahnii are retrieved in “Lithodora I”, while L. oleifolia, L. moroccana and L. rosmarinifolia are retrieved in “Lithodora II”. The molecular data receive clear support from the reexamination of carpological characters: “Lithodora I” is characterised by a broad cicatrix appendage, which does not include a sclerenchymatous tubular channel. However, a sclerenchymatous tubular channel leaves the mericarpid in ventral position directly beneath the keel and continues into the ventral wall of thin-walled cupulate areole (L. fruticosa-type, Fig. 5). In contrast to the depressions of the receptacle found in “Lithodora II”, the cupulate areoles in “Lithodora I” are formed by a hard, thin-walled structure, which rises distinctly above the attachment level of the abortive mericarpids. Johnston (1953b) already described 90

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this syndrome in his treatise of Lithodora as an aberrant condition found only in L. fruticosa. However, neither he nor Seibert (1978) recognised the similarity of this syndrome to the condition found in L. hispidula and L. zahnii and the taxonomic value of this character set. “Lithodora II” is characterised by a peg-like cicatrix appendage which mainly consists of a sclerenchymatous, tubular channel. The areoles are thick-walled depressions of the receptacle and show the remains of the vascular strand, which occupied the tubular channel in the cicatrix appendage before the detachment of the mericarpid (L. prostrata-type, Fig. 6). The morphology of the mericarpids and areoles of L. nitida is exceptional. This species exhibits only a weakly developed, ventrally positioned mericarpid “appendage” and an oblique cicatrix with a weakly developed depression in ventral position. This mericarpid morphology resembles the conditions found in Lithospermum s.str. and might represent the plesiomorphic condition. Floral morphology provides some additional characters differentiating the two clades of Lithodora. All species of Lithospermum s.str., many species of Buglossoides and all species in “Lithodora II” share the presence of threecelled gland-tipped trichomes in the corolla throat. These three-celled glandular trichomes are absent in “Lithodora I” and in the closely allied species Paramoltkia doerfleri, Mairetis microsperma and Halacsya sendtneri. Moreover, the corolla tubes of all species in “Lithodora II” are abaxially pubescent, as are the corollas in Lithospermum s.str. and Buglossoides, whereas the corolla tubes of species of “Lithodora I”, and Paramoltkia, Halacsya and Mairetis are abaxially glabrous. Lithodora falls into two fairly derived clades in mostly herbaceous Lithospermeae and its shrubby, evergreen growth form clearly has to be considered as a derived trait and likely a secondary adaptation to the dry Mediterranean climate. Woody chamaephytes have evolved many times independently from herbaceous ancestors in Boraginaceae, including several taxa within otherwise herbaceous Lithospermum s.str. (e.g., Lithospermum gayanum (Wedd.) I.M. Johnst.). The interpretation of a shrubby habit as indicative of a “primitive type” (Riedl, 1968) clearly has to be rejected on the basis of phylogenetic data (see also Böhle & al., 1996 on Echium). At first glance it seems surprising that stylar polymorphisms are present in both “Lithodora I” and “Lithodora II”, since they represent complex morphological and physiological syndromes. However, stylar polymorphism is already known to have evolved several times independently within Lithospermeae (see review in Ganders, 1979a): They are found in at least seven North American species of Lithospermum s.str. (Johnston, 1952; Ganders, 1979b; Ralston, 1993), and twelve species of Arnebia (Johnston, 1952, 1954a, including Huynhia pulchra (Roem. & Schult.) Greuter & Burdet). Together with the two inde-

