Campanula aureliana (Campanulaceae), a new species from Albania Sandro Bogdanović, Ivana Rešetnik, Salvatore Brullo & Lulëzim Shuka Plant Systematics and Evolution ISSN 0378-2697 Plant Syst Evol DOI 10.1007/s00606-014-1171-0 1 23 Your article is protected by copyright and all rights are held exclusively by SpringerVerlag Wien. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Plant Syst Evol DOI 10.1007/s00606-014-1171-0 ORIGINAL ARTICLE Campanula aureliana (Campanulaceae), a new species from Albania Sandro Bogdanović • Ivana Rešetnik Salvatore Brullo • Lulëzim Shuka • Received: 10 June 2014 / Accepted: 30 October 2014 Ó Springer-Verlag Wien 2014 Abstract Campanula is a species-rich genus with high variability of the morphological traits, controversial taxonomic treatments within the Mediterranean basin as a species diversity center. One of the monophyletic groups in the genus is the Campanula series Garganicae Trinajstić distributed in the amphi-Adriatic and Ionian region. The group as currently delimited encompasses 11 taxa, mostly with highly restricted distributional ranges. In the present study plants previously named as Campanula garganica Ten. var. albanica Markgr. are described and illustrated as an isophyllous species from central Albania, for which name Campanula aureliana Bogdanović, Rešetnik, Brullo & Shuka is proposed. The results of phylogenetic analyses based on nuclear ITS and chloroplast trnL–trnF data support C. aureliana as a clearly distinct taxon within the Handling editor: Sylvain Razafimandimbison. S. Bogdanović and I. Rešetnik have contributed equally to this work. S. Bogdanović (&) Department of Agricultural Botany, Faculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Zagreb, Croatia e-mail: sbogdanovic@agr.hr I. Rešetnik Department of Botany, Faculty of Science, University of Zagreb, Marulićev trg 9a, 10000 Zagreb, Croatia S. Brullo Dipartimento di Scienze Biologiche, Geologiche e Ambientali, Università degli Studi di Catania, via A. Longo 19, 95125 Catania, Italy L. Shuka Department of Biology, Faculty of Natural Sciences, Tirana University, Bld. ZOG I, Tirana, Albania Campanula ser. Garganicae. Its description is given, and diagnostic morphological and SEM seed micromorphological characters of closely related species are compared and discussed. Morphologically C. aureliana shows close affinity with C. fenestrellata Feer, mainly for the habit, shape and size of the corolla, but differs in a lot of significant features. Information on the ecology and conservation status of the newly described species is presented. Keywords Campanula
Balkan Peninsula
Endemic
Isophyllous bellflowers
Phylogeny
Taxonomy Introduction Campanula L. is the largest genus of the family Campanulaceae, and includes ca. 580–600 species (Mansion et al. 2012) distributed in the Northern Hemisphere, with the Mediterranean basin as a major center of species diversity, where about 250 species occur (Damboldt 1965; Podlech 1965; Kovanda 1970a, b, 1977; Geslot 1984; Park et al. 2006). The annual and perennial Campanula taxa grow in various habitats, such as meadows, grasslands, garrigues, woodlands, and mainly in rupestrian stands (Kovačić 2004; Roquet et al. 2008). The high variability of the morphological traits provided numerous classification proposals (De Candolle 1830; Boissier 1875; Gadella 1966a, b; Contandriopoulos 1984; Kolakovsky 1994), but none of them reflect the phylogenetic relationships recently obtained with molecular data, which showed that Campanula and many closely related genera are not monophyletic (Eddie et al. 2003; Park et al. 2006; Roquet et al. 2008, 2009; Borsch et al. 2009; Cellinese et al. 2009; Haberle et al. 2009; Mansion et al. 2012; Crowl et al. 2014). Nevertheless, molecular data identified several well- 123 Author's personal copy S. Bogdanović et al. supported monophyletic groups within Campanula, e.g., the garganica clade (Park et al. 2006; Frajman and Schneeweiss 2009; Bogdanović et al. 2014a, b), the fragilis clade, the pyramidalis clade (Lakušić et al. 2013), and closely related lineages, e.g., Phyteuma (Schneeweiss et al. 2013). All of these monophyletic groups that were further explored revealed ambiguous interspecific relationships and discrepancies with traditional circumscription and taxonomy. The Balkan Peninsula has long been known for its high level of biodiversity and endemic taxa, refugial character and importance for the European phytogeography. However, some parts of the Balkan Peninsula, e.g., Albania, are still insufficiently explored. That fact is manifested in many new taxa