TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides Non-monophyly of Buglossoides (Boraginaceae: Lithospermeae): Phylogenetic and morphological evidence for the expansion of Glandora and reappraisal of Aegonychon Lorenzo Cecchi,1 Andrea Coppi,2 Hartmut H. Hilger3 & Federico Selvi2 1 Università degli Studi di Firenze, Museo di Storia Naturale, sezione botanica “Filippo Parlatore”, Via G. La Pira 4, 50121 Florence, Italy 2 Università degli Studi di Firenze, Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente (DISPAA), Laboratori di Botanica, Piazzale delle Cascine 28, 50144 Florence, Italy 3 Freie Universität Berlin – Institut für Biologie – Systematische Botanik und Pflanzengeographie, Altensteinstr. 6, 14195 Berlin, Germany Author for correspondence: Federico Selvi, federico.selvi@unifi.it DOI http://dx.doi.org/10.12705/635.4 Abstract The phylogeny of the small Old World genus Buglossoides and its position in tribe Lithospermeae was investigated using nrDNA and cpDNA sequences and morphology. Maximum parsimony and Bayesian analyses of ITS-5.8S and trnL-trnF IGS datasets consistently show that this group is close to Glandora and Lithospermum but not monophyletic. Of the seven species usually included, two were retrieved in the genus Glandora, i.e., B. goulandrisiorum from northern Greece and B. gastonii from the western Pyrenees. Based also on morphology and ecology, the placement of these two rare, rupicolous endemics in Glandora is here advocated and new combinations are made. The rest of Buglossoides includes two early-diverging clades, one with annual taxa of section Buglossoides and one with the three perennials of section Margarospermum. Morphological, palynological and ecological data support the separation of these two groups in distinct genera, Buglossoides s.str. and the old but largely neglected Aegonychon. Within Buglossoides, two main clades correspond to the B. arvensis and B. incrassata complexes. These show a largely sympatric distribution from the south Mediterranean to central and northern Europe. Combined with their strong phenotypic polymorphism, this causes difficulties in the distinction between taxa of the two clades, especially without characteristic cotyledons or fruiting material. Molecular and morphological evidence clearly support the transfer of the west Mediterranean B. arvensis subsp. permixta to the B. incrassata complex. Keywords Aegonychon; Boraginaceae; Buglossoides; Glandora; ITS; morphology; pollen; phylogeny; trnL-trnF IGS Supplementary Material Electronic Supplement (Table S1) and alignment are available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax INTRODUCTION Buglossoides Moench is a small genus originally based on B. ramosissima Moench (= Lithospermum tenuiflorum L.f.) and then circumscribed by the monographer Johnston (1954) as comprising seven species native to the Old World, mostly concentrated in the Mediterranean region. They are distinct from those of the closely related Linnaean genus Lithospermum by mainly the shortly apiculate anthers and the arrangement of glanduliferous hairs in longitudinal bands or inflexed pleats within the corolla tube vs. the gibbose, invaginated faucal scales present in most taxa of Lithospermum, including the type L. officinale L. In the absence of clear phylogenetic evidence for its distinctiveness, however, Buglossoides has remained a poorly defined generic unit with controversial limits (Al-Shehbaz, 1991) and was often included in a broadly defined Lithospermum (Greuter & al., 1984; Zhu & al., 1995; Jeanmonod & Gamisans, 2007). In Johnston’s (1954) opinion Buglossoides comprised two groups that he ranked as sections. The first is B. sect. “Eu”Buglossoides, and contains three annual species including the type (B. tenuiflora (L.f.) I.M.Johnst.), with five longitudinal bands of hairs inside the corolla and trigonous-pyriform nutlets with a strongly tuberculate-verrucose surface. Buglossoides sect. Margarospermum (Rchb.) I.M.Johnst., the second group originally established within Lithospermum (Reichenbach, 1830–1832: 336–337), comprises four perennial species with larger corollas showing strongly inflexed pleats and filaments with glanduliferous hairs, as well as globose-ovoid nutlets with a smooth, whitish surface resembling a droplet-shaped piece of porcelaine. These are L. purpurocaeruleum L. (syn. Buglossoides purpurocaerulea (L.) I.M.Johnst.), L. zollingeri A.DC. (syn. B. zollingeri (A.DC.) I.M.Johnst.), L. calabrum Ten. (syn. B. calabra (Ten.) I.M.Johnst.) and L. gastonii Benth. (“gastoni ”; syn. B. gastonii (Benth.) I.M.Johnst.). The Greek endemic L. goulandrisiorum Rech.f. (“goulandriorum”), Received: 14 Mar 2014 | returned for first revision: 27 Apr 2014 | last revision received: 20 Jun 2014 | accepted: 2 Jul 2014 | not published online ahead of inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014 Version of Record (identical to print version). 1065 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides described later (Rechinger, 1971) and then transferred to Buglossoides (Govaerts, 1996), was also placed in this latter group. However, the general resemblance in habit and fruit morphology of L. goulandrisiorum and the other taxa of B. sect. Margarospermum with species of Lithospermum induced some authors to retain them within the latter genus, therefore restricting the limits of Buglossoides to only the annual species of B. sect. Buglossoides (Edmondson, 1979; Meikle, 1985; Strid & Tan, 1991). A different view was expressed by Holub (1973), who transferred the perennial species of B. sect. Margarospermum to Aegonychon Gray, therefore considering them generically distinct from both Lithospermum and Buglossoides. This view has recently been adopted in the Boraginaceae treatment of Flora Iberica (Pastor, 2012), but not in other works. Consequently, these species still move among Lithospermum, Buglossoides and Aegonychon, while the annual species are still alternatively placed in Lithospermum or Buglossoides. Uncertainties result from the somewhat reticulate variation of morphological characters considered to be of taxonomic value and the lack of a comprehensive phylogenetic study of the whole group. Recent papers on Lithospermeae using molecular data have consistently suggested that Buglossoides should be a genus separate from a monophyletic Lithospermum also including most generic segregates from North America, such as Nomosa I.M.Johnst., Macromeria D.Don and Onosmodium Michx. (Thomas & al., 2008; Cecchi & Selvi, 2009; Ferrero & al., 2009; Weigend & al., 2009). These studies have therefore supported Johnston’s (1954) opinion and the phylogenetic significance of the characters of the corolla that he used to distinguish the two genera. They also suggested generic status for Glandora D.C.Thomas & al., a monophyletic group recently separated from Lithodora Griseb., and have shown its position in the Lithospermum s.l. clade close to Buglossoides (Thomas & al., 2008), therefore adding a third element to the problem of relationships in this group. On the other hand, incomplete taxonomic sampling in these previous studies prevented to address the monophyly of Buglossoides and to draw conclusions about the phylogeny and systematics of this group of Lithospermeae. Using