TAXON 57 (1) • February 2008: 79–97

pendent arisals in “Lithodora I” and “Lithodora II”, stylar polymorphisms appear to have arisen at least four times independently in this tribe alone (see Fig. 2). Stylar polymorphisms are also known in Boraginaceae tribe Boragineae, e.g., in Anchusa L. (Philipp & Schou, 1981; Schou & Philipp, 1984; Selvi & Bigazzi, 2003) and Pulmonaria L. (Olesen, 1979; Richards & Mitchell, 1990), and also in Boraginaceae tribe Eritricheae in several species of Amsinckia Lehm. and in Cryptantha Lehm. ex G. Don (Casper & al., 1988; Schoen & al., 1997; Li & Johnston, 2001). Boraginaceae thus represent an excellent model group for the study of the evolution of stylar polymorphisms and correlated issues (breeding systems in sister groups, life history traits) and our future research will be directed towards addressing some of these questions. Seibert (1978) and Hilger & al. (1993) observed that calcium carbonate (CaCO3) incrustation in the outer pericarp is characteristic of the Lithospermeae and of related taxa, which are now placed in the Echiochileae (Antiphytum, Echiochilon, Ogastemma, Sericostema). This character might, therefore, represent a plesiomorphic state in these two tribes. Seibert sampled 53 species in 23 genera in the Lithospermeae. Within his sample, calcium carbonate incrustations were absent only in a few taxa: Pontechium Böhle & Hilger, Echium L., Echiostachys Levyns, and Lobostemon Lehm., all of which are closely allied (“tribe Echieae Dumort.”, Hilger & Böhle, 2000; Buys, 2006), and in “Lithodora I”, Halacsya and Paramoltkia, which are retrieved in clade A in our molecular analysis. Interestingly, calcium carbonate is also absent in a single species of “Lithodora II”, L. prostrata, but present in others (L. oleifolia, L. rosmarinifolia) and the closely related genera Buglossoides and Lithospermum. Calcium carbonate incrustation seems to have been lost at least three times within Lithospermeae: in the Echium alliance, in “Lithodora I” and closely allied taxa, and in L. prostrata. In the latter species the lack of calcium carbonate in the pericarp might be correlated with edaphic conditions. Lithodora prostrata prefers acidic soils, while L. oleifolia and L. rosmarinifolia are calcicolous (Fernandes, 1972). Calcium carbonate incrustations are also absent in both the calcicolous taxon “Lithodora I” and the closely related obligate Balkan serpentine endemics Halacsya and Paramoltkia. Close relationships between calcicolous taxa and serpentine endemic taxa are typical and have been reported for various Mediterranean plant groups (Stevanović & al., 2003). The phylogenetic data here presented surprisingly confirm the systematic justification of most Old World monotypic Lithospermeae genera recognised in the literature (Neatostema, Mairetis, Halacsya, Paramoltkia). The segregation of Neatostema and Mairetis from Lithospermum (Johnston, 1953b) and of Paramoltkia from Moltkia (Greuter, 1981) on the basis of morphological characters


is vindicated by the molecular analysis presented here. The genera Mairetis and Neatostema (and probably also Moltkiopsis), which have been placed in Trigonotideae (Riedl, 1967, 1968, 1997) and Eritricheae (Johnston, 1954b) in the past, are clearly nested in Lithospermeae and form a well-supported, exclusively Mediterranean clade together with Lithodora s.str., Halacsya and Paramoltkia. These molecular data support the original classification of Johnston (1953a) and the fruit anatomical data of Seibert (1978). Also, the removal of Paramoltkia doerfleri from Moltkia is clearly supported. Paramoltkia is not found to be closely associated to Moltkia at all, but is allied with Halacsya. The fruit anatomical similarities of Paramoltkia and Halacsya were already recognised by Seibert (1978) and Greuter (1981). However, these authors suggested a close affinity of these two genera to Echium and Lobostemon, probably mainly based on the lack of calcium carbonate incrusts in the pericarp in all of these taxa and the zygomorphic flowers of Halacsya. The results of our molecular analysis do not support this placement, neither Halacsya nor Paramoltkia are closely allied with Echium. The zygomorphic flowers of Halacsya arose independently of those of Echium from radially symmetrical ancestors.

TAXONOMIC CONSEQUENCES The molecular and the morphological data presented here clearly necessitate taxonomic changes to accommodate the new phylogenetic insight of Lithodora sensu Johnston (1953b) being polyphyletic. Since L. fruticosa is the type of the generic name (Johnston, 1953b: 259) the genus bearing that name has to be restricted to “Lithodora I” including L. fruticosa, L. hispidula and its subspecies, and L. zahnii. This redefined genus Lithodora is closely allied to the Mediterranean monotypic genera Paramoltkia, Halacsya and Mairetis. Within this group “Lithodora I” is morphologically easily distinguishable by its fruit morphology and growth form, while the whole group is morphologically heterogeneous. Because of the presence of a variety of autapomorphies in all of the monotypic genera (Johnston, 1953a, b; Fernandes, 1972; Greuter, 1981) closely allied with “Lithodora I”, it would be counter-productive to unite them and to create a morphologically heterogeneous taxon solely based on molecular data. “Lithodora II” is retrieved in a clade comprising the large genus Lithospermum s.str., but also Buglossoides and the North American segregates Macromeria and Onosmodium. Extended sampling would very likely show that all New World segregate genera (Lasiarrhenum I.M. Johnst., Nomosa I.M. Johnst., Perittostema I.M. Johnst., Psilolaemus I.M. Johnst.) also fall into this latter clade. To accommodate the phylogenetic data in the classification 91