confirmed for the Albanian flora in recent years (e.g., Barina and Pifkó 2008a, b, 2011; Rakaj 2009; Ball 2011; Barina et al. 2009, 2011, 2013; Meyer 2011; Frajman et al. 2013) including numerous species described new for science (Shuka et al. 2010; Meyer 2011; Tan et al. 2011, 2013; Polatschek 2013; Bogdanović et al. 2014a). The combination of less explored geographical area with frequent cases of old, single and recently unconfirmed literature reports together with taxonomic and phylogenetic complexity of the studied genus indicates the necessity for cautious examination of the studied material. Campanula series Garganicae Trinajstić represents a morphologically, karyologically and phylogenetically wellsupported monophyletic group distributed in the amphiAdriatic and Ionian region (Park et al. 2006; Liber et al. 2008; Frajman and Schneeweiss 2009; Bogdanović et al. 2014a, b). The group as currently delimited includes 11 taxa, while the sister species of the group is the Albanian endemic C. comosiformis (Hayek & Janch.) Frajman & Schneew. Morphologically, the members of this group are characterized by a monopodial growth form, isophyllous and long petiolate leaves, with cordate to ovate blades, elongated and more or less unilateral inflorescence, campanulate or rotate corolla, obtuse hairs at the base of filaments, and brown shiny seeds (Damboldt 1965; LovašenEberhardt and Trinajstić 1978). The phylogeny inferred with plastid and ITS sequence data (Park et al. 2006; Frajman and Schneeweiss 2009; Bogdanović et al. 2014a, b) unambiguously separate all taxa in the group; however, their relationships remain unclear due to low clade support and conflicting signals between plastid and nuclear data. In the course of the current comprehensive study of the ser. Garganicae, detailed investigation on herbarium specimens and literature data indicated the occurrence of an endemic variety of C. garganica Ten., named var. albanica Markgr. in central Albania. In particular, C. garganica was described by Tenore (1827) from Gargano promontory (central Italy), while in Albania, its presence was unlikely 123 and doubtful. This variety was described by Markgraf (1931) on material from a single locality in Albania (Tomori: Kapinova, Kalkfels im Dorf, 800 m, bl., 19.VI.1928) and no type specimens exist to confirm this finding, as the deposited material to Berlin Herbarium (B) was burned and destroyed during the Second World War (Damboldt 1965). We also carried out additional checks in B and BP herbaria, as well as in other herbaria, but neither the type nor any duplicates have been hitherto found. From the nomenclatural aspect, the name Campanula garganica var. albanica Markgr. was described and validly published by Markgraf (1931) and in the protologue Markgraf stated that it differs from C. cephallenica Feer in having denser inflorescence, shorter calyx teeth and denser hairs on the whole plant. Some authors (Damboldt 1968; Fedorov and Kovanda 1976; Geslot 1984) considered C. cephallenica and C. acarnanica Damboldt, respectively, occurring in the Ionian islands and West Greece, to be a subspecies of C. garganica, but this treatment is rejected by molecular data (Park et al. 2006; Frajman and Schneeweiss 2009; Bogdanović et al. 2014a, b). Campanula garganica var. albanica was synonymised with C. debarensis Rech.f. by Damboldt (1965) and not included in the Albanian flora by Qosja et al. (1996). More recently, Park et al. (2006) considered C. debarensis an Albanian–Macedonian endemic, phylogenetically distinct from the others species of this group, but they do not elucidate other distribution in Albania. To test the taxonomic assignment and to determine the phylogenetic position of C. garganica var. albanica individuals from Mt. Tomori, within the ser. Garganicae, we investigated plastid and nuclear ribosomal ITS sequence data and evaluated morphological evidence. Accordingly, here we provide a taxonomic treatment of these individuals as a new species, Campanula aureliana Bogdanović, Rešetnik, Brullo & Shuka (see ‘‘Taxonomic treatment’’), including a comprehensive description accompanied by an illustration plate and diagnostic characters. Finally, we provide information about its ecology and propose an IUCN conservation status. Materials and methods Plant material Investigations were carried out on the specimens collected during field trips in Albania, in July 2012 and 2013. In total, only ten individuals were collected to avoid damage to the current small populations in the villages of Tomori and Kapinovë. The plants of these collections were used for