A taxonomic revision of the Campanula lusitanica complex ( Campanulaceae ) in the Western Mediterranean region by Jara Cano-Maqueda &

En este trabajo se revisa la sistemática de las especies anuales del complejo Campanula lusitanica en el occidente del Mediterráneo usando secuencias ITS, cariología y análisis de los caracteres morfológicos de todas las especies del complejo. La información proporcionada por estas tres fuentes de caracteres es congruente, y las distintas especies se reorganizan en dos grupos principales con categoría de sección, que se corresponden con los dos subclados bien definidos: Campanula sect. Rapunculus Boiss. y Campanula sect. Decumbentes, descrita como nueva en este trabajo. En el Mediterráneo occidental, la sect. Rapunculus está formada por las siguientes especies anuales: C. lusitanica L. y C. matritensis A. DC., con 2n = 18 cromosomas, y C. cabezudoi Cano-Maqueda & Talavera, C. specularioides Coss., C. transtagana R. Fern. y C. broussonetiana Schult., con 2n = 20 cromosomas. En la Península Ibérica la sect. Decumbentes está representada por C. decumbens A. DC., con 2n = 32 cromosomas, y C. dieckii Lange, con 2n = 28 cromosomas, ambas endémicas de la Península Ibérica; de C. decumbens se describe una subespecie nueva: C. decumbens subsp. baetica Cano-Maqueda & Talavera, taxon del valle del Guadalquivir muy bien diferenciado morfológicamente de la subsp. decumbens. En la parte sistemática se proporciona una clave para la identificación de estas especies anuales del oeste del Mediterráneo, así como una descripción y tipificación, fotografías de las flores y frutos, mapas de distribución, y aspectos de la ecología para cada uno de los táxones.