morphological and molecular tools, this paper aims at completing previous work by providing a phylogenetic analysis of a complete sample of both sections of Buglossoides, plus a representative selection of Glandora, Lithospermum and other clades of Old World Lithospermeae. Secondly, this paper provides evidence on relationships within B. sect. Buglossoides that can help to address the unclear taxonomic status of specific and infraspecific taxa in this small but difficult group of the Euro-Mediterranean flora. MATERIALS AND METHODS Plant material and taxon sampling. — Most of the taxa / accessions included in this study were sampled by the authors from native populations during field trips across Mediterranean countries. Herbarium vouchers, silica-gel-dried portions of leaf tissue and glutaraldehyde-fixed samples of reproductive 1066 structures were collected for each accession. Vouchers are kept in the Boraginaceae herbarium collection of the authors in FIAF, and indicated as FI-HB with relative number. Material of the missing taxa and additional accessions for morphological observations were obtained from herbarium collections in ATH, B, C, FI, HCT, KUN, P and VER. As a result, all specific and most infraspecific taxa of Buglossoides s.l. as recognized in the main Euro-Mediterranean floristic and taxonomic literature, especially Flora Europaea (Fernandes, 1972) and Med-Checklist (sub Lithospermum; Greuter & al., 1984), were included in this analysis, with the only exception of B. glandulosa (Velen.) R.Fern. (B. sect. Buglossoides), of which it was not possible to obtain material for DNA isolation. The complete list of taxa included in this investigation is reported in Appendix 1, with vouchers and INSDC (International Nucleotide Sequence Database Collaboration) accession numbers; the geographic distribution of the Buglossoides samples is shown in Fig. 1. For a synopsis of the valid names of taxa originally published in Lithospermum and later variously combined in Buglossoides, Aegonychon, Rhytispermum Link, Margarospermum (Rchb.) Opiz and Glandora see Table S1 in the Electronic Supplement. DNA extraction and amplification. — Genomic DNA was extracted from silica-gel-dried samples of leaf tissue following a modified 2×CTAB protocol (Doyle & Doyle, 1990). The extracted DNA was quantified after agarose gel electrophoresis (0.6% w/v) in TAE buffer (1 mM EDTA, 40 mM Tris-acetate) containing 1 µg/ml of ethidium bromide by comparison with a known mass standard. Amplification of the ITS region of nuclear DNA, including ITS1, 5.8S and ITS2, was done using the primers ITS4 and ITS5 of White & al. (1990), while the plastid trnL-trnF IGS region was amplified with the primers “c” and “f ” of Taberlet & al. (1991). The IGS of L. hancockianum Oliv. could not be amplified because of the low quality of genomic DNA obtained from relatively old herbarium material. Preliminary analysis of sequence variation in the protein-coding plastid region rpoC1 was also performed on a sample of six species of Lithospermum and B. sect. Buglossoides and sect. Margarospermum. This region was tested because it had never been analysed before in Lithospermeae in spite of its potential resolving power of species relationships in angiosperms (Chase & al., 2007). Amplification procedures and primers (rpoC1F, rpoC4R) followed standard protocols retrieved from http://www.kew.org/ barcoding/protocols.html Polymerase chain reactions were performed in a total volume of 25 µl containing 2.5 µl of reaction buffer (Dynazyme II; Finnzyme, Espoo, Finland), 1.5 mM MgCl2, 10 pmol of each primer, 200 µM of each dNTP, 1 U of Taq DNA polymerase (Dynazyme II; Finnzyme) and 10 ng of template DNA. Reactions were performed in an MJ PTC-100 thermocycler (Peltier Thermal Cycler; MJ Research, St. Bruno, Quebec, Canada). Fourty amplification cycles were run with annealing temperature 50°C, annealing time 30 s and final extension for 45 s at 72°C. For trnL-trnF IGS, the PCR cycling conditions were the same as those followed by Moore & Jansen (2006) for the rps16 plastid region, and used in Weigend & al. (2013). Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides Subsequently, 5 µl of each amplification mixture were analysed by agarose gel (1.5% w/v) electrophoresis in TAE buffer containing 1 µg/ml ethidium bromide. Excess salts and primer were removed from the PCR reactions with the PCR Purification Kit (Roche, Mannheim, Germany). Automated DNA sequencing was performed directly from the purified PCR products using BigDye Terminator v.2 chemistry and an ABI310 sequencer (PE-Applied Biosystems, Norwalk, Connecticut, U.S.A.). Sequence alignment, datasets and phylogenetic analyses. — Original sequences from the three genomic regions analysed were edited with BioEdit v.7.0 (Hall & al., 1999) and checked for orthology through comparison with GenBank accessions of most closely related taxa. Multiple alignments were performed with Multalin v.5.4.1 (Corpet, 1988) and MAFFT v.5 (Katoh & al., 2005), and then carefully checked for ambiguous positions based on visual inspection of the sequencer output chromatofiles. For phylogenetic analyses, taxon sampling was expanded with a representative selection of species of the two closely related genera Glandora and Lithospermum, the sequences of which were retrieved from INSDC. Both Old and New World species of the latter genus were included, as well as the Yunnan endemic L. hancockianum Oliv., investigated here for the first time (only ITS-5.8S). Our preliminary analyses of wider datasets also including other North American species of Lithospermum and its segregates Onosmodium, Nomosa and Macromeria (Lithospermum s.l.) fully confirmed that these species are all included in a single and well-supported monophyletic clade, as demonstrated in previous studies (Weigend & al., 2009; Cohen & Davis, 2009). Our preliminary trees were topologically congruent with those obtained from reduced datasets not including the taxa listed above. In consequence, we finally excluded them from the analyses to avoid redundancy. Outgroups close to the Lithospermum clade were selected based on Cecchi & Selvi (2009), and included Moltkia Lehm. (clade D), Arnebia Forssk. (clade E), Cerinthe L. and Neatostema I.M.Johnst. (clade B), and Echium L. (clade A). Three single-marker datasets were prepared, ITS-5.8S, trnL-trnF IGS and rpoC1 which included, respectively, 53, 25 and 6 accessions representing 33, 25 and 6 taxa (Appendix 1); the larger size of the first dataset was mainly due to denser Fig. 1. A, distribution range of the five species of Buglossoides sect. Margarospermum; B, geographic origin of the taxa and accessions of B. sect. Buglossoides used in this study. Version of Record (identical to print version). 