BACKGROUND: The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. METHODOLOGY/PRINCIPAL FINDINGS: Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161-162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. CONCLUSIONS/SIGNIFICANCE: The determination of complete plastid genome and nuclear rRNA cluster sequences in three species of the genus Pulsatilla is an important contribution to our knowledge of the evolution and phylogeography of those endangered taxa. The resulting data can be used to identify regions that are particularly useful for barcoding, phylogenetic and phylogeographic studies. The investigated taxa can be identified at each stage of development based on their species-specific SNPs. The nuclear and plastid genomic resources enable advanced studies on hybridization, including identification of parent species, including their roles in that process. The identified nonsynonymous mutations could play an important role in adaptations to changing environments. The results of the study will also provide valuable information about the evolution of the plastome structure in the family Ranunculaceae.

Department of Biology and Ecology University of Ostrava 71000 Ostrava Czech Republic

Department of Botany and Nature Protection University of Warmia and Mazury 10 728 Olsztyn Poland

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Tamura M. Ranunculaceae. In: Kubitzki K., Rohwer J.G., Bittrich V., editors. The Families and Genera of Vascular Plants. Volume 2. Springer-Verlag; Berlin, Germany: 1993. pp. 563–583.

Wang W.C. Flora of China. Volume 27, 28 Science Press (in Chinese); Beijing, China: 1979. Ranunculaceae.

Xu Q., Shu Z., He W., Chen L., Yang S., Yang G. Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo. Phytomedicine. 2012;19:293–300. doi: 10.1016/j.phymed.2011.08.066. PubMed DOI

Martin M.L., San Roman L., Dominguez A. In vitro activity of protoanemonin on antifungal agent. Planta Med. 1990;56:66–69. doi: 10.1055/s-2006-960886. PubMed DOI

Campbell W., Cragg G.M.L., Powrie A.H. Anemonin, protoanemonin and ranunculin from Knowltonia capensis. Phytochemistry. 1979;18:323–334. doi: 10.1016/0031-9422(79)80083-7. DOI

Akeroyd J.R. Pulsatilla Miller. Flora Eur. 1993;1:264–266.

Uotila P. Pulsatilla patens × vernalis Suomessa. Memo. Soc. Fauna Flora Fenn. 1980;56:111–118.

Uotila P. Decline of Anemone patens (Ranunculaceae) in Finland. Acta Univ. Ups. Symb. Bot. Ups. 1996;31:205–210.

Wójtowicz W. Pulsatilla patens (L.) Mill. Sasanka otwarta. In: Werblan-Jakubiec H., Sudnik-Wójcikowska B., editors. Poradnik ochrony siedlisk i Gatunków Natura 2000—Podręcznik Metodyczny, Gatunki roślin (in Polish) GIOS; Warszawa, Poland: 2004. pp. 168–171.

Bock J.H., Peterson S.J. Reproductive biology of Pulsatilla patens (Ranunculaceae) Am. Mid. Nat. 1975;94:476–478. doi: 10.2307/2424441. DOI

Ordway E. The phenology and pollination biology of Anemone patens (Ranunculaceae) in western Minesota. In: Clambey G.K., Pemble R., editors. The Prairie: Past, Present and Future: Proceedings of the Ninth North American Prairie Conference. University of Wisconsin–Madison Press; Moorhead, MN, USA: 1986.

Pilt I., Kukk Ü. Pulsatilla patens and Pulsatilla pratensis (Ranunculaceae) in Estonia: Distribution and ecology. Proc. Estonian Acad. Sci. Biol. Ecol. 2002;51:242–256.

Kalliovirta M., Ryttäri T., Heikkinen R.K. Population structure of a threatened plant, Pulsatilla patens in boreal forests: Modelling relationships to overgrowth and site closure. Biodivers. Conserv. 2006;15:3095–3108. doi: 10.1007/s10531-005-5403-z. DOI

Grzyl A., Kiedrzyński M., Zielińska K.M., Rewicz A. The relationschip between climatic conditions and generative reproduction of the lowland population of Pulsatilla vernalis the last breath of a relict plant fluctuating cycle of regeneration. Plant Ecol. 2014;215:457–466. doi: 10.1007/s11258-014-0316-0. DOI

Priede G., Klavina D. In vitro cultivation and root initiation of the endangered plant Pulsatilla patens. Environ. Exp. Biol. 2011;9:71–74.