herbarium exsiccata, while fresh leaves were conserved in silica gel for DNA analysis. Floral and vegetative parts Author's personal copy Campanula aureliana, a new species from Albania Fig. 1 Distribution of Campanula series Garganicae were placed in 50 % glycerine-ethyl alcohol solution for further morphological assessment. To obtain a denser sampling in Campanula ser. Garganicae (Fig. 1), we used the data sets of Park et al. (2006), Frajman and Schneeweiss (2009), and Bogdanović et al. (2014a, b), extended with four new sequences from newly found taxon. Voucher data and GenBank accession numbers of the newly sequenced taxa, as well as GenBank accession numbers from previous studies are given in Table 1. Herbarium specimens of the isophyllous species of Campanula were studied from B, BEOU, BM, BP, CAT, CNHM, K, MKNH, NAP, NHMR, PAL, RO, TIR, W, WU, ZA, ZAGR, and ZAHO (abbreviations follow Thiers 2014). Scanning electron microscopy (SEM) The micromorphology of the testa of ten dried mature seeds collected in Tomori village (Albania) was studied using a scanning electron microscope (Zeiss EVO LS10). The preparation of the seeds of C. aureliana was done according to Huttunen and Laine (1983). 0.2 lM of each primer (17SE and 26SE of Sun et al. (1994) for the nuclear ITS; c and f of Taberlet et al. (1991) for the plastid trnL–trnF) and 1.25 U of TaKaRa TaqTM HS polymerase (TaKaRa Bio Inc., Shiga, Japan). The PCR conditions for ITS were the same as described in Park et al. (2006) and for trnL–trnF, the same as described in Bogdanović et al. (2014b). The PCR reactions were performed using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, California). The PCR products were purified with the GenElute PCR clean-up kit (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), according to the manufacturer’s protocol. The products were sequenced by the Macrogen Inc. (Seoul, Korea) using the BigDyeTM terminator cycle sequencing kit (Applied Biosystems, Foster City, California) and analyzed on an ABI PRISM 3730XL automated sequencer (Applied Biosystems, Foster City, California). Sequences were edited and manually aligned using the Geneious Pro 5.3.6 (Drummond et al. 2011). Sequence alignments are available from TreeBASE (study number 16523). Phylogenetic analyses DNA extraction, amplification and sequencing Total genomic DNA was extracted from silica gel dried leaves or herbarium specimens using the DNeasy plant mini kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s instructions. Each reaction mix for polymerase chain reaction (PCR) of 50 lL contained 25 ng of DNA, 1 9 PCR Buffer (TaKaRa Bio Inc., Shiga, Japan), 0.2 mM each dNTP (TaKaRa Bio Inc., Shiga, Japan), Three different datasets (ITS, trnL–trnF, ITS–trnL–trnF combined dataset) were analyzed using maximum parsimony (MP) and Bayesian inference (BI). The trees were rooted using Trachelium caeruleum L. as an outgroup. To assess the degree of phylogenetic congruence between the two different datasets, an incongruence length difference (ILD) test (Farris et al. 1994) implemented as partition homogeneity test in PAUP* 4.0b10 (Swofford 123 Author's personal copy S. Bogdanović et al. Table 1 Alphabetical list of taxa (numbers 1–4 indicate different populations of the same taxon), collection details, voucher information and GenBank accession numbers of Campanulaceae species analyzed in the present study No Taxon Collection details Voucher information ITS GenBank number trnL-F GenBank number 1 Asyneuma campanuloides Bornm. Georgia, Greater Caucasus Schönswetter & Tribsch 4469 (WU) DQ304586 FJ426570 2 Asyneuma limoniifolium Bornm. Greece, Ionian Islands, Lefkada Gutermann 35549 (WU) DQ304587 FJ426571 3 Campanula acarnanica Damboldt Greece, Acarnania, Mt. Akarnanika Ori Karamplianis Th. 1692 (ATHU) KF957752 KF957763 4 Campanula aureliana Bogdanović, Rešetnik, Brullo & Shuka 1 Albania, Tomori Mt, western part of village Kapinovë Bogdanović & Jug Dujaković (ZAGR) KM215787a KM215789a 5 Campanula aureliana Bogdanović, Rešetnik, Brullo & Shuka 2 Albania, Tomori Mt, Tomori village Bogdanović, Rešetnik & Temunović s.n. (ZAGR) KM215788a KM215790a 6 Campanula cephallenica Feer 1 Greece, Ionian Islands, Kefallinı́a Gutermann 28945 (WU) DQ304597 FJ426576 7 Campanula cephallenica Feer 2 Greece, Isola Cephalonia, Mt. Aivos Brullo, S. & Giacalone G. s.n. (CAT) KF957753 KF957764 8 Campanula comosiformis (Hayek & Janch.) Frajman & Schneew. 1 Albania, Gjalica, Mustafe Bogdanović & Jug-Dujaković s.n. (ZAGR) KF957754 KF957765 9 Campanula comosiformis (Hayek & Janch.) Frajman & Schneew. 