1067 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides sampling of B. sect. Buglossoides, that was adopted because of the potential usefulness of ITS-5.8S in solving relationships among closely related species. Phylogenetic analyses were first carried out for the ITS and IGS alignments (excluding rpoC1; see Results), using maximum parsimony and Bayesian methods. Gaps were coded as separate characters according to Simmons & Ochoterena (2000) using FastGap v.1.0.8 (Borchsenius, 2007), and appended at the end of the datasets. Congruence between the two datasets and respective trees was evaluated according to the procedures described by Wiens (1998). Since no conflicting well-supported clade was identified, an additional dataset consisting of concatenated ITSIGS sequences plus coded gaps was prepared for a combined analysis (25 taxa). Tree construction was first performed using PAUP* v.4.0 (Swofford, 2000), running Heuristic searches with “tree-bisection-reconnection” (TBR) branch-swapping with accelerated transformation (ACCTRAN) optimisation to infer branch (edge) lengths; MULTREES option on, ADDSEQ = random, twenty randomised replicates. All characters were weighted equally, and character state transitions were treated as unordered. Bootstrap support for clades was obtained performing a heuristic search with 1000 replicates, using TBR branch-swapping, 10 random taxon entries per replicate and MULTREES option on. The ITS-5.8S and combined ITS-IGS datasets were also analysed using Bayesian inference of phylogeny with MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003). Based on jModelTest (Posada, 2008), the best-fitting models of nucleotide substitution were GTR for ITS-5.8S, with gamma-distributed rate variation across sites, and GTR + I + Γ for trnL-trnF IGS. The analyses were performed using four incrementally heated Markov chains (one cold, three heated) simultaneously started from random trees, and run for one million cycles sampling one tree every ten generations. The stationary phase was reached when the average standard deviation of split frequencies reached 0.01. Trees that preceded the stabilization of the likelihood value (the burn-in) were discarded, and the remaining trees were used to calculate a majority-rule consensus phylogram. The trees were viewed and edited with TreeView v.1.6.6 (Page, 1996), with indication of Bayesian posterior probability (PP) values for the internal tree nodes. Micromorphology (SEM). — Fixed material was dehydrated in an acetone series, critical point-dried with liquid CO2, mounted on aluminium stubs, coated with gold and observed with an FEI ESEM-QUANTA 200 scanning electron microscope (SEM) working at 30 kV. Pollen grains from dry specimens were first rehydrated in a solution of Aerosol-OT 20% (Bigazzi & Selvi, 1998) and then observed with the SEM. RESULTS Nuclear ITS-5.8S dataset. — The aligned dataset of ITS15.8S-ITS2 sequences used for tree calculation was 748 bp long, including the coded gaps which were appended at the end of the matrix (positions 660–748). In the MP analysis, 344 sites were constant, 211 variable but uninformative and 228 parsimony 1068 informative. The most parsimonious trees from the heuristic search had length (L) = 953, consistency index (CI) = 0.61 and retention index (RI) = 0.84. The topology of the resulting strict consensus (not shown) was largely congruent with the 50% majority-rule consensus phylogram from the Bayesian analysis which is described here (Fig. 2); the most relevant difference is described below. The ingroup, including the members of the Lithospermum s.l. clade, was retrieved as a monophyletic assemblage (0.99 PP, 100% BS). This clade was divided in Lithospermum /Glandora on the one hand, and Buglossoides sect. Margarospermum and sect. Buglossoides on the other hand. Both these clades, however, were poorly supported in the Bayesian phylogram (Lithospermum /Glandora 0.66 PP, 67% BS; sect. Margarospermum/sect. Buglossoides 0.73 PP, BS < 50%) and were not found in the MP analysis. Here, the clade of B. sect. Buglossoides instead was sister to the rest of the ingroup, while B. sect. Margarospermum was sister to the Lithospermum /Glandora clade, although with moderate support (76% BS) only. Species of Lithospermum were retrieved in a well-supported clade (0.95 PP, 97% BS), with the rare Yunnan endemic L. hancockianum and L. tschimganicum B.Fedtsch. as successive sisters to the other members of this genus. Species of Glandora were also retrieved in a well-supported clade (0.99 PP, 81% BS), which also included B. goulandrisiorum and B. gastonii of B. sect. Margarospermum. The former species was sister to the rest of the taxa in this group, while the latter was nested among typical Glandora species and sister to G. oleifolia (Lapeyr.) D.C.Thomas and G. nitida (Ern) D.C.Thomas (0.98 PP, 68% BS). The other three members of B. sect. Margarospermum were retrieved in a well-supported clade (0.98 PP, 73% BS), with B. calabra sister to the B. zollingeri /B. purpurocaerulea clade (1.0 PP, 99% BS). The annual taxa of B. sect. Buglossoides were grouped in a strongly divergent clade with a long branch (0.95 PP, 100% BS). This contained two well-supported subclades, one with B. tenuiflora and most accessions of the B. arvensis (L.) I.M.Johnst. complex (0.96 PP, 96% BS), and one (0.96 PP, 96% BS) with all accessions of the B. incrassata (Guss.) I.M.Johnst. complex, including B. incrassata s.str. (corresponding to B. arvensis subsp. gasparrinii (Heldr. ex Guss.) R.Fern. in Flora Europaea), B. incrassata subsp. splitgerberi (Guss.) E.Zippel & Selvi and B. minima (Moris) R.Fern.; B. arvensis subsp. permixta (Jord.) R.Fern. was also included in this second subclade. Relationships within both clades were mostly unresolved and no clear relationships with geographical origin of the accessions could be observed. Plastid rpoC1 dataset. — Sequencing of the rpoC1 coding region was performed for six morphologically distinct species representing the main clades retrieved in the ITS phylogeny: B. arvensis subsp. arvensis (sample no. 1 in Appendix 1; accession no. HG939446), B. incrassata subsp. incrassata (sample 5; accession HG939451), B. gastonii (accession HG939447), B. goulandrisiorum (accession HG939449), B. calabra (accession HG939448) and Lithospermum officinale (sample 2; accession HG939450); sequences were 413 bp long, and showed only four variable positions, three of which were singletons. This low level of variation precluded use of this marker for further analysis. Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides Plastid trnL-trnF IGS dataset. — The trnL-trnF IGS alignment included 713 bp, plus coded gaps from position 714 to 755. In the parsimony analysis 612 characters were constant, 74 variable but non-informative and 69 informative. The strict consensus of most parsimonious trees (L = 171; CI = 0.85; RI = 0.80; not shown) was a large polytomy in which monophyly of Lithospermum, Glandora and B. sect. Buglossoides was supported. Relationships of the five perennial taxa of B. sect. Margarospermum to these clades remained unresolved. Combined ITS-IGS dataset. — The alignment was 1435 bp long; coded gaps appended at the end of the matrix were in position 1355–1435. In the parsimony analysis, 972 sites were constant, 182 variable but uninformative and 281 parsimony informative. The heuristic search retrieved four most parsimonious trees with L = 797, CI = 0.73 and RI = 0.77; the resulting strict consensus was fully consistent with the Bayesian phylogram described here (Fig. 3). The Lithospermum s.l. clade received strong support (1.00 PP, 100% BS), and Lithospermum s.str. (1.00 PP, 100% BS) was sister to the rest of the ingroup. This consisted of two clades which were sister to each other (0.97 PP, 63% BS), one (0.99 PP, 74% BS) with B. goulandrisiorum and B. gastonii as successive sisters to Glandora, and one (1.00 PP, 66% BS) with the other three species of B. sect. Margarospermum (0.99 PP, 90% BS) sister to B. sect. Buglossoides (1.00 PP, 100% BS). The latter included B. arvensis as sister to a group of four accessions of the B. incrassata complex (1.00 PP, 100% BS), also comprising B. arvensis subsp. permixta as sister to the B. incrassata Fig. 2. Bayesian 50% majority-rule consensus phylogram generated by ITS-5.8S sequences showing relationships of Buglossoides in the Lithospermum s.l. clade; posterior probability values and bootstrap support percentages > 50% are shown near nodes. Version of Record (identical to print version). 