Systematic implications from a robust phylogenetic reconstruction of the genus Helianthemum (Cistaceae) based on genotyping-bysequencing (GBS) data

INTRODUCTION

Molecular systematics requires the establishment of a robust phylogenetic framework, including extensive geographical and taxonomic representativeness, well-supported species relationships and high certainty of tree topology. Particularly, for recently diversified lineages, data from Sanger sequencing-based techniques usually provide very low support due to insufficient DNA variability (DeFilippis & Moore 2000DeFilippis V.R. & Moore W.S. 2000. Resolution of phylogenetic relationships among recently evolved species as a function of amount of DNA sequence: an empirical study based on woodpeckers (Aves: Picidae). Molecular Phylogenetics and Evolution 16: 143-160.). Alternatively, reduced-representation sequencing methods such as genotyping-by-sequencing (GBS; Elshire & al. 2011Elshire R.J., Glaubitz J.C., Sun Q., Poland J.A., Kawamoto K., Buckler E.S. & Mitchell S.E. 2011. A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6: e19379. ) have been revealed to be highly resolutive because they allow the discovery of thousands of loci even from non-model species (Fernández-Mazuecos & al. 2018Fernández-Mazuecos M., Mellers G., Vigalondo B., Sáez L., Vargas P. & Glover B.J. 2018. Resolving Recent Plant Radiations: Power and Robustness of Genotyping-by-Sequencing. Systematic Biology 67: 250-268.).

Under this perspective, the Palearctic genus Helianthemum Mill. (Cistaceae Juss.), whose rapid diversification has been driven by the geo-climatic events that impacted the Mediterranean Basin since the Upper Miocene (see Thompson 2020Thompson J.D. 2020. Plant Evolution in the Mediterranean. Insights for Conservation. Oxford University Press, Oxford.), is a challenging case of study due to its complex taxonomy and broad geographical distribution (Janchen 1925Janchen E. 1925. Cistaceae. In Engler A. (ed.), Die Natürlichen Pflanzenfamilien, ed. 2, 21: 289-313. Engelmann, Leipzig.; Quézel & Santa 1962Quézel P. & Santa S. 1962. Nouvelle flore de l’Algérie et des régions désertiques méridionales. Centre Nationale de la Recherche Scientifique, Paris.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; Greuter & al. 1984Greuter W., Burdet H. & Long, G. 1984. Med-Checklist. Pteridophyta, Gymnospermae, Dicotyledones (Acanthaceae-Cneoraceae) 1: 320-328. Conservatoire et Jardin Botaniques, Genève.; López-González 1992López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63.; Raynaud 1999Raynaud C. 1999. Cistaceae. In Fennane M., Ibn Tattou M., Mathez J., Aïcha O., El Oualidi, J. (eds.), Flore pratique du Maroc: Manuel de détermination des plantes vasculaires vol. 1. Travaux de l’Institut Scientifique, Université Mohammed V, Série Botanique 36. Rabat: Institut Scientifique Université Mohammed V, Rabat.). To date, two comprehensive molecular phylogenetic reconstructions of the genus Helianthemum have been attempted, one based on the analyses of nrDNA (ITS) plus cpDNA (ndhF, psbA-trnH, trnL-trnF) concatenated Sanger sequences (Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. ), the other based on GBS data (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ). Previous phylogenetic analyses based on Sanger sequencing (Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. ) provided strong evidence for the generic boundaries in the family Cistaceae as stated by Janchen (1925)Janchen E. 1925. Cistaceae. In Engler A. (ed.), Die Natürlichen Pflanzenfamilien, ed. 2, 21: 289-313. Engelmann, Leipzig., i.e., with Crocanthemum Spach restricted to America and Helianthemum to the Old World (also ruling out the segregation of H. subg. Plectolobum Willk. into the separate genus Rhodax Spach). Aparicio & al. (2017)Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. also showed the monophyly of Helianthemum, so defined, and that this genus was integrated by three clades (I, II and III) with no species occupying an early diverging, isolated or intermediate position in relation to the rest of the species. Overall, this study provided support for the above-species classification of the genus, but it was unable to resolve most phylogenetic relationships since most species and subspecies were retrieved in polytomies.