2 Albania, Šija gorge E of Bicaj Frajman 11089 (WU) FJ426592 FJ426572 10 Campanula debarensis Rech.f. FYR Macedonia, Crni Drin Kovačić 1097 (ZA) DQ304595 FJ426575 11 Campanula debarensis Rech.f. FYR Macedonia, Crni Drim K. Micevski s.n. (MKNH 031830) KF957738 KF957745 12 Campanula elatines L. Italy, Alpi Cozie Schönswetter & Tribsch 6349 (WU) DQ304624 FJ426577 13 Campanula elatinoides Moretti Italy, Southern Alps, Lago d’Iseo Gutermann 1879 (WU) DQ304625 FJ426578 14 Campanula fenestrellata Feer subsp. fenestrellata 1 Croatia, Velebit, Velika Paklenica Kovačić 920 (ZA) DQ304592 FJ426579 15 Campanula fenestrellata Feer subsp. fenestrellata 2 Campanula fenestrellata subsp. istriaca (Feer) Damboldt 1 Croatia, NP Krka, Roški slap Šegota & Hršak s.n.(ZAGR) KF957755 KF957766 Croatia, Krk, Uvala Oprna Schönswetter & Tribsch 6272 (WU) DQ304594 FJ426584 Croatia, Istra, Plomin Bogdanović & Ljubičić s.n. (ZAGR) KF957756 KF957767 16 17 Campanula fenestrellata subsp. istriaca (Feer) Damboldt 2 18 Campanula fragilis Cirillo Italy, Calabria, city of Scalea Gutermann 36164 (WU) DQ304626 FJ426580 19 Campanula garganica Ten. 1 Cult. in Botanical Garden Zagreb (material from Italy); Italy, Foggia Kovačić 1012 (ZA); Aldobrandi 12-VII-96 et al. (MA 625685) DQ304596 EF088725 20 Campanula garganica Ten. 2 Italy, Gargano, Vieste Brullo & Signorello s.n. (CAT 037.237/7) KF957739 KF957746 21 Campanula isophylla Moretti Cult. in Botanical Garden Zagreb (material from Italy) Kovačić 1013 (ZA) DQ304630 FJ426583 22 Campanula persicifolia L. Austria, Northeastern Alps Schönswetter & Tribsch 6288 (WU) DQ304590 FJ426573 23 Campanula pollinensis Podlech Italy, Monte Pollino Brullo, Signorello, Spampinato s.n. (CAT 037.066/30) KF957740 KF957747 24 Campanula portenschlagiana Roem. & Schult. 1 Croatia, Biokovo Kovačić 692 (ZA) DQ304600 FJ426587 25 Campanula portenschlagiana Roem. & Schult. 2 Croatia, otok Brač, Vidova gora M. Ruščić s.n. (ZAGR 26291) KF957741 KF957748 26 Campanula portenschlagiana Roem. & Schult. 3 Bosnia and Herzegovina, Ljubuški Šiljeg s.n. (ZAGR) KF957757 KF957768 27 Campanula portenschlagiana Roem. & Schult. 4 Croatia, Island Hvar, Pitve Rimac s.n. (ZAGR) KF957758 KF957769 123 Author's personal copy Campanula aureliana, a new species from Albania Table 1 continued No Taxon Collection details Voucher information ITS GenBank number trnL-F GenBank number 28 Campanula poscharskyana Degen 1 Croatia, Dubrovnik region Kovačić 690 (ZA) DQ304601 FJ426588 29 Campanula poscharskyana Degen 2 Croatia, Radovčići Kovačić (ZAGR) KF957759 KF957770 30 Campanula pyramidalis L. Croatia, Vratnik pass; Croatia, Rijeka Schönswetter & Tribsch 6243 (WU); Vitek 99440 (MA 641379) DQ304606 EF088754 31 Campanula reatina Lucchese 1 Italy, Turano Valley Kovačić 768 (ZA) DQ304599 FJ426589 32 Campanula reatina Lucchese 2 Italy, Valle del Salto, Ponte Figureto Kirin s.n. (ZAGR) KF957760 KF957771 33 Campanula rotundifolia L. Croatia, Platak—Rijeka region; Andorra Kovačić 784 (ZA); Sáez 6134 (BCB) DQ304615 EF088759 34 Campanula scheuchzeri Vill. Croatia, North Velebit Kovačić 807 (ZA) DQ304614 KF957749 35 Campanula skanderbegii Bogdanović, Brullo & D. Lakušić 1 Albania, Kruje Lakušić, Kuzmanović, Lazarević & Alegro s.n. (ZAGR) KF957761 KF957772 36 Campanula skanderbegii Bogdanović, Brullo & D. Lakušić 2 Albania, Kruje Lakušić, Kuzmanović, Lazarević & Alegro s.n. (ZAGR) KF957762 KF957773 37 Campanula stevenii Bieb. Georgia, Minor Caucasus; Armenia, Vayk Schönswetter & Tribsch 6976 (WU); Oganessian s.n. (ERE 154865) DQ304591 EF088770 38 Campanula teutana Bogdanović & Brullo 1 Croatia, Island of Vis, Oključina, calcareous cliffs near Kraljičina špilja S. Bogdanović s.n. (ZAGR 32628) KF957742 KF957750 39 Campanula teutana Bogdanović & Brullo 2 Croatia, otok Vis, Oključina S. Bogdanović s.n. (ZAGR) KF957743 KF957751 FJ426590 40 Campanula tommasiniana Koch Croatia, Učka Kovačić 775 (ZA) DQ304611 41 Campanula versicolor Andrews Greece, Ionian Islands, Kefallinı́a Gutermann 30067 (WU) DQ304607 FJ426591 42 Petromarula pinnata DC. Greece, Crete Schönswetter & Tribsch 7821 (WU) DQ304582 FJ426585 43 Physoplexis comosa Schur Italy, Southern Alps DQ304585 FJ426586 44 Phyteuma globulariifolium Sternb. & Hoppe Austria, Niedere Tauern Schönswetter & Tribsch 3902 (WU) Schönswetter & Tribsch 4551 (WU) DQ304583 FJ426582 45 Phyteuma spicatum L. Croatia, Gorski Kotar; Spain, Barcelona, Aiguafreda Schönswetter & Tribsch 6233 (WU); Roquet 8-V-05 (BC) DQ304584 EF088787 46 Trachelium caeruleum L. Spain, N of Malaga; Spain, Santander, Liencres Schönswetter & Tribsch 8736 (WU); Aldasoro 3503 (MA) DQ304570 EF088791 a New sequences