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of “Lithodora II” there are two options: A complete taxonomical conflagration, i.e., reduction of the genera named under a very widely defined genus Lithospermum, or the segregation of “Lithodora II” into a new, distinct genus. “Lithodora II” has a variety of morphological characters clearly distinguishing it from Lithospermum and the other taxa of this clade (such as shrubby growth form, lack of faucal appendages and an annulus, and the set of carpological characters described above) and a segregation of the group into a new genus seems a more desirable solution. No published genus name for the group is available for resurrection, since the only possible genus synonym of Lithodora is Gymnoleima Decne., which has been typified on a species of Moltkia (Johnston, 1953b: 270–71). We therefore advocate the creation of a new genus for the clade “Lithodora II”. Glandora D.C. Thomas, Weigend & Hilger, gen. nov. – Type: Glandora prostrata (Loisel.) D.C. Thomas ≡ Lithospermum prostratum Loisel. ≡ Lithodora prostrata (Loisel.) Griseb. Etymology: The generic name is a blend of glandulosus (latinized for glandular, a reference to the glandular corolla throat) and the genus name Lithodora. Differt ab Lithodora cicatricibus nucularum appendiculatis canale funiculi inclusis, fauce corollae glandulosa, corolla extus strigosa vel pubescenta (versus glabra). Differt ab Lithospermo et Buglossoide absentia annuli ad basem tubi corollae, fauce corollae non appendiculata, areolis receptaculi cupulatis vel obliquis; a Buglossoide etiam differt floribus dimorphis. Dwarf shrubs with decumbent, ascending, or rarely erect stems, 5–60 cm high; underground stolons sometimes present. Leaves alternate, sessile, narrowly oblong, narrowly elliptic or narrowly obovate, slightly attenuate at base, 0.5–6.5 cm long and 1–20 mm wide; leaf margin entire, sometimes revolute; indumentum hispid, strigose or tomentose. Inflorescences terminal, 2–10(–14) flowered cymoids (cincinni), frondose-bracteose. Flowers sessile, subsessile or rarely shortly pedicellate (Glandora oleifolia), actinomorphic, pentamerous, distylous; calyx 5-cleft almost to base with linear or narrowly triangular lobes, accrescent at maturity; corolla blue, purple, or rarely white, narrowly funnel-shaped, abaxially hairy, adaxially with glandular hairs in the throat, otherwise glabrous or with 5 patches or a ring of trichomes in the throat or rarely with 5 patches of trichomes along the veins of the corolla lobes (G. oleifolia) or 5 vertical bands of trichomes extending from the base of the corolla throat downward to below the middle of the corolla tube (G. diffusa), 5-lobed, lobes ovate or subcircular, erect, ascending or horizontally spreading, faucal scales absent, annulus absent; stamens 5, included (long-styled morphs) or slightly exserted (short-styled 92

morphs), filaments usually inserted at the same height, rarely at two or three distinctly different levels (G. prostrata), filament insertion at the distal part of the corolla tube (short-styled morphs) or in the median part of the tube (long-styled morphs), anthers oblong or subelliptical, apex and base rounded or emarginate; gynoeceum superior, ovary four-parted, with a diffuse nectary in its proximal portion, gynobase flat, style long and exserted (long-styled morph) or short and included (short-styled morph), stigmas 2, terminal at the minutely two-lobed or notched style apex. Mericarpids usually 1–3(–4) per flower due to abortion, straight or slightly incurved in the distal part, ventrally keeled, dorsal side convex, surface smooth or minutely tuberculate (G. prostrata) or slightly tumulose (G. nitida), whitish, greyish or brownish, cicatrix basal, with one peg-like appendage, which integrates a sclerenchymatous tubular channel, or rarely flat apart from a minutely protruding sclerenchymatous tubular channel in ventral position (G. nitida), areoles formed by a cupulate depression in the receptaculum, or rarely areoles oblique with only a weakly developed depression in ventral position (G. nitida). Key to the species of Glandora, Lithodora and allied taxa