1069 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides Fig. 3. Bayesian 50% majority-rule consensus phylogram generated by combined ITS-IGS sequences showing position and relationships of Buglossoides in the Lithospermum s.l. clade; posterior probability values and bootstrap support percentages ≥ 50% are given near nodes; key characters are shown to the right of the clades (original drawings by L. Cecchi). Fig. 4. Floral and fruit morphology of: A, Buglossoides calabra, opened and intact corolla (Cecchi & Coppi FI-HB 07.56); B, B. purpurocaerulea, opened and intact corolla and mericarpid in lateral and ventral views (Bigazzi FI-HB 91.07; Cecchi, Coppi & Selvi FI-HB 06.18); C, B. gastonii, opened and intact corolla, fruiting pedicel with remains of vascular strand, mericarp in ventral and dorsal views and base of mericarpid showing cicatrix (Boissier & Reuter s.n., 1870, FI; Burnat s.n., 1868, FI); D, B. goulandrisiorum subsp. goulandrisiorum, opened and intact corolla and mericarp in dorsal and ventral views (Cecchi & Selvi FI-HB 08.38; Stamatiadou 21217, ATH). — Scale bar: flowers = 1 cm; nutlets = 0.5 cm. — Original drawings by L. Cecchi. 1070 Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides subsp. splitgerberi /B. minima group, with good support (0.91 PP, 78 BS). Inflorescences and flowers. — Since flower and inflorescence morphology of Lithospermum and Glandora have already been described thoroughly in previous papers and treatments (Johnston, 1954; Weigend & al., 2009; Cohen, 2012) only the most relevant features of Buglossoides s.l. are summarized here, with emphasis on the differences between species or groups of species. Inflorescences are basically frondose-bracteose cincinni (“cymoids”) with two to many flowers, usually distinctly elongating in fruit and with well-spaced, nutlet-bearing calyces; however, B. gastonii and B. goulandrisiorum clearly differ in having short, forked cymes with crowded flowers which remain compact and congested even in fruit. Floral characters show considerable variation among the taxa included, especially in size and internal structure of the corolla; most of the species are illustrated in Figs. 4–5. Large corollas (up to 25–30 mm in length) with a showy cup-shaped limb of blue to purple colour are typical for species in B. sect. Margarospermum. Characteristic is the presence of five longitudinal bands on the adaxial (internal) surface of the corolla, which mainly take the form of thickened hairy pleats possibly serving as guides for pollinating insects. These bands are of variable extent and position, and consist of dense trichomes often mixed with sparser and shorter glandular hairs (Fig. 6A). The trichomes are short, enlarged at the apex and obtuse, except for B. calabra where hairs are longer and acute at the apex (Fig. 6D). Stamens are always enclosed in the corolla, but filaments are inserted close to the throat in B. purpurocaerulea and B. zollingeri, while close to the base of the tube (ca. 1.5 mm above) in B. goulandrisiorum, B. calabra and B. gastonii. Very small, oblong anthers (< 1 mm) are typical of the last species, which also differs from the others in the very weakly rather than distinctly apiculate anthers. Filaments bear glanduliferous hairs which are also found along longitudinal lines below stamen insertion in B. purpureocaerulea and B. zollingeri; in B. calabra, the filament base takes the form of a rounded bulge and is covered with a congregation of such hairs (Fig. 6C). Glandular trichomes are also scattered over the internal surface of the corolla tube in B. gastonii and B. goulandrisiorum (Fig. 6B), but not in the other three species. The base of the corolla tube is never distinctly thickened or provided with hairs as usually found in Lithospermum. Small and narrowly infundibular corollas (max. 6 mm in length and 4 mm in diam.) of white, pink or blue colour are typical for taxa in B. sect. Buglossoides. Five longitudinal bands of short, appressed, papillose trichomes enlarged at the apex run from the throat (base of limb) down to the upper half of the tube (Fig. 6E). In B. tenuiflora, the inside of the lower half of the tube has sparse hairs. In all taxa, anthers are inserted on very short filaments just below the base of the bands and close Fig. 5. Floral and fruit morphology of: A, Buglossoides arvensis subsp. arvensis, fruiting calyx with bract and pedicel, opened and intact corolla and mericarpid in dorsal, ventral and lateral views (Bigazzi FI-HB 90.03); B, B. tenuiflora, fruiting calyx with bract and pedicel, opened and intact corolla and mericarpid in dorsal, ventral and lateral views (Cecchi, Coppi & Selvi FI-HB 07.13); C, B. incrassata subsp. incrassata, thickened fruiting calyx with bract and pedicel, opened and intact corolla, and mericarpid in dorsal, ventral and lateral views (Cecchi, Coppi & Selvi FI-HB 07.40); D, B. minima (Sommier s.n., 1872, FI), fruiting calyx with bract and pedicel, opened and intact corolla, and mericarpid in dorsal, ventral and lateral views. — Scale bar: flowers = 1 cm; nutlets = 0.5 cm. — Original drawings by L. Cecchi. Version of Record (identical to print version). 1071 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides to the base of the corolla tube. The anthers are shortly apiculate at the apex. A thickened annulus with sparse, short hairs is found near the base of the corolla tube (Fig. 6F). Fruits. — Nutlet morphology within Buglossoides shows striking variation in terms of size, shape and surface ornamentation. Taxa of B. sect. Buglossoides all have small (1.7–3.5 × 0.8–1.6 mm) trigonous-pyriform nutlets with a prominently verrucose-tuberculate, brownish surface (Fig. 5A–D, 6H); usually no abortion occurs, resulting in four mature mericarpids per fruit. Species of B. sect. Margarospermum often have only 1–3 mature mericarpids by abortion, and these are larger and basically ovoid (2.5–4.5 × 1.8–3.5 mm), mostly with a smooth and glossy, whitish to greyish surface and an obtuse-rotundate apex, as in most members of Lithospermum (Fig. 4B). Concerning surface ornamentation and apex, however, B. gastonii and B. goulandrisiorum are exceptions. In the former, the stout, plump nutlet is externally rugose-foveolate and has a short, blunt beak (Figs. 4C, 6G), while the latter has a smooth surface and an acute beak (Fig. 4D). The base of the nutlet of B. gastonii and, to a lesser extent, also of B. goulandrisiorum, is almost flat and broader than in the other species; the small tubular