produced in this study 2003) was performed using 1,000 partition replicates, each comprising 100 random sequence addition replicates, and TBR branch swapping. Invariant characters were removed from the data sets prior to performing the ILD test (Cunningham 1997). Unweighted MP analyses were conducted using heuristic search, with 1,000 random addition sequence replicates, and tree bisection reconnection (TBR) branch swapping, as implemented in PAUP* 4.0b10 (Swofford 2003). Bootstrap support values (MPB; Felsenstein 1985) from 1,000 replicates were generated using the heuristic search options as above except for random addition sequence with 100 replicates. The scores between 50 and 74 bootstrap percentages were defined as weak support; scores between 75 and 89 % MPB, as moderate support; and scores above 90 % MPB, as strong support. BI was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The analysis of the combined data set was carried out under partition-specific substitution models (Nylander 2004) as selected for each partition separately 123 Author's personal copy S. Bogdanović et al. using AIC scores in MrModelTest. Thus, all substitution model parameters were allowed to vary across partitions. The Markov Chain Monte Carlo (MCMC) settings consisted of two runs with four chains each for 107 generations, with the sample frequency set to 1,000. The first 2,500 trees (prior to the 2.5 9 106 generation), which was well after the chains had reached stationarity as judged from plots of the likelihood and from the average standard deviation of split frequencies being \0.01, were discarded as burn-in. Convergence of the MCMC procedure was assessed further by calculating the effective sample sizes (ESS) with the program Tracer ver. 1.4 (Rambaut and Drummond 2007). A majority rule consensus tree was constructed from the posterior set of 15,000 trees. Results Morphology Morphological features of C. aureliana are presented in Figs. 2, 3, and a detailed description is provided in the taxonomic treatment section. Seed micromorphology Campanula aureliana shows a seed coat morphologically well differentiated from the closely related C. fenestrellata. The seeds of the former are subglobose-ovoid with weakly striate testa characterized by very elongate fibriform cells, imperceptibly anastomosed, with smooth and slightly raised periclinal walls, fused with the anticlinal walls showing a chain of minute papillae (Fig. 4a, b). The seeds in the latter are ovoid, with a markedly striate testa characterized by shorter cells, evidently anastomosed, with periclinal walls showing a deeply incise linear lumen, while the anticlinal walls are quite prominent and slightly channeled on the back (Fig. 4c, d). Phylogenetic analyses The characteristics of the ITS, trnL–trnF, and ITS–trnL– trnF combined datasets analyzed using MP and BI are summarized in Table 2. As the ILD test revealed no significant difference (p = 0.25) between the selected partitions (trnL–trnF and ITS), the phylogenetic tree of the combined data set obtained by BI analysis is presented in Fig. 5. In all analyzed data sets, the C. aureliana individuals are inferred as distinct members of the garganica clade (Fig. 5) without resolved sister taxa. The individuals of C. fenestrellata also formed a separate clade, while all other members of the group formed a clade supported only with BI analysis (0.71 PP). Within this clade C. reatina formed 123 one maximally supported subclade (100 BS, 1 PP), the second BI supported subclade (0.93 PP) included C. skanderbegii and C. portenschlagiana, and the third subclade also supported only with BI analysis (0.95 PP) included the remaining species. Discussion Phylogenetic analyses (Fig. 5) and comparison of morphological characters confirm C. aureliana as a distinct member of the Campanula ser. Garganicae. Furthermore, none of the other members of the garganica clade is resolved as a supported sister taxa of C. aureliana, according to the separate (data not shown) or combined phylogenetic analysis (Fig. 5). According to the herbarium material and literature (Reichenbach 1860; Feer 1890; Damboldt 1965), Campanula aureliana is morphologically closely related to C. fenestrellata Feer, mainly for the habit and shape and size of the corolla, but differs in a lot of significant traits. In particular, C. aureliana is characterized by stems and leaves always densely hairy, stems slender, leaves with petiole max. 7 cm long, blade smaller (max 30 mm long), dentate, calyx teeth linear–triangular, shorter than corolla tube (sometime subequal), adherent to the