Sauliene I., Brinkyte E. The impact of phytohormones on pasgueflower (Pulsatilla) regeneration in vitro. Acta Biol. Univ. Daugavp. 2009;9:249–254.

Šedivá J. In vitro seed propagation of some Pulsatilla species (Pulsatilla L.) Acta Pruhaniciana. 2002;73:48–51.

Šedivá J., Žlebčík J. Summary of findings from a propagation and ex situ conservation of Pulsatilla vernalis, P. pratensis ssp. bohemica, P. patens and P. grandis. Acta Pruhoniciana. 2012;100:155–160.

Danova K., Bertoli A., Pistelli L., Dimitrov D., Pistelli L. In vitro culture of Balkan endemic and rare Pulsatilla species for conservational purposes and secondary metabolites production. Bot. Serbica. 2009;33:157–162.

Naumovski D., Radič S., Pevalek-Kozlina B. In vitro micropropagation of Pulsatilla pratensis (L.) Miller ssp. nigricans (Störck) Zämelis. Propag. Ornam. Plants. 2009;9:16–20.

Bang S.C., Kim Y., Lee J.H., Ahn B.Z. Triterpenoid saponins from the roots of Pulsatilla koreana. J. Nat. Prod. 2005;68:268–272. doi: 10.1021/np049813h. PubMed DOI

Lee S.H., Lee E., Ko Y.T. Anti-inflammatory effects of a methanol extract from Pulsatilla koreana in lipopolysaccharide-exposed rats. BMP Rep. 2012;45:371–376. doi: 10.5483/BMBRep.2012.45.6.018. PubMed DOI

Hantula J., Uotila P., Saura A., Lokki J. Chloroplast DNA variation in Anemone s. lato (Ranunculaceae) Plant Syst. Evol. 1989;163:81–85. doi: 10.1007/BF00936155. DOI

Hoot S.B. Phylogeny of the Ranunculaceae based on epidermal microcharacters and macromorphology. Syst. Bot. 1991;16:741–755.

Hoot S.B. Phylogeny of the Ranunculaceae based on preliminary atpB, rbcL and 18S nuclear ribosomal DNA sequence data. Plant Syst. Evol. 1995;9:241–251.

Horandl E., Paun O., Johanson J.T., Lehnebach C., Armstrong T., Chen L., Lockhart P. Phylogenetic relationships and evolutionary traits in Ranunculus s.l. (Ranunculaceae) inferred from ITS sequence analysis. Mol. Phyl. Evol. 2005;36:305–327. doi: 10.1016/j.ympev.2005.02.009. PubMed DOI

Tamura M. Angiospermae ordnung Ranunculales Fam. Ranunculaceae. II. Systematic Part. In: Hiepko P., editor. Natürliche Pflanzenfamilien. 17aIV. Duncker & Humblot; Berlin, Germany: 1995. pp. 223–519.

Ehrendorfer F., Samuel R. Contributions to molecular systematic and phylogeny of Anemone and related genera (Ranunculaceae-Anemoninae) Acta Phytotaxon. Sin. 2001;39:293–307.

Hoot S.B., Meyer K.M., Manning J.C. Phylogeny and reclassification of Anemone (Ranunculaceae), with an emphasis on austral species. Syst. Bot. 2012;37:139–152.

Hensen I., Obeprieler C., Wesche K. Genetic structure, population size, and seed production of Pulsatilla vulgaris Mill. (Ranunculaceae) in Central Germany. Flora. 2005;200:3–14. doi: 10.1016/j.flora.2004.05.001. DOI

Ronikier M., Costa A., Aguilar J.F., Feliner G.N., Küpfer P., Mirek Z. Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): Chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia. J. Biogeogr. 2008;35:1650–1664. doi: 10.1111/j.1365-2699.2008.01907.x. DOI

Hedrick P.W., Miller P.S. Conservation genetics: Techniques and fundamentals. Ecol. Appl. 1992;2:30–46. doi: 10.2307/1941887. PubMed DOI

Ouborg N.J., Piquot Y., von Groenendael M. Population genetic, molecular markers ant the study dispersal in plants. J. Ecol. 1999;87:5551–5568. doi: 10.1046/j.1365-2745.1999.00389.x. DOI

Provan J., Powell W., Hollingsworth P.M. Chloroplast microsatellites: New tools for studies in plant ecology and evolution. Trends Ecol. Evol. 2001;16:142–147. doi: 10.1016/S0169-5347(00)02097-8. PubMed DOI