Introduction
The genus Campanula comprises about 420 species distributed mainly in temperate regions of the Northern Hemisphere (Lammers, 2007a, b).They are usually perennial herbs, although some are shrubby, and there are also some annual herbs, the latter mainly in the Mediterranean.This genus has a high morphological complexity that is reflected in the different classifications that have been proposed.
Perhaps the taxonomic treatment with most insight was that by Damboldt (1976) during the preparation of the Flora of Turkey.He divided the genus Campanula into six subgenera: Campanula, Megalocalyx Damboldt, Sicyocodon (Feer) Damboldt, Roucela (Dumort.)Damboldt, Brachycodonia (Fedorov) Damboldt and Rapunculus (Boiss.)Kharadze.The subgenus Campanula includes plants with 3 or 5 stigmas, with or without calycine appendages, and with the capsules opening by basal or middle position pores, or indehiscent.This is the most complex subgenus, with 12-15 recognized sections and it contains most of the species of the genus.According to Sáez & Aldasoro (2003), subgenera Megalocalyx and Sicyocodon are annual herbs with flowers with calycine appendages and with capsules opening by basal valves; the two subgenera differ in the size of style: very long (exceeding 35 mm) in the subgen.Sicyocodon, and short (less than 15 mm) in the subgen.Megalocalyx.The around 15 species of these two subgenera are distributed in the Mediterranean Basin, SW Asia and Macaronesia.The subgenus Roucela comprises annual plants, with flowers without calycine appendages and capsules opening by basal valves; it consists of 5 species distributed throughtout the E Mediterranean and SW Asia, with the exception of C. erinus L. which has a wider distribution.The subgenus Brachycodonia comprises only C. fastigiata Dufour, an annual plant with axillary inconspicuous flowers, without calycine appendages, and with capsules opening by three apical valves; C. fastigiata occurs on gypsum soils in Spain, N Africa, C Asia and Transcaucasia.
The subgenus Rapunculus includes annual and perennial plants, without calycine appendages, with a large campanulate or infundibuliform corolla, and with capsules opening by apical or middle position pores.Damboldt (1976Damboldt ( , 1978) )  Sect.Alaria contains a single species, C. pterocaula Hausskn., endemic to N and C Anatolia (Turkey); it is a biennial glabrous plant, with winged stems, large flowers arranged in spiciform inflorescences, and capsules opening by apical pores.
Sect.Rapunculus contains around 50 species distributed mainly in the Mediterranean region; it comprises perennial, biennial or annual plants, glabrous or tomentose, with wingless stems, the inflorescence a panicle, or sometimes spiciform, the capsules opening by apical or middle position pores, and wingless seeds.
The Campanula core consists of two subclades: one formed by the subgenera Campanula, Megalocalyx and Roucela, together with other genera of Campanulaceae; and a second subclade comprising subgenus Rapunculus (sections Rapunculus and Alaria) and subgenus Brachycodonia, again with some other genera of Campanulaceae.
However, our detailed morpho-geographical analysis of the exsiccata available for this group (which we call the Campanula lusitanica complex) lead us to con-clude that not only are many of the species described by various authors valid, but that several new taxa can be recognised.Moreover, our analyses (Cano- Maqueda & al., 2008 and the present study) based on combined analysis of ITS sequences and trnT-L using samples from most of the annual species of the Western Mediterranean area not only support recognition of these species, but have also revealed that the annual species of sect.Rapunculus sensu Damboldt comprise a polyphyletic assemblage.
In this study we present a formal taxonomic revision of the Campanula lusitanica complex from the Iberian Peninsula and Morocco, based on our studies of exsiccata, karylogical parameters, and molecular markers.And using information provided by the nrDNA ITS for a large number of Campanula species and allied taxa we have also attempted to place the species of Campanula sect.Rapunculus sensu Damboldt within the context of the ongoing molecular systematics of the genus Campanula.
Genomic DNA was extracted from silica-gel-dried leaves collected in the field and from herbarium material using the DNeasy Plant Mini Kit (Qiagen) following the protocols provided by the manufacturer.
Amplification of the ribosomal ITS region (ITS1-5.8S-ITS2)was performed in 25 µL reaction volume with 22.5 µL Thermo-Start ReddyMix Master Mix, 0.5 µL of each primer, 1 µL of DMSO (100%) and 0.5 µL of DNA.Forward ITS5 and reverse ITS4 primers (White & al., 1990) were used in the amplification and sequencing processes.The polymerase chain reaction (PCR) sequence profile was one cycle of 1 min at 96 ºC, followed by 35 amplification cycles of 10 s denaturing step at 96 ºC, 5 s annealing at 50 ºC, and 3 min elongation step at 60 ºC, plus an ending cycle of 8 min at 72 ºC.
Amplified products were purified using the QI-Aquick PCR Purification Kit (Qiagen) according to Taxonomic review of the Campanula lusitanica complex the manufacturers protocols.Purified products were sequenced in both directions.The sequence PCR profile was of a time of incubation of 15 min at 37 ºC and 15 min at 80 ºC.
The direct and reverse sequences of each sample were compared and corrected using the program Geneious 4.8.3, obtaining the respective consensus sequence.Sequences were aligned using the algorithm of the program ClustalX, and then adjusted manually using the options of the program Se-Al v. 1.0 alpha 1 (Rambaud, 1996).Gap indels were coded as binary characters by their presence/absence (0/1 matrix).Only those gaps that were unambiguous and potentially informative (Torrecilla & Catalán, 2002) were added to their correspondent sequence matrix and used for parsimony-based analysis.
In the parsimony analysis, the data matrix was subjected to two heuristic searches (first search: closest, TBR, MULTIPARS ON; second search: randomorder-entry of 10,000 replicates, TBR, MULTIPARS OFF, saving no more than 10 trees of score > 10 per replicate) aimed at finding different putative islands of most-parsimonious trees.Bootstrap support for the best trees found under the parsimony criterion was estimated by heuristic search with 1,000 bootstrap replicates (Felsenstein, 1985) using the TBR and MULTIPARS OFF strategy proposed by DeBry and Olmstead (2000) to reduce the tree-search effort in bootstrap resampling analysis.Initial MaxTrees setting was 300,000 with an auto-increase of 100.Previous to the Bayesian inference search, 24 models of nucleotide substitution were tested for which the optimal model GTR + G + I.The Bayesian analysis was performed through 1,100,000 generations using the Markov chain Monte Carlo (MCMC), sampling trees every 100 generations and burn-in all sampled point from generations previous to convergence to a stable likelihood value (Huelsenbeck & Ronquist, 2002;Leaché & Reeder, 2002).From each search, a 50% majority-rule consensus tree that showed de posterior-probability values of branches was constructed.