1 1* 2

2*

3

3*

4

4*

Corolla tube abaxially hairy and/or corolla throat with glandular hairs . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Corolla tube abaxially completely glabrous, corolla throat without glandular hairs . . . . . . . . . . . . . . . . 13 Stamens inserted just above the base of the corolla. Flowers small ( < 7 mm), yellow. Corolla lobes glanduliferous on both sides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neatostema apulum (L.) I.M. Johnst. Stamen inserted higher up in corolla tube, or when inserted near the base, then flowers distinctly larger ( > 11 mm) and blue. Corolla lobes not glanduliferous, or only glanduliferous at the base of the adaxial surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Stamens attached at two different levels at the corolla tube: Two stamina with shorter filaments attached slightly lower than three stamina with slightly longer filaments, the two shorter, lower stamina separated by a longer one. Annulus well developed as a hairy ring. Calyx circumscissile at the base. Mericarpids slightly asymmetrical with oblique ventral keel . . . . . . . . . . . . Moltkiopsis ciliata (Forssk.) I.M. Johnst. Stamens attached at the same level, or when stamina attached at different levels, then calyx not circumscissile and annulus missing (Glandora prostrata, see 6 and 6*). Mericarpids with straight ventral keel . . 4 Areoles flat to slightly concave. Mericarpid appendage missing or minute. Corolla with annulus and/or faucal scales or vertical pleats . . . . . . . . . . . . . . . 11 Areoles cupulate or rarely oblique with ventral depres-

TAXON 57 (1) • February 2008: 79–97

5

5* 6

6*

7

7*

8 8*

9

9*

10

sion. Mericarpids with distinct peg-like appendage or rarely mericarpid appendage only minute. Annulus and corolla invaginations missing. Glandora . . . 5 Stamina inserted at different levels in the corolla tube. Mericarpids minutely tuberculate. Glandora prostrata (Loisel.) D.C. Thomas . . . . . . . . . . . . . . . . . 6 Stamina inserted at one level in the corolla tube. Mericarpids smooth or slightly tumulose . . . . . . . . . . 7 Prostrate to ascending dwarf shrubs. Adaxial corolla with a ring of trichomes in the upper corolla tube. Indumentum of abaxial leaf surface monomorphic with a continuum of shorter to longer, appressed or semi-erect, acroscopic trichomes . . . . . . . . . . . . . . . . .Glandora prostrata (Loisel.) D.C. Thomas subsp. prostrata Erect or ascending dwarf shrubs. Adaxial corolla surface with 5 vertical, small fields of trichomes in the upper corolla throat, or throat nearly glabrous. Indumentum of abaxial leaf surface often dimorphic with longer, straight, semi-erect to appressed acroscopic trichomes and much shorter, often bend, appressed to spreading trichomes . . . . . . . . . . . . . . . . . . . . . . . . . .Glandora prostrata (Loisel.) D.C. Thomas subsp. lusitanica (Samp.) D.C. Thomas Leaves adaxially and abaxially with a dense strigosetomentose indumentum. Trichomes of both the adand abaxial leaf surface predominantly acroscopic but some trichomes basiscopic or pointing in various directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Glandora moroccana (I.M. Johnst.) D.C. Thomas Leaves adaxially with a strigose or hispid indumentum. Trichomes, at least of the adaxial leaf surface, strictly acroscopic . . . . . . . . . . . . . . . . . . . . . . . . 8 Leaves obovate, narrowly obovate or narrowly elliptic, not more than four times longer than wide . . 9 Leaves narrowly oblong, narrowly elliptic or rarely narrowly obovate, the larger leaves more than four times longer than wide . . . . . . . . . . . . . . . . . . . . . 10 Leaves silvery because of a dense strigose indumentum on both sides. Mericarpids straight. Flowers very shortly pedicellate ( –1 mm). Cicatrices almost flat, apart from a minute, ventrally protruding funicular channel. Areoles oblique, with only a minor depression in ventral position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Glandora nitida (Ern) D.C. Thomas Adaxial leaf surface with a moderately dense, strigose indumentum, abaxial leaf surface whitish, with a dense strigose-tomentose indumentum. Flowers evidently pedicellate (1–8 mm). Cicatrices with a conspicuous peg-like appendage. Areoles cupulate . . . . . . . . . . . . . . . . . . Glandora oleifolia (Lapeyr.) D.C. Thomas Ascending to erect dwarf shrubs. Corolla purple, adaxially glabrous apart from stipitate glands in the corolla throat. Leaves with a strigose indumentum of