Chiang T-Y., Chiang Y-C., Chen Y-J., Chou C-H., Havanond S., Hong T-N., Huang S. Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs. Mol. Ecol. 2001;10:2697–2710. doi: 10.1046/j.0962-1083.2001.01399.x. PubMed DOI

Kanno M., Yokoyama J., Suyama Y., Ohyama M., Itoh T., Suzuki M. Geographical distribution of two haplotypes of chloroplast DNA in four oak species (Quercus) in Japan. J. Plant Res. 2004;117:311–317. doi: 10.1007/s10265-004-0160-8. PubMed DOI

Birky C.W. The inheritance of gens in mitochondria and chloroplasts: Laws, mechanisms and models. Annu. Rev. Genet. 2001;35:125–148. doi: 10.1146/annurev.genet.35.102401.090231. PubMed DOI

Cruzan M.B., Templeton A.R. Paleoecology and coalescence: Phylogeographic analysis of hypotheses from the fossil record. Trends Ecol. Evol. 2000;15:491–496. doi: 10.1016/S0169-5347(00)01998-4. PubMed DOI

Orive M.E., Asmussen M.A. The effects of pollen and seed migration on nuclear-dicytoplasmic systems. II. A new method for estimating plant gene flow from joint nuclear-cyto plasmic data. Genetics. 2000;155:833–854. PubMed PMC

Petit R.J., Pineau E., Demeseure B., Bacilieri R., Ducousso A., Kremer A. Chloroplast DNA footprints of postglacial recolonization by oaks. Proc. Natl. Acad. Sci. USA. 1997;94:9996–10001. doi: 10.1073/pnas.94.18.9996. PubMed DOI PMC

Honjo M., Ueno S., Tsumura Y., Washitani I., Ohsawa R. Phylogeographic study based on intraspecific sequence variation of chloroplast DNA for the conservation of genetic diversity in the Japense endangered species Primula sieboldii. Biol. Conserv. 2004;120:211–220. doi: 10.1016/j.biocon.2004.02.016. DOI

Yang J.-B., Tang M., Li H.-T., Zhang Z.-R., Li D.-Z. Complete chloroplast genome of the genus Cymbidium: Lights into the species identification, phylogenetic implications and population genetic analyses. BMC Evol. Biol. 2013;13 doi: 10.1186/1471-2148-13-84. PubMed DOI PMC

Demesure B., Comps B., Petit R.J. Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolution. 1996;50:2515–2520. doi: 10.2307/2410719. PubMed DOI

Taberlet P., Fumagalli L., Wust-Saucy A.-G., Cosson J.-F. Comparative phylogeography and postglacial colonisation routes in Europe. Mol. Ecol. 1998;7:453–464. PubMed

Hewitt G.M. Post-glacial re-colonization of European biota. Biol. J. Linn. Soc. 1999;68:87–112. doi: 10.1111/j.1095-8312.1999.tb01160.x. DOI

Newton A.C., Allnutt T.R., Gillies A.C.M., Lowe A.J., Ennos R.A. Molecular phylogeography, intraspecific variation and the conservation of tree species. Trends Ecol. Evol. 1999;14:140–145. doi: 10.1016/S0169-5347(98)01555-9. PubMed DOI

Palme A.E., Vendramin G.G. Chloroplast DNA variation, postglacial recolonisation and hybridisation in hazel, Corylus avellana. Mol. Ecol. 2002;9:1769–1780. doi: 10.1046/j.1365-294X.2002.01581.x. PubMed DOI

Taberlet P., Gielly L., Pautou G., Bouvet J. Universal primers for amplification of three noncoding regions of chloroplast DNA. Plant Mol. Biol. 1991;17:1105–1109. doi: 10.1007/BF00037152. PubMed DOI

Demesure B., Sodzi N., Petit R.J. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 1995;4:129–131. doi: 10.1111/j.1365-294X.1995.tb00201.x. PubMed DOI

Shaw A.J., Cox C.J. Variation in “biodiversity value” of peatmoss species in Sphagnum section Acutifolia (Sphagnaceae) Am J. Bot. 2005;92:1774–1783. doi: 10.3732/ajb.92.11.1774. PubMed DOI

Sawicki J., Szczecińska M. A comparison of PCR-based markers for molecular identification of Sphagnum species of the section Acutifolia. Acta Soc. Bot. Pol. 2011;80:185–192. doi: 10.5586/asbp.2011.017. DOI

Gerbi S.A. Evolution of ribosomal rRNA. In: MacIntyre R.J., editor. Molecular Evolutionary Genetics. Plenum; New York, NY, USA: 1985. pp. 419–517.