Karyological study
Plants or seeds of each taxon of the Campanula lusitanica complex sensu Cano-Maqueda & al. (2008), were collected from natural populations (Table 1) and cultivated in the greenhouses of the University of Seville.Chromosomes were observed from meristematic cells of root tips or meiosis in the anthers of flower buds.The root tips were treated with 8-hidroxiquinoleine 0,002M for three hours and a half at 4 ° C. Subsequently, roots and flower buds were fixed with Carnoy solution (3:1 ethanol 96%: glacial acetic acid) for a minimum of 24 hours.Staining of chromosomes was performed with-alcoholic-hydrochloric carmine (Snow, 1963).The images were taken with a Leica DC 300 inserted in a Zeiss Axiophot microscope with Plan-apochromatic objective 63/1.4 and an increase of 1.25.Levan & al. (1964) were followed for the morphological terminology and Stebbins (1938) for size terminology of the chromosomes.

Systematic Treatment
A morpho-geographical analysis of herbarium specimens of all relevant taxa associated with the C. lusitanica complex was performed, based on the following Herbaria: C, COI, FCO, G, HVR, LISE, LISU, MA, MGC, P, SALA, SALAF, SEV, W. All recognized species and most heterotypic synonyms have been typified.The initials of the provinces of Spain and Portugal which are cited in the description of each taxon follow those used in "Flora iberica".

Molecular Phylogeny
The ITS region comprised 726 aligned nucleotide positions of which 469 were variable and 368 were J. Cano-Maqueda & S. Talavera parsimony informative.The first heuristic search found 101,914,528 equally parsimonious trees that were 2,480 steps long, with a consistency index of 0.353, excluding uniformative characters, and a retention index of 0.748.The second search did not find any other island of most-parsimonious trees, and trees from the first search were used to compute the strict consensus tree.The Bayesian analysis sampled 7,639 trees, which reached a stable likelihood value after burn-in 1,500 trees.The 50% majority-rule consensus tree of all sampled trees showed a topology totally concordant with that recovered from the parsimony analysis.The Bayesian tree was better resolved than the parsimony-based tree, so only the Bayesian tree with both bootstrap and posterior probability support values for branches is shown in Fig. 1.
Topology within the ITS tree is consistent with results obtained in previous phylogenetic studies of the genus Campanula and allies (Eddie & al., 2003;Park & al., 2006;Roquet & al., 2008Roquet & al., , 2009;;Cellinese & al., 2009;Haberle & al., 2009;Stefanović & Lakušić, 2009).These show that the genus is paraphyletic and divided into two major clades, the 'Campanula s.str 1).All species studied have small or moderately small chromosomes (1.2-4.1 µm), with the centromere located in the middle or submid region (Fig. 2).In C. specularioides, one pair of chromosomes with the centromere in the submiddle region has a satellite on the short arm.Chromosomes form bivalents in the meiotic metaphase.

Systematic treatment
After reviewing all specimens we found that the different species can be separated in two groups on basis on the morphology of the style and stigma: one formed by C. decumbens and C. dieckii, with the style glabrous and with three stigmas with arms straight and erect or erect-patent; and another formed by all other species, with the style hairy in the upper half, and a trifid stigma, with the stigmatic arms curved or circinate.These two groups are consistent with the molecular results and karyology, so that they are probably natural, and the most appropriate taxonomic treatment is to recognise them as sections.The second group, which includes Campanula rapunculus L., would constitute the sect.Rapunculus.The first group would establish a new section, sect.Decumbentes which is formally described below.Within these sections, the most significant characters are: the shape of the corolla (campanulate or infundibuliform), middle cauline leaves (petiolate or sessile), capsule morphology (obpyramidal, subglobose or subovoid), and ovary indumentum (setose, glabrous or papillose).
We used this combination of characters to create a key for the identification of all annual species of the C. lusitanica complex in the Western Mediterranean.The section is monophyletic (see lusitanica clade in Fig. 1).