corolla, which is outside ciliate along the midribs, with lobes 2.5–3 mm wide al the base, stamen filaments 1.5–2 mm long, with basal blade subcircular, anthers shorter (3–3.2 mm), capsule subglobose, 2.6–3 mm in diameter, with calyx teeth suberect, seeds subcircular– ovoid, smaller (0.5 9 0.4 mm). Conversely, C. fenestrellata is glabrous (rarely tomentose above), more robust, with petiole up to 9 cm long, leaf blade up to 40 mm long, biserrate, calyx teeth linear-lanceolate, thin, longer than corolla tube, detached from the corolla or deflexed, corolla glabrous or ciliate at the base, with lobes 3–4 mm wide al the base, stamen filaments 3.5 mm long, with basal blade long ovate, anthers longer (4 mm), capsule ovoid, 3.5 mm long, with calyx teeth patent to deflexed, seeds elliptical– ovoid, bigger (0.65–0.8 9 0.4 mm). The features of seed micromorphology are also used in the genus Campanula to differentiate species, and two main seed coat patterns, the reticulate and striate types, are present (Geslot 1980; Murata 1992; Toniuc 1999; Buss et al. 2001; Akcin 2009). Each type shows a specific variability when the seeds of a taxon are analyzed by SEM, and specifically in the Campanula ser. Garganicae, including only isophyllous taxa, the seed coat is usually striate, with elongated cells and lumen essentially linear, but quite variable in shape, size and arrangement (Bogdanović et al. 2014a, b). These differences are also present between C. aureliana and C. fenestrellata (Fig. 4) providing further evidence for the separation of the two taxa. Furthermore, the morphological Author's personal copy Campanula aureliana, a new species from Albania Fig. 2 Campanula aureliana. a Habit, b leaves, c leaf apex, d flower and buds in verticillaster. Drawing by Salvatore Brullo resemblance with C. fenestrellata or with C. garganica and C. debarensis as postulated in the past (Markgraf 1931; Damboldt 1965), is not supported by molecular data. Although the overall phylogenetic relationships between taxa in the group are not fully resolved, some indications are provided. The morphologically similar Croatian endemic C. portenschlagiana and the Albanian endemic C. skanderbegii are resolved as sister taxa according to the BI (0.93 PP, Fig. 5). The rest of the southern Balkan taxa and a coastal Apennine taxon form a weakly supported group (0.71 PP) inferred in the Bayesian analysis. In that group maximally supported grouping is present among the Greek endemics C. cephallenica and C. acarnanica, indicating a very close relationship which is not surprising considering the close geographic proximity of the two species. More interesting is the moderately to strongly supported (81 MPB, 0.95 PP) grouping between the southern Adriatic endemics C. poscharskyana and C. garganica distributed on the opposite coasts of the Adriatic Sea (Fig. 5). As the other Apennine member of the garganica clade, C. reatina, is phylogenetically distinct, this provides further evidence for two dispersal events across the Adriatic as already suggested by Park et al. (2006) and Frajman and Schneeweiss (2009). Such amphi-Adriatic distributions are well known in plants (Turrill 1929), and interestingly, there is increasing evidence showing such independent dispersals 123 Author's personal copy S. Bogdanović et al. Fig. 3 Campanula aureliana. a Flower, b flower (lateral view), c flower (dorsal view), d corolla open (dorsal view), e bud, f and g stamens, h and i style and stigma, j fructiferous calyx and capsule, k seeds (ZAGR!, holotype). Drawing by Salvatore Brullo within the same genera or groups (Knautia, Frajman et al. submitted; Edraianthus, Surina et al. 2014). In conclusion, all lines of evidence presented in this paper strongly support the recognition of plants previously named as C. garganica var. albanica at species level, and without close relationship to Italian endemic C. garganica. As the epithet ‘‘albanica’’ is unavailable in combination with Campanula at species level because of previously validly published name Campanula albanica Witasek, the name C. aureliana is proposed for this new species. 123 Taxonomic treatment Campanula aureliana Bogdanović, Rešetnik, Brullo & Shuka, sp. nov. (Figs. 2, 3, 4) =Campanula garganica var. albanica Markgr., Denkschr. Kaiserl. Akad. Wiss., Wien. Math.