channel in the ventral position of the cicatrix area is occupied Fig. 6. SEM micrographs of floral and fruit characters. A–B, B. goulandrisiorum (Cecchi & Selvi FI-HB 08.38): A, trichomes of the longitudinal hairy bands above throat; B, lower part of corolla tube showing stamen position, glanduliferous hairs on filaments and adaxial corolla surface. C–D, B. calabra (Cecchi & Coppi FI-HB 07.56): C, gibbous base of stamen filament with glanduliferous hairs; D, trichomes of the longitudinal hairy bands above throat. E–F, B. incrassata subsp. incrassata (Cecchi, Coppi & Selvi FI-HB 07.42): E, abaxial surface of corolla showing upper part of longitudinal hairy bands at throat, with short, obtuse trichomes; F, short trichomes of the thickened annulus at the base of corolla tube. G, B. gastonii (Döbbeler FI-HB 07.58), whole mericarpid in lateral view. H, B. incrassata subsp. incrassata (Cecchi, Coppi & Selvi FI-HB 07.42), whole mericarpid in lateral view. — Scale bars: A, H = 1 mm; B–E = 200 µm; F = 100; G = 2 mm. 1072 Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides by a strand of vascular tissue the remains of which often arise as a bristle-like protrusion from the almost flat detachment areola of the gynobase (Fig. 4C). Pollen. — The main stereostructural characters of pollen within Buglossoides are summarized in Table 1 and illustrated in Fig. 7. Significant differences exist between the two sections. Grains of taxa of B. sect. Buglossoides are isopolar, while those of B. sect. Margarospermum usually show a slight asymmetry in the size of the two poles, and can be defined as subisopolar (Díez & al., 1986) or “slightly heteropolar” (Clarke, 1977). There are usually four apertures in the grains of the B. sect. Margarospermum species (Fig. 7A–D), but usually six (more rarely up to eight) in those of B. sect. Buglossoides. In the latter, a microgranulose-spinulose equatorial band connecting the ectoapertures is often visible (Fig. 7F), which is always lacking in B. sect. Margarospermum. Consequently, the rhombic shape of the well-spaced ectoapertures is more distinct in the taxa of the latter group. The small pore-like endoapertures lie in the Fig. 7. SEM micrographs of pollen grains of: A, B. zollingeri (Silvestri s.n., FI); B, B. calabra (Cecchi & Coppi FI-HB 07.56); C, B. gastonii (Doassans s.n., FI); D, B. goulandrisiorum (Cecchi & Selvi FI-HB 08.38); E, Glandora prostrata (Bicknell s.n., FI); F, B. tenuiflora (Cecchi, Coppi & Selvi FI-HB 07.15). — Scale bar = 5 µm. 1.53 isopolar 6(–7) rhombic 7.1 median granular psilate Italy B. incrassata subsp. incrassata 11.2 6.2 1.80 isopolar 6(–7) rhombic 5.1 median granular psilate Israel B. minima 13.1 9.1 1.44 isopolar 6 rhombic 7.3 median granular psilate Italy B. tenuiflora 14.0 9.8 1.43 isopolar 6 rhombic 6.5 median granular psilate Syria 11.9 8.5 1.40 subisopolar 4 rhombic 6.9 median granular psilate Italy B. purpurocaeruleum 12.6 9.5 1.32 subisopolar 4 rhombic 9.6 polar granular psilate Italy B. zollingeri 12.5 9.4 1.33 subisopolar 4 rhombic 8.3 median granular psilate Taiwan B. gastonii 11.6 8.1 1.40 isopolar 4 rhombic 7.5 median granular psilate France B. goulandrisiorum 12.0 7.9 1.40 subisopolar 4 rhombic 7.5 median granular psilate Greece Colpus membrane 8.5 Endoaperture position 13.1 Colpus length [µm] B. arvensis subsp. arvensis Shape Ectoaperture shape Equator. diam. (E) [µm] P/E Taxon Apertures Polar diam. (P) [µm] Origin of material examined (vouchers in FI) Table 1. Main pollen characters of members of Buglossoides sect. Buglossoides and sect. Margarospermum. Tectum B. sect. Buglossoides B. sect. Margarospermum B. calabra Version of Record (identical to print version). 1073 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides centre of the ectoapertures in the taxa of B. sect. Buglossoides but closer to the larger pole in those of B. sect. Margarospermum, in line with their slightly heteropolar structure. DISCUSSION Evidence for the placement of Buglossoides gastonii and B. goulandrisiorum in Glandora. — Unlike previous broad- scale phylogenetic studies of Boraginaceae (e.g., Långström & Chase, 2002; Thomas & al., 2008; Cecchi & Selvi, 2009; Weigend & al., 2009, 2013; Cohen, 2014), this work includes a nearly complete taxonomic sampling of taxa described under Buglossoides, allowing to better understand the relationships in this Old World group of Lithospermeae. First, all species of this genus as circumscribed by Johnston (1954) were confirmed to be outside Lithospermum s.str., implying that the taxonomic treatments in some reference Floras are not in line with phylogenetic evidence (Edmondson, 1979; Meikle, 1985; Strid & Tan, 1991; Jeanmonod & Gamisans, 2007). Lithospermum is a morphologically diverse monophyletic group (Weigend & al., 2009, 2010), here shown to include also L. hancockianum from the Yunnan region in south China. Pollen and fruit characters had already suggested its position in this genus (Seibert, 1978; Riedl, 1993; Zhu & al., 1995), and the present work suggests it is sister to all other species, including L. tschimganicum (≡ Ulugbekia tschmiganica (B.Fedtsch.) Zakirov) also from central Asia and of similar morphology (Johnston, 1954). On the other hand, evidence is provided that Buglossoides is not monophyletic (or “holophyletic” sensu Hörandl & Stuessy, 2010). This confirms results by Weigend & al. (2009), where the members of Buglossoides investigated were sister to Glandora and Lithospermum and did not form a single clade. Combined cpDNA and nrDNA sequence data showed that the majority of species of Buglossoides form a well-supported group, but also that the two rare endemics B. goulandrisiorum (Greece) and B. gastonii (France and Spain) are outside this group and sister to members of the central-western Mediterranean genus Glandora. This implies a noteworthy east–west Mediterranean disjunction, suggesting that the ancestor of Glandora may have been a rupicolous species formerly more widely distributed across the Mediterranean mountains, including the southern Balkans. ITS sequence data alone retrieved B. gastonii as sister to G. oleifolia and G. nitida, both restricted to the mountains of eastern and southern Spain, respectively (Pastor, 2012). Ecological characters support a relationship of B. goulandrisiorum and B. gastonii to Glandora, as they are the only two species of Buglossoides that live in open, rocky habitats similar to species of Glandora. Although morphological traits such as the shortly apiculate anther tips and the longitudinal hairy bands in the corolla would support placement of these two endemics in B. sect. Margarospermum as proposed by previous authors (Johnston, 1954; Rechinger, 1971; Aldén, 1976), other characters show their relationship to Glandora. This is particularly true for B. gastonii and G. nitida, one of the most differentiated species of 1074 the genus. In both these Pyrenean endemics the fruit surface is not smooth but slightly tumulose (G. nitida) or foveolate-rugose (B. gastonii), and the broad basal cicatrix is almost flat and without the peg-like appendage (elaiosome) found in most members of Glandora; as a consequence, the detachment areola has only a weakly developed depression instead of being cup-shaped as in the rest of the latter