KEY TO
Experimental manual crosses showed that all annual species of the W Mediterranean had hybridization barriers at different levels: (1)-fruits were not produced, (2)-seeds do no germinated, (3)-germinated seeds formed chlorotic seedlings which did not survive and (4)-no chlorotic seedlings survived after the cotyledon state but they did not complete their development (Cano-Maqueda unpublished studies).Illustrations: Fig. 4 A, B.
Habitat, phenology and distribution: Wet grasslands on generally acid substrates, sometimes draining and river banks; 15-1800 m. (V)VI-VIII.• Endemic to the NW of the Iberian Peninsula, common in Galicia and N Portugal, and also occurring in S Portugal, Monchique (Fig. 5).Portugal: Ag BA BL DL Mi TM.Spain: C Le Lu O Or Po Sa Za.

Selected specimens
Habitat, phenology and distribution: Pine, cork oak and holm oak woodlands or their shrubby secondary communities, often on sandy substrates, but also on clay; 0-2200 m.V-VI(VII).• Endemic to the Iberian Peninsula.Distributed almost throughout the entire peninsula, but rare in limestone areas of C, E and N of Spain, and absent in NE of Spain (Fig. 5).Observations: This species is very variable in size, flower size, indumentum and shape of the calyx, and Taxonomic review of the Campanula lusitanica complex indumentum and size of the capsules.The plants that live in very shady banks are usually delicate, have small flowers and, in general, asymmetric capsules due to lack of fertilization in one or two of the three locules, probably caused by deficient pollination.In contrast, plants living in sunny and humid areas are vigorous, highly branched, with large flowers and perfectly symmetrical obpyramidal capsules, with all locules full of seeds, denoting a very efficient fertilization.On shallow soils, in montane areas, plants are very small, slightly branched, with small flowers and sometimes with ± ovoid and malformed capsules due to a deficient pollination, and these may be confused with C. transtagana or even with C. lusitanica.
Habitat, phenology and distribution: Fissures of calcareous rocks; 400-1600 m.VI-VII.• Endemic to the Sierra Subbética from Seville to Jaén, and the Penibética, where it is very frequent, especially in the Sierra de Loja and Tejeda (Fig. 8).Spain: Ca Co Gr J Ma Se.
Observations are similar in C. cabezudoi and C. decumbens, but C. cabezudoi has hairy style in the upper half and a single trifid stigma with curved and patent stigmatic lobes, while C. decumbens has a glabrous style and three straight and erect-patent stigmas.Moreover, in the topology of the ITS analysis, C. cabezudoi and C. decumbens are in two different clades (Fig. 1) and they have different chromosomes number (Table 1, Fig. 2).
Observations: Despite its narrow distribution, this species is markedly variable in the colour of anthers and stigmas, and in the indumentum of calyx and capsule.since both can be very hairy or completely glabrous, even in the same population, although more commonly all plants of a population have glabrous calyx, and likewise the capsule.Within populations there are plants with white anthers and stigmas, and others with blue anthers and stigmas, or even plants with white anthers and blue stigma or vice versa.All these colour morphs, which certainly have a genetic basis, seem to be interfertile so this trait has little onomic value.Based on the middle position of the pores in the capsule, this species was included by Fedorov (1976) in the sect.Campanula.The phylogenetic tree of nr DNA ITS shows that C. transtagana and C. broussonetiana are sister species of C. specularioides (see Fig. 1).