Conversely, the phylogenetic analyses of GBS data (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ) (i) provided highly resolved phylogenetic trees with very strong support even at the most external nodes, (ii) robustly confirmed the three clades previously identified by Aparicio & al. (2017)Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. , and (iii) showed striking similar topologies among them, each one consisting of one species-rich subclade that corresponded with one of the three largest sections in the genus (H. sects. Eriocarpum Dunal, Pseudocistus Dunal and Helianthemum) plus a few very poorly diversified ones: H. sects. Argyrolepis Spach, Lavandulaceum G.López and Pseudomacularia Grosser in clade I; Caput-felis G.López, Macularia Dunal and Atlanthemum (Raynaud) G.López, Ortega Oliv. & Romero García in clade II; and Brachypetalum Dunal in clade III. On the one hand, this phylogenetic hypothesis (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ) allowed the reconstruction of the biogeographic history of Helianthemum showing that the three largest taxonomic sections represent three evolutionary radiations which synchronously diversified during the Pliocene and Pleistocene, and that clades I, II and III diverged under pre-Mediterranean environmental conditions in northern Africa during the Middle Miocene. Then, clade II expanded throughout arid and semiarid ecosystems in northern Africa, Middle East and central Asia (to a considerably lesser extent to Macaronesia) whereas clades I and III expanded to Mediterranean and temperate regions through much of the Mediterranean Basin, central and northern Europe and the Canary Islands (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ; 2021Martín-Hernanz S., Albaladejo R.G., Lavergne S., Rubio E., Grall A. & Aparicio A. 2021. Biogeographic history and environmental niche evolution in the Palearctic genus Helianthemum (Cistaceae). Molecular Phylogenetics and Evolution 163: 107238.).

On the other hand, the resulting non-monophyletic taxa advised further systematic changes at different taxonomic ranks. For example, at subgenus level, H. subg. Helianthemum as usually considered by taxonomists (i.e., clade II plus clade III) (Spach 1836Spach E. 1836. Conspectus monographiae Cistacearum. Annales des Sciences Naturelles, Botanique, ser. 2, 6: 357-375.; Willkomm 1856Willkomm M. 1856. Icones et descriptiones plantarum novarum criticarum et rariorum Europae Austro-Occidentalis praecipue Hispaniae vol. 2. Sumtibus A.H. Payne, Leipzig.; Grosser 1903Grosser W. 1903. Cistaceae. In Engler H.G.A. (ed.), Das Pflanzenreich vol. 4. Berlin, Leipzig.; Janchen 1925Janchen E. 1925. Cistaceae. In Engler A. (ed.), Die Natürlichen Pflanzenfamilien, ed. 2, 21: 289-313. Engelmann, Leipzig.; Quézel & Santa 1962Quézel P. & Santa S. 1962. Nouvelle flore de l’Algérie et des régions désertiques méridionales. Centre Nationale de la Recherche Scientifique, Paris.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; López-González 1992López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63.) was retrieved as a non-monophyletic (paraphyletic) group since clade III (H. sects. Brachypetalum and Helianthemum) and clade I (H. subg. Plectolobum) shared a recent-most common ancestor compared to clade II (see Fig. 1A). At the species level, the most common and taxonomically complex species, such as H. apenninum (L.) Mill., H. cinereum (Cav.) Pers., H. marifolium (L.) Mill., H. nummularium (L.) Mill. or H. oelandicum (L.) Dum. Cours. were also retrieved as non-monophyletic groups. These are young species probably impacted by the Pleistocene glacial cycles, whose ample variability is usually described by taxonomists as subspecies arrays or species aggregates (e.g., Janchen 1907Janchen E. 1907. Helianthemum canum (L.) Baumg. und seine nächsten Verwandten. Abhandlungen der K. K. Zool.-Botan. Gesellschaft in Wien 4: 1-67.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; Greuter & al. 1984Greuter W., Burdet H. & Long, G. 1984. Med-Checklist. Pteridophyta, Gymnospermae, Dicotyledones (Acanthaceae-Cneoraceae) 1: 320-328. Conservatoire et Jardin Botaniques, Genève.). The biological and taxonomic complexity of these species is also reflected in the low statistical support retrieved for their intraspecific relationships as well as in the existence of topological incongruences for some nodes of the phylogenetic trees (see Fig. 4 in Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ), further revealing that trait convergence, incomplete lineage sorting or hybridization and introgression could be playing an essential role in the differentiation of these lineages (Soubani & al. 2014aSoubani E., Hedrén M. & Widén B. 2014a. Phylogeography of the European rock rose Helianthemum nummularium (Cistaceae): Incongruent patterns of differentiation in plastid DNA and morphology. Botanical Journal of the Linnean Society 176: 311-331., 2014bSoubani E., Hedrén M. & Widén B. 2014b. Genetic and morphological differentiation across a contact zone between two postglacial immigration lineages of Helianthemum nummularium (Cistaceae) in southern Scandinavia. Plant Systematics and Evolution 301: 1499-1508.; Volkova & al. 2016Volkova P.A., Schanzer I.A., Soubani E., Meschersky I.G. & Widén B. 2016. Phylogeography of the European rock rose Helianthemum nummularium s.l. (Cistaceae): Western richness and eastern poverty. Plant Systematics and Evolution 302: 781-794.). Overall, these findings emphasize that, beyond the systematic implications, studies at microevolutionary scale are necessary to evaluate diversification dynamics in these complexes and to understand why most species of Helianthemum are endemic or have very restricted distribution ranges (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. , 2019bMartín-Hernanz S., Martínez-Sánchez S., Albaladejo R.G., Lorite J., Arroyo J. & Aparicio A. 2019b. Genetic diversity and differentiation in narrow versus widespread taxa of Helianthemum (Cistaceae) in a hotspot: The role of geographic range, habitat, and reproductive traits. Ecology and Evolution 9: 3016-3029. ).