Wendel F.F., Schnabel A., Seelanan T. Bidirectional interlocus concerned evolution following allopolyploid speciation in cotton (Gossypium) Proc. Natl. Acad. Sci. USA. 1995;92:280–284. doi: 10.1073/pnas.92.1.280. PubMed DOI PMC

Karvonen P., Szmidt A.E., Savolainen O. Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species. Theor. Appl. Genet. 1994;89:969–974. doi: 10.1007/BF00224526. PubMed DOI

Schoch C.L., Seifert K.A., Huhndorf S., Robert V., Spouge J.L., Levesque C.A., Chen W. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. USA. 2012;109:6241–6246. doi: 10.1073/pnas.1117018109. PubMed DOI PMC

Bednarek-Ochyra H., Ochyra R., Sawicki J., Szczecińska M. Bucklandiella seppeltii, a new species of Grimmiaceae from Australasia, and its phylogenetic position based on molecular data. Turk. J. Bot. 2014;38:1214–1228. doi: 10.3906/bot-1405-26. DOI

Denduangboripant J., Cronk Q.C.B. High intraindividual variation in internal transcribed spacer sequences in Aeschynanthus (Gesneriaceae): Implications for phylogenetics. Proc. Biol. Sci. 2000;267:1407–1415. doi: 10.1098/rspb.2000.1157. PubMed DOI PMC

Won H., Renner S.S. The internal transcribed spacer of ribosomal DNA in the gymnosperm Gnetum. Mol. Phyl. Evol. 2005;36:581–597. doi: 10.1016/j.ympev.2005.03.011. PubMed DOI

Volkov R.A., Komarova N.Y., Hemleben V. Ribosomal DNA in plant hybrids: Inheritance, rearrangement, expression. Syst. Biodivers. 2007;5:261–276. doi: 10.1017/S1477200007002447. DOI

Small R.L., Cronn R.C., Wendel J.F. Use of nuclear genes for phylogeny reconstruction in plants. Aust. Syst. Bot. 2004;17:145–170. doi: 10.1071/SB03015. DOI

Sun Y.-L., Park W.-G., Oh H.-K., Hong S.-K. Genetic diversity and hybridization of Pulsatilla tongkangensis based on nrDNA ITS region sequence. Biologia. 2014;69:24–31. doi: 10.2478/s11756-013-0284-1. DOI

Raubeson L.A., Peery R., Chuley T.W., Dziubek C., Fourcade H.M., Boore J.L., Jansen R.K. Comparative chloroplast genomics: Analyses including new sequences from the angiosperms Nuphar advena and Ranunculus macranthus. BMC Genom. 2007;8:174. doi: 10.1186/1471-2164-8-174. PubMed DOI PMC

Fajardo D., Senalik D., Ames M., Zhu H., Steffan S.A., Harbut R., Polashock J., Vorsa N., Gillespie E., Kron K. Complete plastid genome sequence of Vaccinium macrocarpon: Structure, gene content and rearrangements revealed by next generation sequencing. Tree Genet. Genomes. 2012;9:489–498. doi: 10.1007/s11295-012-0573-9. DOI

Szczecińska M., Gomolińska A., Szkudlarz P., Sawicki J. Plastid and nuclear genomic resources of a relict and endangered plant species—Chamaedaphne calyculata (L.) Moench. (Ericaceae) Turk. J. Bot. 2014;38:1229–1238. doi: 10.3906/bot-1405-80. DOI

Parks M., Cronn R., Liston A. Increasing phylogenetic resolution at a low taxonomic levels using massively parallel sequencing at chloroplast genomes. BMC Biol. 2009;7 doi: 10.1186/1741-7007-7-84. PubMed DOI PMC

Zhang Y.-J., Ma P.-F., Li D.-Z. High-throughput sequencing of six bamboo chloroplast genomes: Phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae) PLoS ONE. 2011;6:e20596. doi: 10.1371/journal.pone.0020596. PubMed DOI PMC

Wu C.-S., Wang Y.-N., Hsu C.-Y., Lin C.-P., Chaw S.-M. Loss of different inverted repeat copies from the chloroplast genomes of Pinaceae and Cupresophytes and influence of heterotachy on evolution of Gymnosperms phylogeny. Genome Biol. Evol. 2011;3:1284–1295. doi: 10.1093/gbe/evr095. PubMed DOI PMC