tightly appressed trichomes . . . . . . . . . . . . . . . . . . . . . . . . Glandora rosmarinifolia (Ten.) D.C. Thomas 10* Prostrate to ascending dwarf shrubs. Corolla blue, with 5 vertical lines of trichomes in the upper part of the corolla tube, and stipitate glands in the corolla throat. Leaves with a hispid indumentum of appressed to semi-erect trichomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glandora diffusa (Lag.) D.C. Thomas 11 Corolla blue or purple, 15–20 mm long. Corolla throat and tube exhibiting 5 distinct vertical pleats bearing hairs and/or stipitate glands, and 5 congregations of glandular hairs directly beneath the attachment of the anthers. Perennial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buglossoides Moench sect. Margarospermum (Rchb.) I.M. Johnst. 11* Corolla yellow, orange, whitish or when blue or purple corolla either much shorter (–10 mm) or corolla invaginations completely lacking. Corolla invaginations lacking, or 5 faucal scales present, or when 5 weakly developed vertical invaginations present, then plants annual to biennial, corolla at the most 10 mm long and congregations of glandular hairs directly beneath the attachment of the anthers missing . . . . . . . . . . . . 12 12 Plants annual or biennial. Corolla inside bearing 5 vertical lines of simple or glandular hairs. Mericarpids tuberculate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buglossoides Moench sect. Buglossoides 12* Herbaceous perennials, rarely subshrubs, shrubs or biennials to annuals. Corolla inside frequently bearing 5 faucal scales, or invaginations lacking. Mericarpids usually smooth, or smooth with few to numerous punctate depressions, rarely tuberculate, rugose or tumulose. . . . . . . . . . . . . . . . . . . . . Lithospermum 13 Corolla distinctly zygomorphic, yellow. Corolla throat with 5 non-invaginate, rounded to triangular appendages borne at or slightly below the level of the filament attachment . . . . . Halacsya sendtneri (Boiss.) Dörfl. 13* Corolla actinomorphic or with only a very weak tendency to zygomorphy, blue, purplish, pinkish or rarely white. Corolla throat without appendages . . . . . . 14 14 Annuals. Corolla very small ( < 7 mm in length). Annulus formed by a villose ring. Calyx at anthesis tubular for ca. 1/3 of its length, greatly accrescent at maturity and then tubular for ca. 1/2 to 2/3 of its length . . . Mairetis microsperma (Boiss.) I.M. Johnst. 14* Subshrubs or dwarf shrubs. Corolla larger ( > 10 mm in length). Annulus absent. Sepals free nearly to the base, calyx at most very shortly tubular . . . . . . . 15 15 Subshrub, producing simple, erect stems from a thick rhizome. Mericarpid cicatrix without a distinct appendage, only exhibiting two minute protrusions. Areoles almost flat . . . . . . . . . . . . . . . . . . . . . . . . . . . Paramoltkia doerfleri (Wettst.) Greuter & Burdet 15* Much-branched dwarf shrubs or shrubs, rhizomes 93