In this paper, we update the overall systematic scheme of the genus Helianthemum using a robust molecular phylogenetic framework. To that aim, we considered the intrageneric systematic overview and the set of taxa studied by Aparicio & al. (2017)Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. and Martín-Hernanz & al. (2019a)Martín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. which targeted a broad taxonomic and geographic representation of the genus including two subgenera, ten sections, 73 species and 25 subspecies (i.e., about 75% of the genus) from the entire geographic distribution range (see Fig. 1 in Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. and Table S2 in Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ). Considering the overall statistical robustness of the phylogenetic relationships retrieved, particularly those based on GBS data, the subsequent systematic arrangements entail the establishment of a new subgenus, novel re-ascriptions of sections and species, and several nomenclatural novelties. We are aware that numerous species and subspecies relationships are still unknown and that many taxonomic and nomenclatural questions remain unresolved. Addressing these would require a microevolutionary approach, with detailed population level studies and an increased number of samples per population and taxa.

MATERIAL AND METHODS

The systematic changes proposed here are based on phylogenetic trees obtained by maximum likelihood (RaxML 7.2.8; Stamatakis 2006Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688-2690.) and Bayesian (ExaBayes 1.4.1; Aberer & al. 2014Aberer A.J., Kobert K., & Stamatakis, A. 2014. ExaBayes: massively parallel Bayesian tree inference for the whole-genome era. Molecular Biology and Evolution 31: 2553-2556. ) analyses of a GBS assembly, applying the MaxResol configuration and the minimum 15% taxon coverage (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ). Briefly, the MaxResol configuration was defined as the parameter set configuration that provided the highest number of supported nodes in the phylogenetic analyses (both Bayesian and Maximum likelihood) as opposed to the MinError configuration, which was designed to minimizing allele and SNP error rates and provided more accurate branch length estimates in the phylogenetic trees (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ). Hence, MaxResol configuration retrieves trees suited for molecular systematic inferences whereas MinError configuration yields trees adequate for downstream analyses concerning the evolutionary history of the genus (molecular dating, ancestral area reconstructions or diversification rates). Minimum taxon coverage refers to the minimum number of samples at a given locus required to be retained in the final dataset. Details about the implications of parameter configuration for the phylogenetic reconstructions are in Martín-Hernanz & al. (2019a)Martín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. .