Chen X., Qiushi L., Ying L., Qian J., Han J. Chloroplast genome of Aconitum barbatum var. pubreulum (Ranunculaceae) derived from CCS reads using the PacBio RS platform. Front. Plant Sci. 2015;6 doi: 10.3389/fpls.2015.00042. PubMed DOI PMC

Yang J.B., Yang S.X., Li H.T., Yang J., Li D.Z. Comparative chloroplast genomes of Camellia species. PLoS ONE. 2013;8:e73053. doi: 10.1371/journal.pone.0073053. PubMed DOI PMC

Yang M., Zhang X., Liu G., Yin Y., Chen K., Yun Q., Zhao D., Al-Mssallem I.S., Yu J. The complete chloroplast genome sequence of date Palm (Phoenix dactylifera L.) PLoS ONE. 2010;15 doi: 10.1371/journal.pone.0012762. PubMed DOI PMC

Frazer K.A., Pachter L., Poliakov A., Rubin E.M., Dubchak I. VISTA: Computational tools for comparative genomics. Nucleic Acids Res. 2004;1:W273–W279. doi: 10.1093/nar/gkh458. PubMed DOI PMC

Korotkova N., Nauheimer L., Ter-Voskanyan H., Allgaier M., Borsch T. Variability among the most rapidly evolving plastid genomic regions is lineage-specific: Implications of pairwise genome comparisons in Pyrus (Rosaceae) and other angiosperms for marker choice. PLoS ONE. 2014;9:e112998. doi: 10.1371/journal.pone.0112998. PubMed DOI PMC

Nie X., Lv S., Zhang Y., Du X., Wang L. Complete chloroplast genome sequence of a major invasive species, crofton weed (Ageratina adenophora) PLoS ONE. 2012;7:e36869. doi: 10.1371/journal.pone.0036869. PubMed DOI PMC

Dong W., Liu J., Yu J., Wang L., Zhou S. Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PLoS ONE. 2012;7:e35071. doi: 10.1371/journal.pone.0035071. PubMed DOI PMC

Jukes T.H., King J.L. Deleterious mutations and neutral substitutions. Nature. 1971;231:114–115. doi: 10.1038/231114a0. PubMed DOI

Kimura M. The Neutral Theory of Molecular Evolution. Cambridge University Press; Cambridge, UK: 1983.

Anastasi G., Cutroneo G., Santoro G., Arco A., Rizzo G., Bramanti P., Rinaldi C., Sidoti A., Amato A., Favaloro A. Costameric proteins in human skeletal muscle during muscular inactivity. J. Anat. 2008;213:284–295. doi: 10.1111/j.1469-7580.2008.00921.x. PubMed DOI PMC

Ganapathi M., Srivastava P., Das Sutar S.K., Kumar K., Dasgupta D., Pal Singh G., Brahmachari V., Brahmachari S.K. Comparative analysis of chromatin landscape in regulatory regions of human housekeeping and tissue specific genes. BMC Bioinform. 2005;6:126. doi: 10.1186/1471-2105-6-126. PubMed DOI PMC

Rose A.B. Intron-mediated regulation of gene expression. Curr. Top. Microbiol. Immunol. 2008;326:277–290. PubMed

Dong W., Xu Ch., Li Ch., Sun J., Zuo Y., Shi S. ycf1, the most promising plastid DNA barcode of land plants. Sci. Rep. 2015;5:8348. doi: 10.1038/srep08348. PubMed DOI PMC

Krawczyk K., Sawicki J. The uneven rate of the molecular evolution of gene sequences of DNA-dependent RNA polymerase I of the genus Lamium L. Int. J. Mol. Sci. 2013;14:11373–11391. doi: 10.3390/ijms140611376. PubMed DOI PMC

Zhang C.Y., Wang F.Y., Yan H.F., Hao G., Hu C.M., Ge X.J. Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae) Mol. Ecol. Res. 2012;12:98–108. doi: 10.1111/j.1755-0998.2011.03076.x. PubMed DOI

Krawczyk K., Szczecińska M., Sawicki J. Evaluation of 11 single-locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae) Mol. Ecol. Res. 2014;14:272–285. doi: 10.1111/1755-0998.12175. PubMed DOI

Sloan D.B., Alverson A.J., Wu M., Palmer J.D., Taylor D.R. Recent acceleration of plastid sequence and structural evolution coincides with extreme mitochondrial divergence in the angiosperm genus Silene. Genome Biol. Evol. 2012;4:294–306. doi: 10.1093/gbe/evs006. PubMed DOI PMC

Erixon P., Oxelman B. Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene. PLoS ONE. 2008;3:e1386. doi: 10.1371/journal.pone.0001386. PubMed DOI PMC

Frankham R., Briscoe D.A., Ballou J.D. Introduction to Conservation Genetics. Cambridge University Press; New York, NY, USA: 2002.