absent. Mericarpids with one distinct pyramidal to asymmetrical cicatrix appendage. Areoles cupulate. Lithodora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 16 Leaves narrowly obovate. Mericarpids muriculate. The branches which are terminated by an inflorescence persisting after the flowering period as leafless shoot thorns. Lithodora hispidula (Sibth. & Sm.) Griseb. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 16* Leaves linear, narrowly oblong or narrowly elliptical. Mericarpids rugulose (macroscopically smooth). Shoot thorns absent . . . . . . . . . . . . . . . . . . . . . . . 19 17 Dwarf shrub, 10–35 cm tall. Cincinni reduced, flowers mostly solitary (1[–4]flowered cincinni) . . . . . . . . Lithodora hispidula (Sibth. & Sm.) Griseb. subsp. hispidula 17* Shrubs, up to 100 cm or more. Inflorescences: cincinni or condensed double cincinni to condensed, depauperate thyrsoids, (1–)3–6(–more)-flowered . . . . . 18 18 Corolla dark blue. Inflorescences 4–6(–more)-flowered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lithodora hispidula (Sibth. & Sm.) Griseb. subsp. cyrenaica (Pamp.) Brullo & Furnari 18* Corolla with conspicuous ontogenetic colour transition from white to pinkish to purple, plants often exhibiting flowers of different colours at once on the same plant. Inflorescences (1–)3(–5)-flowered . . . . . . .Lithodora hispidula (Sibth. & Sm.) Griseb. subsp. versicolor Meikle 19 Corolla purple or blue. Leaves 6–25 mm long. Mericarpids straight or slightly incurved, suprabasally constricted, with a thicker collar at the base. Flowers exhibiting a stigma height dimorphism . . . . . . . . . . . . . . . . . . . . . . . . . . Lithodora fruticosa (L.) Griseb. 19* Corolla light blue or rarely white. Leaves 20–40 mm long. Mericarpids strongly incurved, suprabasally constricted, with a short, stout neck. Flowers distylous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lithodora zahnii (Heldr. ex Halácsy) I.M. Johnst. Glandora diffusa (Lag.) D.C. Thomas, comb. nov. ≡ Lithospermum diffusum Lag., Varied. Ci. 4: 39. 1805 ≡ Lithodora diffusa (Lag.) I.M. Johnst. in Contr. Gray Herb. nov. ser. 73: 56. 1924 – Lectotype (designated by Valdés, 1981: 1337): [Spain, Asturia], Arvas, VIIVIII, Lagasca s.n. (MA). Glandora moroccana (I.M. Johnst.) D.C. Thomas, comb. nov. ≡ Lithodora moroccana I.M. Johnst. in J. Arnold Arbor. 34: 264. 1953 – Type: [Morocco, Eastern region], Monte Bu-Ibdiren, Mauricio & Sennen 7946 (BM). = Lithospermum diffusum Lag. var. micranthum Faure & Maire. in Bull. Soc. Hist. Nat. Afrique N. 22: 56. 1931 ≡ Lithodora diffusa (Lag.) I.M. Johnst. var. 94