-Naturwiss. Kl. 102: 356 (1931). Campanula fenestrellata similaris sed scapis et foliis semper dense pilosis, folii petiolo usque ad 7 cm longo, lamina foliorum max. 30 9 25 mm, dentata, dentibus calycinis lineari-triangularibus, tubo corollino plerumque brevioribus et corolla adhaerentibus, corolla extus ciliata Author's personal copy Campanula aureliana, a new species from Albania Fig. 4 Scanning electron micrographs of seed surface of Campanula aureliana (a, b) from holotype ZAGR! and C. fenestrellata (c, d) from Mt Velebit ZAGR!. a, c Seed (full view); b, d testa detail Table 2 Characteristics of cpDNA, ITS and cpDNA–ITS combined datasets analyzed using maximum parsimony (MP) and Bayesian inference (BI) Region cpDNA ITS Combined Alignment length Number/ % of parsimonyinformative characters Number/length of MP trees CI/RI Substitution model used in BI Harmonic mean of the posterior likelihood scores (ln L) of BI trees Effective sample size (ESS) 978 118/12.06 766 208/27.15 1744 326/18.69 13256/278 18/581 102/864 0.7644/0.935 GTR?G 0.613/0.842 SYM?G -3201.98 -4227.94 0.651/0.874 GTR?G (cpDNA), SYM?G (ITS) -7433.22 14987.00 14247.89 13514.82 CI consistency index (excluding uninformative characters), RI retention index secum costam, lobis 2.5–3 mm latis basi, filamentis staminorum 1.5–2 mm longis, lamina basali subcirculari, anthera breviore (3–3.2 mm), capsula subglobosa, dentibus calicis suberectis, seminibus subcirculari-ovoideis, minoribus, differt. TYPE: ALBANIA. Tomori village calcareous rocky places in the village, 23 June 2013, 807 m alt., S. Bogdanović, I. Rešetnik & M. Temunović s.n. (holotype: ZAGR!; isotypes: CAT!, ZA!, ZAGR! and TIR!). Description Plant perennial, densely hairy, with rigid and erect-patent hairs. Rootstock woody, branched, naked, with numerous prostrate or ascending stems, simple or branched at the base. Stems herbaceous, 10–30 cm long, leafy, ending in many flowered racemes. Leaves arranged in basal rosettes, densely covered by rigid hairs, 0.4–1 mm long; petiole 2–7 cm long, densely covered by patent hairs; blade cordate, dark green, 7–30 9 6–25 mm, cordate at the base, acute at the apex irregularly dentate at the margin (6–11 acute to obtuse teeth for side), with pinnate venations; cauline leaves similar to the basal, gradually decreasing in size upwards, with petioles 4–25 mm long, blade 4–25 9 3–20 mm. Flowers usually solitary or 2–4 arranged in raceme at leaf axil, 15–35 mm long; pedicel 5–20 mm long, densely hairy, with 0–1 bracteoles. Calyx green, densely hairy, with teeth entire, linear–triangular, 1-nerved, 1.5–2 (2.5) 9 0.8–1 mm, patent, acute at the apex. Corolla blue–violet, rotate-infundibular, 8–10 mm long, 14–16 mm in diameter, glabrous inside, outside 123 Author's personal copy S. Bogdanović et al. Fig. 5 Phylogenetic relationships of Campanula series Garganicae and relatives inferred from Bayesian analysis of combined nuclear ITS and plastid trnL–trnF data. Values above branches are Bayesian posterior probabilities (PP) and values below branches are maximum parsimony (MPB) bootstrap percentages (only shown if at least 50 %) ciliate on the principal veins; tube cup-shaped, 1.5–2 mm long; lobes 6.5–8 9 2.5–3 mm, oblong-elliptical, divaricate to subpatent, with 1 midrib and various secondary veins, apex acute. Style with stigma exerted from corolla, 7.5–9 mm long, white and glabrous below, violet and papillose-hairy above, with 3 stigmas, ventrally whitish, each 1.5 mm long. Stamens 5, with filaments widened at base into a subcircular blade (or disk), 1 mm in diameter, densely ciliate in the upper part and margin; filaments glabrous, slightly violet, 1.5–2 mm long; anthers pale-blue to violet, 3–3.2 mm long, apiculate at the apex; pollen white to pale yellow. Capsule subglobose, 2.6–3 mm in diameter, 5-ribbed, densely hairy (hairs 0.2–0.3 mm long), without pores, with suberect calyx teeth, 3–3.5 mm long. Seeds subglobose-ovoid, 0.5 9 0.4 mm, brown, shiny (Figs. 2, 3, 4a, b, 6a, b). Beratit), Tomori Mt (Mali i Tomorrit), western part of village Tomorr i Vogël, in the valley of River Tomorri (Lumi i Tomorrit); on limestone rock, near a spring, alt: 565 m, Barin, Z. & Nemet, Cs., no. 6201, 24 May 2004 (BP746929!). North-western slopes of Tomori Mt, the north-eastern side of Tomori castle; on limestone rock crevices that occur between clearings of Pinus heldreichii and Juniperus foetidissima woods and in rocky calcareous cliff faces, partly shading by Fagus sylvatica woods, alt: 1200-1300 m, Shuka L. & Xhulaj M., no. 5832–5836, 25 June 2013. Northern slopes of Tomori Mt, below the peak of Çuka e Partizanit; in shady crevices and alcoves of limestone cliffs with NW exposition and surrounded sparsely by Pinus heldreichii and Juniperus communis subsp. alpina woods, alt: 1950 m, Shuka L. & Xhulaj M., Hoda P. & Mahmutaj E., no. 6238, 13 July 2014. Additional specimens examined Etymology ALBANIA. Tomori Mt, western part of village Kapinovë, on limestone rocky crevices, in shady places. Bogdanović S. & Jug-Dujaković M., 13 July 2012 (ZAGR32635!, ZAGR32634!, ZAGR32633!). District of Berat (Rrethi i The specific epithet refers to the Latin name Aurelius (in Croatian Zlatko), and it is dedicated to the botanists Prof. Zlatko Liber and Prof. Zlatko Šatović from University of Zagreb (Croatia). 