Kapralov M.V., Smith J.A.C., Filatov D.A. Rubisco evolution in C4 eudicots: An analysis of Amaranthaceae sensu lato. PLoS ONE. 2012;7:e52974. doi: 10.1371/journal.pone.0052974. PubMed DOI PMC

Hu S., Sablok G., Wang B., Qu D., Barbaro E., Viola R., Li M., Varotto C. Plastome organization and evolution of chloroplast genes in Cardamine species adapted to contrasting habitats. BMC Genom. 2015;16:306. doi: 10.1186/s12864-015-1498-0. PubMed DOI PMC

Gilley L., Yong-Ming Y., Küpfer D., Taberlet P. Phylogenetic use of noncoding regions in the genus Gentiana L. Chloroplast trnL (UAA) intron versus nuclear ribosomal internal transcribed spacer sequences. Mol. Phyl. Evol. 1996;3:460–466. doi: 10.1006/mpev.1996.0042. PubMed DOI

Sawicki J., Plášek V., Szczecińska M. Molecular evidence do not support the current division of Orthotrichum subgenus Gymnoporus. Plant Syst. Evol. 2009;279:125–137. doi: 10.1007/s00606-009-0153-0. DOI

Szczecińska M., Kwaśniewski M., Chwiałkowska K., Sawicki J. Isolation and characterization of microsatellite loci in Pulsatilla patens (L.) Mill. (Ranunculaceae) a rare and endangered plant species in Europe. Conserv. Genet. Resour. 2013;5:421–423. doi: 10.1007/s12686-012-9818-z. DOI

Wyman S.K., Jansen R.K., Boore J.L. Automatic annotation of organellar genomes with DOGMA. Bioinformatics. 2004;20:3252–3255. doi: 10.1093/bioinformatics/bth352. PubMed DOI

Darling A.C., Mau B., Blattner F.R., Perna N.T. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–1403. doi: 10.1101/gr.2289704. PubMed DOI PMC

Krzywinski M., Schein J., Bird I., Connors J., Gascoyne R., Horsman D., Jones S.J., Marra M.A. Circos: An information aesthetic for comparative genomics. Genome Res. 2009;19:1639–1645. doi: 10.1101/gr.092759.109. PubMed DOI PMC

Faircloth B.C. MSATCOMMANDER: Detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol. Ecol. Res. 2008;8:92–94. doi: 10.1111/j.1471-8286.2007.01884.x. PubMed DOI

Edgar R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. PubMed DOI PMC

Golenberg E.M., Clegg M.T., Durbin M.L., Doebley J.D.P. Evolution of a noncoding region of the chloroplast genome. Mol. Phylogenet. Evol. 1993;2:52–64. doi: 10.1006/mpev.1993.1006. PubMed DOI

Morton B.R., Clegg M.T. A chloroplast DNA mutational hotspot and gene conversion in a noncoding region near rbcL in the grass family (Poaceae) Curr. Genet. 1993;24:357–365. doi: 10.1007/BF00336789. PubMed DOI

O’Donnell K. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris) Curr. Genet. 1992;22:213–220. doi: 10.1007/BF00351728. PubMed DOI

Akaike H. A new look at the statistical model identification. IEEE Trans. Autom. Control. 1974;19:716–723. doi: 10.1109/TAC.1974.1100705. DOI

Posada D., Crandall K.A. Modeltest: Testing the model of DNA substitution. Bioinformatics. 1998;14:817–818. doi: 10.1093/bioinformatics/14.9.817. PubMed DOI

Huelsenbeck J.P., Ronquist F.R. MRBAYES: Bayesian inference of phylogeny. Bioinformatics. 2001;17:754–755. doi: 10.1093/bioinformatics/17.8.754. PubMed DOI

Rambaut A., Drummond A.J. Tracer v1.3. 2003. [(accessed on 17 February 2012)]. Available online: http://evolve.zoo.ox.ac.uk.

Mitogenomic analyses support the recent division of the genus Orthotrichum (Orthotrichaceae, Bryophyta)