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micrantha (Faure & Maire) Stroh in Beih. Bot. Centralbl. 58: 212. 1938 – Type: not seen. Glandora nitida (Ern) D.C. Thomas, comb. nov. ≡ Lithospermum oleifolium Lapeyr. subsp. nitidum Ern in Senckenberg. Biol. 49: 79. 1968 ≡ Lithodora nitida (Ern) R. Fern. in Bot. J. Linn. Soc. 64: 73. 1971 – Type: [Spain, Andalusia], Sierra de Mágina, Provinz Jaén, Spanien, 1900 m Höhe, 6.6.1963, Ern s.n. (FR!). Glandora oleifolia (Lapeyr.) D.C. Thomas, comb. nov. ≡ Lithospermum oleaefolium Lapeyr., Hist. Pl. Pyrénées, Suppl.: 28. 1818 ≡ Lithodora oleifolia (Lapeyr.) Griseb., Spicil. Fl. Rumel. 2: 531. 1846 – Type: not seen. Glandora prostrata (Loisel.) D.C. Thomas, comb. nov. ≡ Lithospermum prostratum Loisel., Fl. Gall. 1: 105. 1806 ≡ Lithodora prostrata (Loisel.) Griseb., Spicil. Fl. Rumel. 2: 85. 1846 – Lectotype (designated here): [France, Aquitania], sur le bord des chemins et dans les landes aux environs de Bayonne, 3 juin 1803, Loiseleur-Deslongchamps s.n. (AV [digital photograph!; “lectotype” noted by Aizpuru & Catalan on the herbarium sheet, dated 16. 12. 1988]; isotype: AV [digital photograph!]). Glandora prostrata (Loisel.) D.C. Thomas subsp. lusitanica (Samp.) D.C. Thomas, comb. nov. ≡ Lithospermum lusitanicum Samp., Herb. Port.: 123. 1913 ≡ Lithodora diffusa (Lag.) I.M. Johnst. subsp. lusitanica (Samp.) P. Silva & Rozeira in Agron. Lusit. 24: 170. 1962 ≡ Lithodora prostrata subsp. lusitanica (Samp.) Valdés in Bol. Soc. Brot. ser. 2. 53.2: 1336. 1981 ≡ Lithodora lusitanica (Samp.) Holub in Preslia 58: 302. 1986 – Type: not seen. = Lithospermum prostratum Loisel. var. erectum Coss., Notes Pl. Crit. 1: 42. 1849 ≡ Lithospermum diffusum Lag. var. erectum (Coss.) Rouy, Fl. France 10: 314. 1908 ≡ Lithospermum fruticosum L. subsp. diffusum var. erectum (Coss.) Rouy ex Jahand. & Maire, Cat. Pl. Maroc. 3: 602. 1934 ≡ Lithodora diffusa (Lag.) I.M. Johnst. var. erecta (Coss.) Stroh in Beih. Bot. Centralbl. 58: 212. 1938 – Type: [Spain, Andalusia], Au Picacho de Alcalá de los Gazules, 20 Avril 1849, E. Bourgeau 345 (P-CO, as “Bourgeau 245” [photocopy!]). Cosson (1848: 42) cites a collection with the number 345, the number 245 on a new herbarium label with otherwise identical data is almost certainly a transcription error. Glandora rosmarinifolia (Ten.) D.C. Thomas, comb. nov. ≡ Lithospermum rosmarinifolium Ten., Fl. Napol. Vol. 1. Suppl. 2: 66. 1811–15 ≡ Lithodora rosmarinifolia (Ten.) I.M. Johnst in Contr. Gray Herb. nov. ser. 73: 56. 1924 – Type: not seen.

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Appendix. Voucher data and GenBank accession numbers. An asterisk identifies Genbank accessions reported in previous studies (Böhle & al., 1996; Hilger & al., 2004).

Taxon: voucher number or collection date (herbarium): collection location; Genbank accession numbers: ITS1, trnLUAA intron. Outgroup species Tribe Boragineae: Anchusa officinalis L.: H. Hilger 25.06.2000 (BSB): Germany; AY045710*, AY045703*. Borago officinalis L.: H. Hilger 25.06.2000 (BSB): Germany; AY383283*, AY383245*. Tribe Echiochileae: Echiochilon fruticosum Desf.: H. Hilger 11/94 (BSB): Israel; EU044843, EU044881. Ogastemma pusillum (Coss. & Durieu ex Bonnet & Barratte) Brummitt: M. Bigazzi & F. Selvi 04.35 (BSB): Tunisia; EU044842, EU044880. Ingroup species Tribe Lithospermeae: Alkanna sieberi A. DC.: A. Kagiampaki 2000/10 (BSB): Greece; EU044844, EU044882. Alkanna tuberculata (Forssk.) Meikle: Hilger 11.1.00 (BSB): culta HB Graz; EU044845, EU044883. Arnebia coerulea Schipcz.: K. Rechinger 33894 (M): Afghanistan; EU044856, EU044894. Arnebia decumbens (Vent.) Coss. & Kralik: D. Podlech 32857 (M): Tunisia; EU044857, EU044895. Buglossoides arvensis (L.) I.M. Johnst. subsp. arvensis: M. & K. Weigend 2000/991 (BSB, M, MO): Germany; EU044865, EU044903. Buglossoides incrassata (Guss.) I.M. Johnst.: H. Hilger 17.05.99 (BSB): Greece, Crete; EU044866, EU044904. Bu96

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Appendix. Continued.