123 Author's personal copy Campanula aureliana, a new species from Albania Fig. 6 Campanula aureliana. a Habit, b inflorescence, c and d habitat (photos by Sandro Bogdanović and Lulëzim Shuka) Phenology Flowering from May to July, depending on the altitude and fruiting late July to August. Distribution and ecology Campanula aureliana is only known from the single population in Tomori Mt that is fragmented into four subpopulations, Tomori and Kapinovë villages, Castle of Tomori and the northern peak of Tomori Mt in central Albania (Fig. 1). It grows on limestone in rocky crevices of shady places at an altitude of 500–1950 m asl (Fig. 6d). This species is a true chasmophyte exclusive of fresher niches, where it is a member of a rupestrian community characterized by Aethionema saxatile (L.) R.Br., Ajuga chamaepytis (L.) Schreb., Asplenium ceterach L., A. ruta-muraria L., A. trichomanes L., Bupleurum veronense Turra, Campanula ramosissima Sibth. & Sm., Desmazeria rigida (L.) Tutin, Galium rubrum L., Geranium robertianum L., Leontodon crispus Vill., Micromeria juliana (L.) Benth. ex Rchb., Minuartia verna (L.) Hiern, Petrorhagia saxifraga (L.) Link, Putoria calabrica (L.f.) DC., Satureja montana L., Sedum acre L., Sedum ochroleucum Chaix., and Teucrium polium L. among others. In the central part of distribution range (1200–1300 m asl), C. aureliana occurs in rocky cervices, within openings of Fago-Pinetum leucodermis, association of the order Fagetalia sylvaticae (Mahmutaj et al. 2013). In this habitat (Fig. 6c), it occurs in rupestrian communities dominated by Juniperus foetidissima Willd. and J. oxycedrus L., shrubs associated with Acinos alpinus (L.) Moench., Alkanna pindicola Hausskn., Asplenium trichomanes L., Carex sp., Dactylis glomerata L., Globularia cordifolia L., Melica uniflora Retz. Micromeria cristata (Hampe) Griseb., M. juliana (L.) Benth. ex Rchb., Onosma echioides L., Parietaria officinalis L., Pterocephalus perennis Coulter, Ramonda serbica Pančić, Satureja montana L., Sedum acre L., Teucrium polium L., and others. In the upper limit of distribution it is usually found in association with Amphoricarpos autariatus Blečić & E.Mayer, Campanula spatulata subsp. spruneriana (Hampe) Hayek, Carex sp., Crepis baldaccii Halácsy, Festuca varia Haenke, Geranium macrorrhizum L., Heliosperma pusillum (Waldst. & Kit.) Rchb., Lamium garganicum L., Leontodon crispus Vill., Potentilla speciosa Willd., Saxifraga marginata Sternb., Saxifraga paniculata Mill. and others. 123 Author's personal copy S. Bogdanović et al. Conservation status Campanula aureliana is known from one population, distributed in four localities, all in western and north-western slopes of Mt. Tomori, in Berati district (central Albania), where approximately not more than 1000 mature individuals grow in a very small area of 8 km2. Since the species is fragmented in four locations, half of which occurs in settled area, the habitat is under human influence and it could be threatened by human activities. Therefore, according to the IUCN Red list category (IUCN 2014), this species for its rarity, number of mature individuals and restricted population distribution, should be included on the list of threatened plants as Vulnerable—VU D1?2. Acknowledgments We thank the curators of the following herbaria B, BEOU, BM, BP, CAT, CNHM, K, MKNH, NAP, NHMR, PAL, RO, TIR, W, WU, ZA, ZAGR, and ZAHO for the examination of Campanula specimens. Thank to Nicholas Turland (Germany) for kind search of type material of C. garganica var. albanica in B, as well as to Zoltán Barina (Hungary) for access of Albanian Campanula material in BP. We also thank Theophanis Constantinidis (Greece) for sending the material of Campanula acarnanica, and Marija Jug-Dujaković and Martina Temunović (Croatia) for assistance in the field trip. We are grateful to Katherine Challis (England) for nomenclatural advice. This study was financially supported by project no. 119-1191193-1232 of the Ministry of Science, Education and Sports (Zagreb, Croatia) and by the Croatian Academy of Science project ‘‘Genetic diversity of Croatian endemic Campanula’’. 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