RESULTS AND DISCUSSION

The 50% majority-rule consensus tree resulting from the Bayesian inference analysis of GBS data shows that most nodes received high statistical support with full posterior probability (PP = 1) and bootstrap (BS) values higher than 70% (Fig. 1; see also Fig. S1a in Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. for bootstrap values). Thus, this tree provides compelling evidence that the genus Helianthemum is integrated by three main lineages (clades I, II and III). Furthermore, it gives very strong support for the non-monophyletic status of H. subg. Helianthemum as traditionally considered: i.e., clades II plus III (Spach 1836Spach E. 1836. Conspectus monographiae Cistacearum. Annales des Sciences Naturelles, Botanique, ser. 2, 6: 357-375.; Willkomm 1856Willkomm M. 1856. Icones et descriptiones plantarum novarum criticarum et rariorum Europae Austro-Occidentalis praecipue Hispaniae vol. 2. Sumtibus A.H. Payne, Leipzig.; Grosser 1903Grosser W. 1903. Cistaceae. In Engler H.G.A. (ed.), Das Pflanzenreich vol. 4. Berlin, Leipzig.; Janchen 1925Janchen E. 1925. Cistaceae. In Engler A. (ed.), Die Natürlichen Pflanzenfamilien, ed. 2, 21: 289-313. Engelmann, Leipzig.; Quézel & Santa 1962Quézel P. & Santa S. 1962. Nouvelle flore de l’Algérie et des régions désertiques méridionales. Centre Nationale de la Recherche Scientifique, Paris.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; López-González 1992López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63.). Therefore, we are proposing a new subgenus coinciding with clade II, making all three subgenera monophyletic. Thus, H. subg. Eriocarpum (clade II) includes sects. Argyrolepis, Lavandulaceum, Pseudomacularia and Eriocarpum; H. subg. Plectolobum (clade I) contains sects. Caput-felis, Atlanthemum, Macularia and Pseudocistus; and, finally, H. subg. Helianthemum (clade III) is formed by sects. Brachypetalum and Helianthemum. Additional nomenclatural combinations and the re-ascription of taxa to different taxonomic levels are further proposed based on strongly supported phylogenetic relationships.

Helianthemum Mill., Gard. Dict. Abr. ed. 4 (1754). Lectotype: H. nummularium (L.) Mill. (Cistus nummularius L.).

Helianthemum subg. Eriocarpum (Dunal) Martín-Hernanz, Velayos, Albaladejo & Aparicio, stat. & comb. nov. [≡ Helianthemum sect. Eriocarpum Dunal in DC., Prodr. 1: 273 (1824Dunal F. 1824. Cistineae. In Candolle A.P. de (ed.), Prodromus systematis naturalis regni vegetabilis, vol. 1: 263-288. Sumptibus sociorum Treuttel et Würtz, Parisiis [Paris]. ), basion.].

Helianthemum subg. Eriocarpum is mostly integrated by fruticose deserticolous plants with the upper leaves alternate inhabiting arid and semiarid ecosystems from the Maghreb to the Middle East, the Horn of Africa and Central Asia. Helianthemum subg. Helianthemum and H. subg. Pseudocistus are typical fruticulose or suffruticose plants (rarely therophytic) with all leaves opposite inhabiting Mediterranean and Eurosiberian ecosystems from the Mediterranean Basin to northern Europe.

Included taxon

Included taxa

Grosser (1903)Grosser W. 1903. Cistaceae. In Engler H.G.A. (ed.), Das Pflanzenreich vol. 4. Berlin, Leipzig. described this section on page 62 of the Engler’s Pflanzenreich. Subsequently, on page 92 the author listed the species included without specifying any of them as type of the section. We choose H. songaricum as lectotype.

A recent Sanger-based phylogenetic reconstruction which included six out of the seven species of Helianthemum endemic from the Horn of Africa (Martín-Hernanz & al. 2021Martín-Hernanz S., Albaladejo R.G., Lavergne S., Rubio E., Grall A. & Aparicio A. 2021. Biogeographic history and environmental niche evolution in the Palearctic genus Helianthemum (Cistaceae). Molecular Phylogenetics and Evolution 163: 107238.) retrieved the large H. sect. Eriocarpum as non-monophyletic due to the inclusion in the same clade of the species that conform H. sect. Pseudomacularia, albeit with low statistical support. This is a remarkable result that would require the re-ascription of H. sect. Pseudomacularia within H. sect. Eriocarpum, but at present we keep these two sections as separate entities until the confirmation of this result by means of GBS (i.e., highly resolutive) data, when available. Nevertheless, the GBS-based phylogenetic tree here considered (Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. ) confirmed the phylogenetic relationship among the Turkey endemics H. antitauricum P.H.Davis & Coode and H. germanicopolitanum Bornm. (which were previously ascribed to sects. Helianthemum and Lavandulaceum, respectively; Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. ) and the Asiatic species H. ordosicum Y.Z.Zhao, ZongY.Zhu & R.Cao and H. songaricum Schrenk ex Fisch. & C.A.Mey., hence their ascription to H. sect. Pseudomacularia.