genus (see Thomas & al., 2008). Unlike in the other members of B. sect. Margarospermum, the remains of the vascular strand which enter the funicular canal in the cicatrix during development of the mericarpid arise bristle-like in a ventral position from the areole in B. gastonii. Although these characters are less evident in B. goulandrisiorum, this species shares with B. gastonii and G. nitida the acute, nearly beaked apex of the nutlet, and with most other Glandora species the lack of slender, long-procumbent stems (typical of B. purpurocaerulea, B. calabra and B. zollingeri), cymes which do not elongate in fruit and the scattered glandular hairs on the adaxial surface of the corolla (lacking in the other taxa of B. sect. Margarospermum). In addition, the slight pollen heteropolarity of G. nitida (Díez & al., 1986) provides another connection to species of B. sect. Margarospermum. Altogether, this suggests B. gastonii and B. goulandrisiorum to be palaeoendemics with plesiomorphic characters that have been partly retained in species of B. sect. Margarospermum and partly in species of Glandora, especially G. nitida and G. diffusa (which also has vertical hairy bands inside the corolla; Thomas & al., 2008). However, the two species are clearly early-diverging members in the Glandora clade. Reappraisal of Aegonychon. — The three remaining species of B. sect. Margarospermum form a well-supported group with B. calabra sister to the B. purpurocaerulea /B. zollingeri clade. This fits with the morphological distinctiveness of this narrow endemic of the southern Apennines, which has longitudinal bands of long, acute trichomes in the corolla, stamens inserted in the lower half of the tube (as in B. goulandrisiorum and B. gastonii) and a rounded bulge with stipitate glands at the base of the filaments. Accordingly, B. calabra is possibly the closest relative to the ancestor of the other two wide-ranging but sharply allopatric Eurasian species, which are clearly close to each other also in view of their common morphological traits (Popov, 1953; Johnston, 1954). Previous investigations did not clearly resolve relationships between members of B. sect. Margarospermum and those of sect. Buglossoides or other groups in the Lithospermum s.l. clade (Thomas & al., 2008; Weigend & al., 2009; Cohen, 2014). This study shows a deep phylogenetic divergence between the three species discussed above and those of B. sect. Buglossoides, which finds support in their morphological, palynological, and ecological differentiation. Here, we largely corroborate the observations by Johnston (1954) and provide additional palynological evidence for their separation. The shift from subisopolar, usually 4-aperturate grains to isopolar, 5–8-aperturate grains has likely been a major change from B. sect. Margarospermum to B. sect. Buglossoides (see also Díez & al., 1986). Furthermore, habit, fruit and flower characters allow an even more immediate distinction between the species of the two sections. While B. calabra, B. purpurocaerulea and Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides B. zollingeri have conserved the plesiomorphic fruit structure of most Old World Lithospermum s.str., apomorphic traits have originated in the annual taxa of B. sect. Buglossoides, such as the smaller size, the trigonous-pyriform shape, the strongly tuberculate-verrucose mericarpid surface and the frequent synaptospermic dispersal (L. Cecchi & F. Selvi, pers. obs.). These three species also have smaller flowers, as well as a broader range of corolla colours (e.g., white) and an internal rearrangement consisting mainly in the loss of glanduliferous hairs on the stamen filaments and the development of a thickened annulus at the base of the corolla tube. Reduction has also occurred in plant size, habit and life-cycle, leading to the shift from robust, often rhizomatous perennials to small, strictly therophytic (winter annual) plants. Most likely, this has proceeded in parallel with the ecological shift from mesophilous forest habitats to xerophilous, open environments such as fields (B. sect. Buglossoides). In view of the strong correlation between our phylogenetic findings, morphology and ecology, we advocate separation of B. sect. Buglossoides from B. calabra, B. zollingeri and B. purpurocaerulea into two different genera distinct from Lithospermum and Glandora. Previous authors had already restricted Buglossoides to only the annual species, but retained the perennial species in Lithospermum as originally described (Edmondson, 1979; Meikle, 1985; Strid & Tan, 1991). Generic separation of the two sections was proposed by Berchtold & Opiz (1839: 73–74) who placed L. purpurocaeruleum L. in Margarospermum (Rchb.) Opiz (see also Pouzar, 1964), although Gray (1821) had already described Aegonychon based on both L. repens Stokes (= L. purpurocaeruleum L.) and L. arvense L. This problem was resolved by Holub (1973) who showed that L. purpurocaeruleum, later typified by Selvi (in Cafferty & Jarvis, 2004), has to be accepted as the type of Aegonychon. Holub (1973) transferred all five species of B. sect. Margarospermum to Aegonychon so that the necessary nomenclatural combinations are already available: A. calabrum (“calabricum”) (Ten.) Holub, A. gastonii (Benth. ex A.DC) Holub, A. goulandrisiorum (Rech.f.) Holub, A. purpurocaeruleum (L.) Holub and A. zollingeri (A.DC.) Holub. In recent treatments, Holub’s (1973) taxonomy has been adopted in the Flora Iberica treatment (Pastor, 2012) for A. purpurocaeruleum and A. gastonii. As described above, however, we propose to place B. gastonii and B. goulandrisiorum in Glandora, therefore restricting Aegonychon to the three woodland species which form a well-supported monophyletic clade. Such treatment formally recognizes phylogenetic relationships and the major patterns of morphological diversity in the group. Relationships within Buglossoides s.str. — This work includes a broad geographical-taxonomic sampling of B. sect. Buglossoides to better understand relationships in this small but polymorphic and difficult group of annual species that are commonly found especially in dry, synanthropic habitats of the Mediterranean area and Europe (Fernandes, 1972, 1973). In line with two previous studies of this group in Alto Adige / South Tyrol (Zippel & Wilhalm, 2003) and central Europe (Clermont & al., 2003), both based on ITS 1 sequences and morphology, our analysis retrieved two well-supported sister clades: one with accessions of the B. arvensis complex and one with accessions of the B. incrassata complex. The two clades differ in 14 and 5 positions in the ITS and trnL-trnF IGS regions, respectively (see also Clermont & al., 2003). Notably, there is no geographic separation of the two clades which largely overlap in most of their ranges from North Africa to Central Europe (see Fig. 1B). The B. arvensis clade includes the typical B. arvensis subsp. arvensis and B. arvensis subsp. sibthorpiana (Griseb.) R.Fern., a weakly distinct race from mainly SE Europe and the Middle East (Strid, 2000). Buglossoides tenuiflora from arid habitats of the southeast Mediterranean is also nested in this clade, but morphological characters such as yellowish hairs on the calyx and distinctly 2-gibbous nutlets