Selected spcimens
Habitat, phenology and distribution: Wet grasslands on sandy or slate-rich substrates; 50-800 m.V-VI (VII).• Endemic to SW Iberian Peninsula, from the River Tajo to the Guadiana, Sierra de Guadalupe and almost all Sierra Morena (Fig. 12).Portugal: AAl Ag BAl BB.Spain: Ba Cc Co H J Se.
Observations: C. transtagana is rare in Portugal and in Sierra de Guadalupe but very common throughout the Sierra Morena, especially in shady slopes and valley bottoms, where it normally co-habits with C. matritensis.The profuse branching, small corollas with white background tube and petiolate leaves of the stem, clearly differentiate it from C. matritensis.
The holotype of C. transtagana consists of five cultivated plants that are very branched from the base, between 20 and 35 cm, and placed on three sheets in Coimbra herbarium.Illustrations: Fig. 10 E, F.
Habitat, phenology and distribution: Shady hollows and the understory of cork woodlands, on limestone, or slate-rich or sandy substrates, from sea level to high mountains; 0-2000 m.IV-VII(VIII).• Endemic to W and N Morocco, along most of the Atlantic coastlands from Sidi Ifni to the Mamora forests; also the Rif, Middle Atlas and Great Atlas (Fig. 12).
Observations: This species has the same color morphs for anthers and stigmas as those found in C. specularioides, and similary, the morphs seem to be interfertile.Alphonse De Candolle (1830) synonymized this species with C. loeflingii Brot., possibly influenced by the illustration 18 of the "Phytographia Lusitaniae Selector" (Brotero, 1816) under this name.Certainly, C. lusitanica and C. broussonetiana are very similar morphologically.Both have long hairy indumentum that is straight and patent on the stem, and wide leaves, but in C. broussonetiana the middle cauline leaves are attenuate at the base or petiolate and in C. lusitanica are sessile and subauriculate.The molecular phylogeny has shown that C. broussonetiana is more closely related to C. transtagana than any other species of sect.Rapunculus (see Fig. 1).
The sheet of type material of C. broussonetiana (W03798) contains a complete highly branched plant with many flowers.Schultes (1819-1820: 104) already indicated that the type material was in the Willdenow herbarium under the name Campanula ramosissima.In the herbarium of the Prodromus of De Candolle there are two sheets (G 138472 and G 138271-G 138478), one (G 138472) with a complete plant of c. 30 cm and the other (G 138271, G 138478) with three smaller plants (15-22 cm), all belonging to the expedition by Broussonet in 1804, collected at Mogador (Essaouira, Morocco).This last sheet contains two labels.In one (G 138478), handwritten by A. De Candolle, is indicated "Voyage de Broussonet 1804" at the base of two of the plants, and in the other (G 138271), with orthography of Broussonet, is written: "Campanula/Mogador" and also, with orthography of Alphonse De Candolle, "Campanula loeflingii Brot./ _ Broussonetiana Roem.Et Sch./ A. DC.".These materials were probably not studied by Schul tes, and therefore they can not be considered type material (Fig. 13).