Included taxa

Taxon not included in the phylogenetic analysis

Dunal (1824)Dunal F. 1824. Cistineae. In Candolle A.P. de (ed.), Prodromus systematis naturalis regni vegetabilis, vol. 1: 263-288. Sumptibus sociorum Treuttel et Würtz, Parisiis [Paris]. published H. sect. Eriocarpum in De Candolle’s Prodromus. After the diagnosis of the new section, the author listed the species included, but did not specify a type of the section. For this reason we selected H. kahiricum as lectotype.

Included taxa

Taxa not included in the phylogenetic analysis

The assignation of the endemic species from the Horn of Africa to this section is based on Aparicio & al. (2017)Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. and Martín-Hernanz & al. (2021)Martín-Hernanz S., Albaladejo R.G., Lavergne S., Rubio E., Grall A. & Aparicio A. 2021. Biogeographic history and environmental niche evolution in the Palearctic genus Helianthemum (Cistaceae). Molecular Phylogenetics and Evolution 163: 107238..

Helianthemum subg. Plectolobum Willk., Icon. Descr. Pl. 2: 139 (1862). Rhodax Spach, Ann. Sci. Nat. Bot. Ser. 2, 6: 363 (1836Spach E. 1836. Conspectus monographiae Cistacearum. Annales des Sciences Naturelles, Botanique, ser. 2, 6: 357-375.).

The GBS-based phylogenetic tree provides strong support for the inclusion of H. sect. Caput-felis G.López in H. subg. Plectolobum. López-González (1992)López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63. considered this section to be somewhat intermediate among both subgenera but ascribed it to H. subg. Helianthemum despite emphasizing the Plectolobum-like seed morphology of H. caput-felis.

Included taxon

Dunal (1824)Dunal F. 1824. Cistineae. In Candolle A.P. de (ed.), Prodromus systematis naturalis regni vegetabilis, vol. 1: 263-288. Sumptibus sociorum Treuttel et Würtz, Parisiis [Paris]. described this section in De Candolle’s Prodromus. After the diagnosis of the section, that author did not specify the type of the section. We choose H. lunulatum as lectotype. Helianthemum lunulatum is a species restricted to high-altitude ecosystems in the Maritime Alps and the only species usually ascribed to H. sect. Macularia (e.g., Grosser 1903Grosser W. 1903. Cistaceae. In Engler H.G.A. (ed.), Das Pflanzenreich vol. 4. Berlin, Leipzig.; Janchen 1925Janchen E. 1925. Cistaceae. In Engler A. (ed.), Die Natürlichen Pflanzenfamilien, ed. 2, 21: 289-313. Engelmann, Leipzig.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.). However, the analyses retrieved an unexpected sister-relationship among this species and H. pomeridianum Dunal (formerly ascribed either to H. sect. Eriocarpum or to H. sect. Chamaecistus Willk. in different subgenera; Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. ), a species restricted to northern Algeria and the Moroccan Atlas Mountains range. This relationship reveals a major intercontinental disjunction happened during the early diversification of H. subg. Plectolobum in the Upper Pliocene (Aparicio & Albaladejo 2017Aparicio A. & Albaladejo R.J. 2017. On the identity of Helianthemum mathezii and H. pomeridianum (Cistaceae). Anales del Jardín Botánico de Madrid 74: e060.).

Included taxa

Taxon not included in the phylogenetic analysis

Similarly, the phylogenetic reconstructions considered in this paper provide strong support for the inclusion of H. sanguineum (Lag.) Lag. in the monotypic H. sect. Atlanthemum as proposed by López-González (1992)López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63., ruling out the consideration of the separate genus Atlanthemum Raynaud (Raynaud 1987Raynaud C. 1987. Atlanthemum Raynaud, un nouveau genre pour la famille des Cistaceae. Anales del Jardín Botánico de Madrid 44: 309-317.).