with very fragile pericarp show that this is a separate species. The B. incrassata clade includes typical B. incrassata and subsp. splitgerberi, both originally described from Sicily (Selvi & Cecchi, 2009). The two subspecies are widely distributed and largely sympatric in the Mediterranean region and the southern Alpine area (Zippel & Wilhalm, 2003), but subsp. splitgerberi extends more to the north and also occurs as a weed in Central Europe (Clermont & al., 2003, sub B. arvensis subsp. sibthorpiana). In addition, B. arvensis subsp. permixta from the western Mediterranean and B. minima endemic to Sardinia, Sicily and, possibly, south Italy, were also retrieved in this group. This clear phylogenetic result is partly matched by morphological evidence. The two synapomorphic traits for the B. incrassata complex are the circular cotyledons without secondary venation and the obliquely thickened fruit pedicel (Fig. 5C), whereas the B. arvensis complex is characterized by oblong cotyledons with distinct secondary venation and the pedicel remaining thin in fruit (Fig. 5B; Clermont & al., 2003; Zippel & Wilhalm, 2003). In B. incrassata subsp. splitgerberi, however, thickening of fruit pedicels is only partial, causing considerable difficulty in the distinction from B. arvensis s.l. (Selvi & Cecchi, 2009). Distinctly thickened fruit pedicels and circular cotyledons can instead be easily observed in material of B. arvensis subsp. permixta from southern France and Spain (L. Cecchi & F. Selvi, pers. obs. in cultivated material from the French Maritime Alps; see also Pastor, 2012), which nicely fits the position of this taxon in the B. incrassata clade. Notably, Jordan (1855: 344–346) himself noticed that the fruit pedicel of the specimens he used to describe the species was shorter and more distinctly thickened than in typical L. arvense. This character is also visible in one of the specimens (WAG 323, the only specimen with fruits that we could trace) that Jordan grew, after description of the species in 1855, from seeds of the type collection from the Hautes Alpes. This further corroborates the placement of this taxon in the B. incrassata complex, rather than in the B. arvensis complex as proposed by Fernandes (1971, 1972). Partial discrepancy between morphology and phylogenetic relationships is caused instead by the rare B. minima, which has circular cotyledons without secondary venation (L. Cecchi & F. Selvi, pers. obs. on cultivated material from Sardinia) but non-thickened fruiting pedicels (Fig. 5D), perhaps as a consequence of character loss or reversal. The presence of three 1-bp insertions in the ITS 1 sequence of this taxon and its peculiar Version of Record (identical to print version). 1075 TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides combination of characters (type depicted in Selvi & Cecchi, 2009) suggest to keep it as a separate species, at least until more data on population variation are available. The strongly polymorphic B. incrassata subsp. splitgerberi resembles B. arvensis (both subspecies) in habit and flower characters, and can only be distinguished by the partially thickened fruiting pedicels or the circular, unveined cotyledons (Zippel & Wilhalm, 2003). These characters cannot be observed in many herbarium specimens, which therefore often are virtually impossible to identify correctly. Although the distribution range of B. incrassata subsp. splitgerberi includes large parts of the east and central Mediterranean, the Middle East and Central Europe as shown here, this taxon still remains imperfectly known and deserves further investigation. Buglossoides incrassata subsp. permixta (Jord.) L.Cecchi & Selvi, comb. nov. ≡ Lithospermum permixtum Jord. in Schultz, Arch. Fl. France Allem.: 344. 1855 ≡ Buglossoides arvensis subsp. permixta (Jord.) R.Fern. in Bot. J. Linn. Soc. 64: 374. 1971 ≡ Buglossoides permixta (Jord.) Holub in Preslia 58: 301. 1986 – Type not designated, Ind. loc.: [France] “Gap (Hautes Alpes)”. Material grown by Jordan himself after 1855 from seeds of the type collection that was sent to him by Blanc from the Hautes Alpes is kept under “Lithospermum permixtum Jord.” in the herbaria of Montpellier and Wageningen (MPU019782!, MPU019783!, WAG0000323!; http://plants.jstor.org); one of these specimens could be designated as a neotype in case no original material collected before publication in 1855 is found. The thickened fruit pedicels are visible in especially WAG0000323, which includes fruiting material collected in June 1858. NEW COMBINATIONS Based on the discussion above, the following new combinations are made: Glandora gastonii (Benth.) L.Cecchi & Selvi, comb. nov. ≡ Lithospermum gastonii Benth. in Candolle, Prodr. 10: 83. 1846 (“gastoni ”; correction of the epithet’s original spelling mandated by ICN Art. 60.12) ≡ Buglossoides gastonii (Benth.) I.M.Johnst. in J. Arnold Arbor. 35: 45. 1954 ≡ Aegonychon gastonii (Benth.) Holub in Folia Geobot. Phytotax. 8: 164. 1973 – Holotype: [FRANCE]. “Rochers de Balourde en montant des Eaux-Bonnes au Pic de Gers”, Gaston (G-DC barcode G00148824!; isotype: “Rochers de Balourde en montant Pic de Gers”, Aug 1839, FI-W No. 130145!). Glandora goulandrisiorum (Rech.f.) L.Cecchi & Selvi, comb. nov. ≡ Lithospermum goulandrisiorum Rech.f. in Bot. Not. 124: 355. 1971 (“goulandriorum”; correction of the epithet’s original spelling mandated by ICN Art. 60.12). ≡ Aegonychon goulandrisiorum (Rech.f.) Holub in Folia Geobot. Phytotax. 8: 165. 1973 ≡ Buglossoides goulandrisiorum (Rech.f.) Govaerts, World Checkl. Seed Pl. 2: 14. 1996 – Holotype: “Graecia, Epirus: Montes Tymphi, in praeruptis calc. ad austro-orientem lacus Drakolimni, 1900–2000 m”, 12 Aug 1969, Stamatiadou 7244 (W!; isotype: ATH!). Glandora goulandrisiorum subsp. thessalica (Aldén) L.Cecchi & Selvi, comb. nov. ≡ Lithospermum goulandrisiorum subsp. thessalicum Aldén in Bot. Not. 129: 305. 1976 ≡ Aegonychon thessalicum (Aldén) Holub in Preslia 58: 301. 1986 ≡ Buglossoides goulandrisiorum subsp. thessalica (Aldén) Govaerts, World Checkl. Seed Pl. 2: 14. 1996 ≡ Aegonychon goulandrisiorum subsp. thessalicum (Aldén) Valdés in Willdenowia 34: 61. 2004 – Holotype: “Graecia, Thessalia: Mons Koziakas, supra pagum Elati, in praeruptis calcareis, 1900 m”, 7 Jul 1972, Aldén 151 (LD!). Paratypes: “[Graecia, Thessalia:] Mt. Koziakas, 11 km NW of Pili (near Elati), ca. 1800 m”, A[ldén] 1202; “[Graecia, Thessalia:] 5 km NE of Pertoulion, 1750–1900 m”, A[ldén] 1205. 1076 Revised key to Old World genera of the Lithospermum s.l. clade 1. Annual herbs, usually small. Corolla ≤ 7 mm long. Mericarpids trigonous-pyriform, strongly verrucose-tuberculate, up to 3.5 mm long, usually 4 per fruit ...... Buglossoides 1. Herbaceous perennials, subshrubs or shrubs (rarely biennials). Corolla ≥ 8 mm long. Mericarpids mainly ovoid, up to 4.5 mm long, frequently smooth and shiny, rarely slightly rugose-foveolate, tumulose or minutely tuberculate, usually 1–3 through abortion .................................... 