glossoides purpureocaerulea (L.) I.M. Johnst.: O. Mohr 582 (BSB): Romania, culta HB Berlin-Dahlem; EU044864, EU044902. Buglossoides tenuiflora (L. f.) I.M. Johnst.: M. Ristow & B. Seitz 12.03.98 (BSB): Israel; EU044867, EU044905. Echium vulgare L.: U.-R. Böhle s.n. (BSB): Germany; L43310*, L43312*. Echium wildpretii H. Pearson: U.-R. Böhle s.n. (BSB): Spain, Tenerife; L43314*, L43316*. Halacsya sendtneri (Boiss.) Dörfl.: D. Uzunov & al. 17.05.2003 (BSB): Yugoslavia; EU044847, EU044885. Lithodora diffusa (Lag.) I.M. Johnst.: H. Scholz & P. Hiepko 947 (B): Spain; EU044862, EU044900. Lithodora fruticosa (L.) Griseb. E. Zippel 02–26 (BSB): Spain; EU044853, EU044891. Lithodora hispidula (Sibth. & Sm.) Griseb. subsp. hispidula: A. Kagiampaki 2000/9 (BSB): Greece, Crete; EU044852, EU044890. Lithodora moroccana I.M. Johnst.: D. Podlech 51478 (MSB): Morocco; EU044860, EU044898. Lithodora nitida (Ern) R. Fern.: D. Thomas 1–05 (BSB): Spain, culta HB Berlin-Dahlem; EU044859, EU044897. Lithodora oleifolia (Lapeyr.) Griseb.: D. Thomas 9–05 (BSB): Spain, culta HB Berlin-Dahlem; EU044858, EU044896. Lithodora prostrata (Loisel.) Griseb. subsp. prostrata: P. Garin 17735 (B): Spain; EU044863, EU044901. Lithodora rosmarinifolia (Ten.) I.M. Johnst.: E. di Gristina 15.02.03 (BSB): Italy; EU044861, EU044899. Lithodora zahnii (Heldr. ex Halacsy) I.M. Johnst.: D. Thomas 2–05 (BSB): Greece, culta HB Marburg; EU044851, EU044889. Lithospermum afromontanum Weim.: M. Richards 25843 (M): Tanzania; EU044873, EU044911. Lithospermum caroliniense (J.F. Gmel.) MacMill.: R. Thomas & S. Thomas 148354 (NY): U.S.A.; EU044876, EU044914. Lithospermum cinereum DC.: O. Leistner 2109 (M): South Africa; EU044874, EU044912. Lithospermum distichum Ortega: R. Fernández 2529 (NY): Mexico; EU044879, EU044917. Lithospermum gayanum (Wedd.) I.M. Johnst.: M. Weigend & C. Schwarzer 8060 (BSB, HUT, USM): Peru; EU044878, EU044916. Lithospermum latifolium Michx.: S. Hill 30330 (NY): USA; EU044887, EU044915. Lithospermum mirabile Small: R. Worthington 10288 (NY): U.S.A.; EU044875, EU044913. Lithospermum officinale L.: A. Werres & M. Ristow 15.7.98 (BSB): Germany; EU044872, EU044910. Macromeria longiflora Sessé & Moç. ex D. Don: J. Rzedowski 34254 (NY): Mexico; EU044871, EU044909. Macromeria viridiflora DC.: E. Lehto & al. 20212 (NY): U.S.A.; EU044870, EU044908. Mairetis microsperma (Boiss.) I.M. Johnst.: D. Podlech 48277 (MSB): Morocco; EU044849, EU044887. Moltkia petraea (Tratt.) Griseb.: H. Hilger 22.09.1999 (BSB): culta HB Berlin-Dahlem; EU044854, EU044892. Moltkia suffruticosa (L.) Brand in Koch: F. Selvi 01.28 (BSB): Italy; EU044855, EU044893. Neatostema apulum (L.) I.M. Johnst.: M. Bigazzi & F. Selvi 01.09 (BSB): Greece; EU044850, EU044888. Onosmodium bejariense A.DC. in DC.: B. Ertter 5317 (NY): U.S.A.; EU044868, EU044906. Onosmodium molle Michx.: R. Thomas & al. 128151 (NY): U.S.A.; EU044869, EU044907. Paramoltkia doerfleri (Wettst.) Greuter & Burdet: H. Hilger 25.2.2000 (BSB): Albania, culta HB Berlin-Dahlem; EU044848, EU044886. Podonosma orientalis (L.) Feinbrun: H. Hilger 26/94 (BSB): Israel; EU044846, EU044884.

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