Included taxon

Some complex species in this section as currently defined have been retrieved non-monophyletic. The GBS data provide support for the consideration of H. frigidulum Cuatrec. and H. raynaudii Ortega Oliv., Romero García & C. Morales at species level, as originally described, i.e., not subordinated either to H. marifolium or to H. viscidulum Boiss., respectively. Conversely, the consideration of H. marifolium and H. origanifolium (Lam.) Pers. as different species (e.g., Guinea 1954Guinea E. 1954. Cistáceas españolas (con exclusión del género Cistus). Instituto forestal de investigaciones y experiencias, Madrid.; Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; Greuter & al. 1984Greuter W., Burdet H. & Long, G. 1984. Med-Checklist. Pteridophyta, Gymnospermae, Dicotyledones (Acanthaceae-Cneoraceae) 1: 320-328. Conservatoire et Jardin Botaniques, Genève.; Crespo & al. 2016Crespo M.B., Alonso M.A., Vicente A. & Villar, J.L. 2016. A new North African subspecies in the Helianthemum origanifolium aggregate (H. subg. Plectolobum, Cistaceae). Phytotaxa 252: 263-272.) resulted firmly unsupported. Helianthemum oelandicum is an extremely polymorphic complex usually considered as a species’ aggregate (Janchen 1907Janchen E. 1907. Helianthemum canum (L.) Baumg. und seine nächsten Verwandten. Abhandlungen der K. K. Zool.-Botan. Gesellschaft in Wien 4: 1-67.; Yuzepchuk 1974Yuzepchuk S.V. 1974. Helianthemum Adans. In Schishkin B.K. (ed.), Flora of the U.S.S.R. 15: 248-260. Trans. from Russian by N. Landau. Israel Program for Scientific Translations, Jerusalem.; Tzvelev 2006Tzvelev, N. 2006. Flora of Russia: The European part and bordering regions vol. 9. CRC Press/Balkema, Rotterdam.; Soubani 2010Soubani E. 2010. Systematics, phylogeography and multiple origins of morphs in two species complexes belonging to Cistaceae, Helianthemum oelandicum and H. nummularium. Ph.D. dissertation, Lund University, Lund.); very interestingly, to this complex is to be ascribed a gypsophile specialist plant endemic to central Spain which was subordinated to H. marifolium (H. marifolium subsp. conquense Borja & Rivas Goday ex G.López) albeit its morphological relationship with H. oelandicum was originally suspected (López-González 1992López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63.). For a comprehensive insight of the polymorphic species H. cinereum, H. marifolium and H. polyanthum (Desf.) Pers., it is a requisite to take into account the ample variability that exists at both sides of the strait of Gibraltar.

Included taxa

Taxa not included in the phylogenetic analysis H. baschkirorum (Juz. ex Kupat.) Juz.