7.
Habitat, phenology and distribution: Grassland and wet meadows, on basic substrates (limestone and dolomite); 10-1250 m.VI-VII(VIII).• Endemic to the S of Spain, in the Guadalquivir valley, Sierra de Grazalema and Serranía de Ronda, cited only once from Aranjuez.Spain: Ca M? Ma, Se.
Observations: The identity of C. decumbens has been discussed by various authors (Pau, 1896;Cuatrecasas, 1929;Caballero, 1942;Fedorov, 1976;López-González, 1979-1980); but in fact most authors do not treat with the decumbent plant from Aranjuez described by Alphonse De Candolle.These authors looked for this species near Aranjuez, but they only found an upright and very hairy plant with crenate leaves.This upright plant is the species described by Lange (1893) as C. dieckii and later by Pau (1896) as C. semisphaerica.Only Fedorov (1976) indicated that C. decumbens A. DC. is indeed decumbent.From the type he thought it endemic to Aranjuez, and that it might be better treated as subspecies or variety of C. patula L., as proposed by Cuatrecasas (1929).
The type material of Campanula decumbens (G 138256) contains two complete plants of 20 and 25 cm and two stems, all with flower buds and only one plant with an open flower.This flowering plant is chosen as lectotype.The other materials are isolectotypes (see Fig. 14).In the general herbarium at Geneva there is another sheet (G 104141), also from the Ventenat herbarium, containing an incomplete plant of 20 cm with two flowers.The label indicates "Campanula/ affinis vincaeflorae/ Aranjuez près Madrid".The Taxonomic review of the Campanula lusitanica complex plant contained in this sheet may also form part of type material.
The type material has petiolate middle leaves of the stem, and a small and infundibuliform corolla, in which it resembles C. cabezudoi [described by López-González (1979-1980) as C. decumbens var.pseudospecularioides].But the style with three ± straight stigmas is similar to that of C. dieckii and so, it is clear that C. decumbens A. DC.belongs to sect.Decumbentes.Cano-Maqueda & al. (2008) indicated that this species, apparently only known only from the Delessert type materials, could be extinct.However, several populations have been located in Sierra de Grazalema and Serrania de Ronda, with the same characters as the type material of C. decumbens A. DC.Plants from two of these populations were cultivated in the greenhouses of the University of Seville.They retained the decumbent habit, are self-incompatible (Cano-Maqueda & al., unpublished data) and the meiotic number of chromosomes is n = 16.
Due to this discovery that C. decumbens occurs on diverse localities on alkaline substrates in the Serranía de Ronda and surrounding areas, it is likely that the material studied by Alphonse de Candolle came from the Southern Betic Cordillera and not from Aranjuez as stated in the sheet of the type.
Subsequently, a number of exsiccata by previous collectors in this area and also from the Guadalquivir valley and Cádiz coastland were also identified as C. decumbens.However, the Guadalquivir valley plants are morphologically rather different from those of the Sierras Béticas, although the molecular phylogeny has shown that both groups of plants do not present any changes in the nucleotide sequence (Fig. 1).They also have the same chromosome number (n = 16; Table 1 and Fig. 2H).However, since in addition to the morphological differences, the populations present a different ecology and distribution, we have considered it most appropriate to treat these two groups of populations as two subspecies.4 mm; lobes 9-10 × 5-6 mm, broadly triangular.Stigmas 2-3.2 mm.Capsule (2.2)3-4(4.4)× 2.4-4 mm, ovoid.2n =32.
We have seen material of this species from two collections from Sintra (Estremadura, Portugal).One collected by F. Fernandes in V-1914 (G 104249, 104150;LISU 36374;MA 121500), distributed in exiccata by F. Sennen (in F. Sennen Pl. Esp. n.º 6006), and another by W. Rothmaler on 13-V-1938 (G 104148;LISE 4368).Since this plant has not been collected again at this locality, the presence of this species as a native in Portugal may be considered doubtful.However, this kind of disjunction is not uncommon in other species of the Iberian Peninsula, i.e., Silene distichia Willd., a common species in the Eastern half of Spain and rare in the W Portugal (Talavera, 1990).
The type material of Campanula dieckii is composed by 7 whole plants at anthesis of 10-20 cm with sessile, elliptic, crenate cauline leaves, infundibuliform corolla, glabrous style, straight stigma and densely setose ovary.The sheet also contains two flowers in an envelope.The first plant, in the upper left corner of the sheet is chosen as lectotype, because it is the one that best fits the description of the author.The other six plants are isolectotypes (see Fig. 18).In the Geneva herbarium there is a sheet (G104244) with three plants that are also isolectotypes.
The sheet of type material of C. matritensis var.nevadensis contains 6 plants in flower.We have chosen as lectotype the plant placed at the top right, about 10 cm, very hairy, with crenate leaves and two flowers in anthesis.The remaining plants are isolectotypes.
The type material of C. semisphaerica consists of two very small plants, each with one flower open.The plant on the right is the lectotype.On the sheet there is a typed label by Ginés López-González dated 13-11-1979 indicating the material contained on the sheet as holotype.Pau's indication "genuine" may be understood that this material was chosen as type by the author of the binomial.

Selected specimens
divided this subgenus in three sections: Pterophyllum Damboldt, Alaria Damboldt and Rapunculus.Sect.Pterophyllum includes three species: C. primulifolia Brot.(Iberian Peninsula), C. alata Desf.(Algeria and Morocco), and C. peregrina L. (E Mediterranean, including Cyprus, Lebanon and SW Turkey and S Anatolia); they are hispid perennial plants with large infundibuliform flowers, arranged in spikes or panicles, with capsules opening by three pores of middle position, and with winged seeds.
also provided a brief description of C. loeflingii, but like Brotero, they could be referring to both C. lusitanica and C. matritensis.Boissier (1839) described C. duriaei based on the characters of plants collected by Durieu in Cangas de Tineo (Asturias) distributed as the Exiccata No 280 in 1835.In the herbarium at Geneva there are three sheets of this collection (G 104093, G 104120 y G 104116).The first (G 104093) consists of 4 complete plants mounted on two sheets, and comes from the Boissier Herbarium.One of the sheets contains two plants of 30 cm and 22 cm respectively, and a Boissier handwritten label indicating "C.Duriaei/284".We have chosen as lectotype the 30 cm plant because it is the closest to the description; the other plant, of 22 cm, is an isolectotype.The other sheet also contains two plants without any annotation by Boissier; they are also likely isolectotypes.The other two sheets (G 104120 and G 104116) are from the herbarium of Moïse-Etienne Moricand which were incorporated into the herbarium of the Conservatoire et Jardín Botanique de Genève in 1908; the plants contained in them, which were not seen by Boissier, are not part of the type material.Boissier was the first author to clearly describe the characteristic hairs of the Asturian plants: "Toute la plante est d'une consistance délicate et couverte de poils long et étalés, tandis que la C. loeflingii est u glabre ou hérissée de poils rudes et courts".Boissier's concept of C. loeflingii was very wide, since he included in this species most of the taxa of this group which had been collected by him in southern Spain or that he had seen in the Fouche or De Candolle herbaria.We identify Fouche herbarium plants as C. decumbens subsp.baetica Cano-Maqueda & Talavera and those in the De Candolle herbarium as C. broussonetiana, and the plants that he collected in Southern Spain as C. matritensis and C. dieckii (see these species below).
Portugal: AAl Ag BA BAl BB BL E Mi R TM. Spain: Ab Al Av Ba Bu C Ca Cc Co CR Cu Gr Gu H J Le Lo Lu M Ma O S Sa Se Sg So Te To Va Vi Z Za.