2 2. Corolla frequently with gibbose, invaginated faucal scales, always without longitudinal hairy bands. Hairy annulus at the base of tube often present. Mericarpids usually smooth and shiny, often with sparse, punctate, pit-like depressions, scattered or along ventral keel, rarely rugose .............. .................................................. Lithospermum 2. Corolla without faucal scales, with or without longitudinal hairy bands. Hairy basal annulus always absent. Mericarpids smooth and shiny or rarely slightly tumulose to tuberculate, always without pit-like depressions ........ 3 3. Dwarf shrubs or cespitose perennials of open, rocky habitats, without long-procumbent, slender stems. Fruiting cymes contracted, with calyces closely appressed to nutlets. Often heterostylous. Vertical hairy bands inside corolla present or absent. Glanduliferous hairs on adaxial side of corolla tube always present. Cicatrix with a peg-like appendage, rarely flat with a minutely protruding channel (G. gastonii, G. goulandrisiorum, G. nitida); areole a cupshaped depression in the flat gynobase, or areole oblique to nearly planar with only a weakly developed depression in ventral position (G. gastonii, G. nitida) ...... Glandora 3. Perennial herbs of forest habitats, with numerous slender, long-procumbent and leafy stems. Fruiting cymes strongly elongated with well-spaced calyces. Homostylous. Vertical bands of trichomes in corolla and patches of glanduliferous hairs on stamen filaments always present. Glanduliferous hairs inside corolla tube absent. Mericarpid cicatrix without peg-like appendages; detachment areoles flat ......... .................................................... Aegonychon Version of Record (identical to print version). TAXON 63 (5) • October 2014: 1065–1078 Cecchi & al. • Non-monophyly of Buglossoides ACKNOWLEDGEMENTS Doyle, J.J. & Doyle, J.L. 1990. Isolation of plant DNA from fresh tissue. Focus (Washington, DC) 12: 13–15. Edmondson, J.R. 1979. Lithospermum L., Buglossoides Moench. Pp. 313–317 in: Davis, P.H. (ed.), Flora of Turkey and the East Aegean islands, vol. 6. Edinburgh: University Press. Fernandes, R.B. 1971. Buglossoides. P. 379 in: Heywood V. (ed.), Flora Europaea: Notulae Systematicae ad Floram Europaeam spectantes, 11. Bot. J. Linn. Soc. 64: 353–381. Fernandes, R.B. 1972. Buglossoides Moench. Pp. 87–88 in: Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine, D.H., Walters S.M. & Webb D.A. (eds.), Flora Europaea, vol. 3. Cambridge: Cambridge University Press. Fernandes, R.B. 1973. Notes sur le genre Buglossoides Moench. Acta Bot. Acad. Sci. Hung. 19: 93–101. 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Voucher information is given only for specimens originally analysed here (marked with an asterisk) or in Cecchi & Selvi (2009) (marked with a ); INSDC numbers of taxa analysed for the rpoC1 region are given in the “Results” section. OUTGROUP: Alkanna tinctoria (L.) Tausch: FJ763250, FJ763304; Arnebia decumbens (Vent.) Coss. & Kralik: Syria around Al Qaryatayn, Cecchi & al. (FIHB 07.05), KJ394991*, HG939444*; Arnebia linearifolia DC.: Syria, around An-Nasyriah, Cecchi & al. (FI-HB 07.03), EU919580a, HG939445*; Cerinthe major L. subsp. major: Italy, Sardinia, near Osilo, Cecchi & Coppi (FI-HB 08.01), EU919583a; Echium vulgare L.: FJ763247; Moltkia angustifolia DC.: Syria, around Deir-ez-Zor, Cecchi & al. (FI-HB 07.18), EU919593a, FJ763306; Moltkia petraea (Tratt.) Griseb.: FJ763194, FJ763258; Moltkia suffruticosa (L.) Brand subsp. suffruticosa: Italy, Veneto, Mt. Summano, Cecchi & Coppi (Herb. Cecchi 624), KJ394993*; Neatostema apulum (L.) I.M.Johnst.: FJ763198, FJ763262; Paramoltkia doerfleri (Wettst.) Greuter & Burdet: Albania, Kukes, Mt. Pastrik, Cecchi & al. (FI-HB 06.20), EU919605a; Podonosma orientalis (L.) Feinbrun: Syria near Palmyra, Jebel-et-Tar, Cecchi & al. (FI-HB 07.16), EU919607a, FJ763307. — INGROUP: Buglossoides arvensis (L.) I.M.Johnst. subsp. arvensis, 1: Italy, Tuscany, Marina di Grosseto, Selvi (FI-HB 07.64), KJ394968*, HG939439*; 2: Italy, Latium, around Viterbo, Cecchi & Selvi (FI-HB 06.42), KJ394965*; 3: Italy, Umbria, Castelluccio di Norcia, Dal Lago (MNAV-Dal Lago), KJ394961*; 4: Italy, Veneto, Piane di Schio, Scortegagna (MNAV), KJ394962*; 5: Tunisia, around Feriana, Selvi & Bigazzi (FI-HB 04.48), KJ394963*; 6: Turkey, around Burdur, Cecchi & Selvi (FI-HB 13.50), KJ394960*; B. arvensis subsp. permixta (Jord.) R.Fern., 1: Spain, Jaén, Sierra de la Sagra, Cecchi & al. (FI-HB 11.13), KJ394970*; 2: France, Maritime Alps, Valle Roya, Andrieu (FI-HB 10.94), KJ394971*; 3: France, Maritime Alps, Caussols, Tison (FI-HB 13.63), KJ394982*, HG939442*; B. arvensis subsp. sibthorpiana (Griseb.) R.Fern., 1: Greece, Crete, Thripti, Hilger (FI-HB, 13.84), KJ394964*; 2: Syria, around Yabroud, Cecchi & al. (FI-HB 07.37), KJ394966*; B. calabra (Ten.) I.M.Johnst.: Italy, Calabria, Villaggio Mancuso, Cecchi & Coppi (FI-HB 07.56), KJ394986*, FJ763305; B. gastonii (Benth.) I.M.Johnst.: Germany, Schachen Alpine Garden (culta), Döbbeler (FI-HB 07.58), KJ394988*, HG939437*; B. goulandrisiorum (Rech.f.) Govaerts subsp. goulandrisiorum: Greece, Ipiros, Mt. Timfi around lake Drakolimni, Cecchi & Selvi (FI-HB 08.38), KJ394989*, HG939443*; B. incrassata (Guss.) I.M. Johnst. subsp. incrassata, 1: Syria, Damascus, around Zebdani, Cecchi & al. (FI-HB 07.42), KJ394981*; 2: Turkey, Antalya, Termessos, Cecchi & Selvi (FI-HB 10.12), KJ394972*; 3: Turkey, Antalya-Akseki, Gembos Yayla, Cecchi & Selvi (FI-HB 10.03), KJ394983*; 4: Spain, Granada, Sierra Nevada; Cecchi & al. (FI-HB 11.16), KJ394973*; 5: Greece, Ipiros, Mt. Timfi near Astraka, Cecchi & Selvi (FI-HB 08.37), KJ394979*; 6: FJ763191, FJ763255; B. incrassata subsp. splitgerberi (Guss.) E.Zippel & Selvi, 1: Italy, South Tyrol, Kompatsch, Wilhalm (BOZ PVASC5518), KJ394974*; 2: Italy, South Tyrol, Paulsner Feld, Wilhalm (BOZ PVASC5526), KJ394976*; 3: Italy, South Tyrol, Faslar, Wilhalm (BOZ PVASC5524), KJ394985*; 4: Italy, Umbria, Mt. Subasio, Selvi (FI-HB 06.01), KJ394969*; 5: Italy, Sicily, Mt. Etna, Bianchini 11499 & Di Carlo (VER), KJ394984*; 6: Turkey, Bursa, Uludag, Cecchi & Selvi (FI-HB 10.44), KJ394975*, HG939440*; 7: Germany, Brandenburg, Neutornow, Hand 5773 (B), KJ394978*; 8: Italy, South Tyrol, Mals, Hand 5633 (B), KJ394980*; B. minima (Moris) R.Fern.: Italy, Sardinia, Mt. Tului, Coppi & Selvi (FI-HB 09.22), KJ394977*, HG939441*; B. purpurocaerulea (L.) I.M.Johnst., 1: FJ789859, FJ763308; 2: AJ555897; B. tenuiflora (L.f.) I.M.Johnst.: Syria, ruins of Palmyra, Cecchi & al. (FI-HB 07.15), KJ394967*; B. zollingeri (A.DC.) I.M.Johnst.: Taiwan, Yehai, s.coll. (HCT, TFRI 80) , KJ394987*, HG939438*; Glandora diffusa (Lag.) D.C.Thomas: FJ763246, FJ763300; G. moroccana (I.M.Johnst.) D.C.Thomas: FJ789867; Glandora nitida (Ern) D.C.Thomas: FJ763245, FJ763299; G. oleifolia (Lapeyr.) D.C.Thomas: FJ789869, FJ789887; G. prostrata (Loisel.) D.C.Thomas: Japan, cultivated (commercial material), Fukunaga (FI-HB 08.62bis), KJ394992*, FJ763277; G. rosmarinifolia (Ten.) D.C.Thomas: FJ763236, FJ763291; Lithospermum cinereum DC.: FJ763240, FJ763295; L. erythrorhizon Siebold & Zucc.: EF199861, FJ763309; L. hancockianum Oliv.: China, Yunnan, Yunnanfu, Handel-Mazzetti 6058 (KUN), KJ394990*; L. officinale L., 1: FJ763189, FJ763254; 2: Italy, Abruzzo, Gran Sasso-Laga, Bigazzi & Selvi (FI-HB 03.03) [rpoC1 sequence accession no. in Materials and Methods]; L. peruvianum DC.: FJ763216; L. tschimganicum B.Fedtsch.: FJ763220. 1078 Version of Record (identical to print version).