Fig. 6 .
Fig. 6.Lectotype of Campanula matritensis (plant located in the lower left corner) (G 138402, indicated by the arrow).The lectotype is the plant of the center, the other two plants are isolectotypes.The other two plants with their respective labels are not material type.

Fig. 13 .
Fig. 13.Materials of Campanula broussonetiana.Collected by Broussonet in Mogador (Essaouira, Morocco) during his campaign in Morocco in 1805.The type material of this collection is located in Willdenow herbarium (B).

Fig. 14 .
Fig. 14.Lectotype and isolectotype of Campanula decumbens (G 138256).The lectotype is the plant located in the center, on the right, the only one with a flower in anthesis.The other plants are isolectotypes.

Table 1 .
Chromosome numbers for annual species of the Campanula luisitanica complex in the W Mediterranean area.Origin of material, gametic (n) or somatic (2n) chromosome number of and authors who have studied the different annual species in this complex.
Fig. 1.Bayesian 50% MR concensus tree topology of Campanulaceae ITS.Bootstrap and posterior probability values are indicated on corresponding branches.Symbols indicate the habit of the species: •, annual; ࡗ, biennial; ᮡ , perennial.The two sections that are the focus of this article are marked in gray.The chromosome numbers shown for the species that are not are specified in the text, derive from indices of plant chromosome number (Castroviejo, S. & Valdés-Bermejo, E. (eds.).1991.Archivos de Flora iberica I: números cromosomáticos de plantas vasculares ibéricas.CSIC.Madrid; Index to plant chromosome numbers.Monographs in Systematic Botany from the Missouri Botanical Garden [www.tropicos.org]; Moore, D.M. 1982.Flora Europaea.Checklist and chromosome index.Cambridge University press).
THE SPECIES 1. Style 2.1-4(4.8)mm, glabrous, with three stigmas; stigmas Lower leaves oblanceolate or spathulate, petiolate.Without calycine appendages.Corolla campanulate or infundibuliform.Stamens with white or blue anthers.Style with numerous pollen collecting hairs on the upper half, with a trifid stigma; stigmatic arms curved or circinate, patent, white or blue, with numerous pollen collecting hairs on the abaxial surface glabrous, and receptive on the adaxial surface.Capsule subspherical, ovoid or obpyramidal, dehiscing by three apical or middle position pores.x = 9, 10 (see karyology).This section includes: Campanula rapunculus and C. patula of the Mediterranean region; C. lusitanica, C. matritensis, C. cabezudoi, C. specula rioides, C. transtagana and C. broussonetiana from the W Mediterranean; and C. sparsa and possibly C. spa tulata Sibth.& Sm. of the E Mediterranean.
This subspecies differs from Campanula decumbens subsp.decumbens in its non-petiolate, sessile cauline leaves, erect rather than decumbent stems, and obpyramidal capsule, not ovoid.It differs of Campanula dieckii by its entire or toothed and glabrescent leaves, and by the larger size of the calyx, corolla and stigmas.