Helianthemum subg. Helianthemum

Included taxa

Taxon not included in the phylogenetic analysis

The complex species H. apenninum and H. nummularium, as currently delimited, have been retrieved as non-monophyletic making necessary some nomenclatural combinations and taxonomic re-ascriptions. Future studies are required to evaluate the variability of H. apenninum and to ascertain its relationships with H. croceum (Desf.) Pers. (see López-González 1992López-González G. 1992. Apuntes para justificar el tratamiento del género Helianthemum Miller, s.l. (Cistaceae), en Flora Iberica. Anales del Jardín Botánico de Madrid 50: 35-63.) and H. neopiliferum Muñoz Garm. & C. Navarro. On the other hand, a huge ecological and morphological variation is inherent to H. nummularium (Proctor & Heywood 1968Proctor M. & Heywood V. 1968. Helianthemum Miller. In Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M & Webb D.A. (eds.), Flora Europaea 2: 286-292. Cambridge University Press, Cambridge.; Volkova & al. 2016Volkova P.A., Schanzer I.A., Soubani E., Meschersky I.G. & Widén B. 2016. Phylogeography of the European rock rose Helianthemum nummularium s.l. (Cistaceae): Western richness and eastern poverty. Plant Systematics and Evolution 302: 781-794.) and the results that we analyse in this paper increase the complexity of this taxon. For example, we have unexpectedly found that the sample identified in the phylogenetic tree as H. apenninum subsp. stoechadifolium 236, which represents a few populations restricted to maritime sandy soils around the Doñana National Park in the coast of southern Spain (see Table S2 in Martín-Hernanz & al. 2019aMartín-Hernanz S., Aparicio A., Fernández-Mazuecos M., Rubio E., Reyes-Betancort A., Santos-Guerra A., Olangua-Corral M. & Albaladejo R.G. 2019a. Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae). Frontiers in Plant Science 10: 1416. and Fig. 1), clustered within the clade of H. nummularium. Although these plants can be readily identified as H. apenninum subsp. stoechadifolium (Brot.) Samp., the facts that this taxon is not cited for this geographic area by López-González (1993)López-González G. 1993. Helianthemum Mill. In Castroviejo S., Aedo C., Cirujano S., Laínz M., Montserrat P., Morales R., Muñoz Garmendia F., Navarro C., Paiva J. & Soriano C. (eds.), Flora iberica 3: 365-421. Real Jardín Botánico, CSIC, Madrid. in Flora iberica and the retrieved phylogenetic relationship, stress that a detailed study is necessary to ascertain the real taxonomic identity of these populations. Similarly, the stenochorous H. apenninum subsp. urrielense (M.Laínz) G.López and H. apenninum subsp. cantabricum (M.Laínz) G.López were retrieved within the clade of H. nummularium together with H. morisianum Bertol., an endemic taxon from Sardinia, which was previously considered either included in H. nummularium subsp. berteroanum (Bertol.) Breistr. (Pignatti 1982Pignatti S. 1982. Flora d’Italia vol. 2. Edagricole, Bologna.) or as H. nummularium subsp. morisianum (Bertol.) Zangh. (Greuter & al. 1984Greuter W., Burdet H. & Long, G. 1984. Med-Checklist. Pteridophyta, Gymnospermae, Dicotyledones (Acanthaceae-Cneoraceae) 1: 320-328. Conservatoire et Jardin Botaniques, Genève.).

Also, in this section a cluster of species whose relationships are not fully supported is retrieved [H. guerrae Sánchez-Gómez, J.S.Carrion & M.A.Carrión, H. viscarium Boiss. & Reut., H. violaceum (Cav.) Pers., H. asperum Lag. ex Dunal, H. marminorense Alcaraz, Peinado & Mart.Parras and H. fontqueri Sennen plus H. hirtum (L.) Mill. and H. scopulicola L.Sáez, Rosselló & Alomar]. Indeed, this is an extremely complex group of species almost restricted to eastern and southern Spain where phenotypic plasticity and hybridization is very common (e.g., Mateo Sanz 2012Mateo Sanz, G. 2012. Nuevos taxones del género Helianthemum Miller en la zona oriental de la Península Ibérica, I. Flora Montiberica 50: 30-43.; Pérez Da Costa & Mateo Sanz 2012Pérez Da Costa J.M. & Mateo Sanz G. 2012. Nuevos taxones del género Helianthemum Miller en la zona oriental de la Península Ibérica, II. Flora Montiberica 50: 44-61.). Again, the study of this complex of species would require a population level approach with an enlarged number of samples per population and species. This last consideration can be extended to the lineage integrated by species mostly from south-eastern Spain and northern Maghreb such as H. almeriense Pau, H. alypoides Losa & Rivas Goday, H. virgatum (Desf.) Pers., H. pergamaceum Pomel, H. ciliatum (Desf.) Pers. and H. vesicarium Boiss. where hybridization is also common and the species limit result many times unclear (Quézel & Santa, 1962Quézel P. & Santa S. 1962. Nouvelle flore de l’Algérie et des régions désertiques méridionales. Centre Nationale de la Recherche Scientifique, Paris.; Raynaud 1999Raynaud C. 1999. Cistaceae. In Fennane M., Ibn Tattou M., Mathez J., Aïcha O., El Oualidi, J. (eds.), Flore pratique du Maroc: Manuel de détermination des plantes vasculaires vol. 1. Travaux de l’Institut Scientifique, Université Mohammed V, Série Botanique 36. Rabat: Institut Scientifique Université Mohammed V, Rabat.). Finally, the analysed GBS data firmly support the existence of the ‘Canary Island clade’, a monophyletic lineage endemic to the Canary Islands explosively diversified during the Pleistocene (Albaladejo & al. 2021Albaladejo R., Martín-Hernanz S., Reyes-Betancort J.A., Santos-Guerra A., Olangua-Corral M. & Aparicio A. 2021. Reconstruction of the spatiotemporal diversification and ecological niche evolution of Helianthemum (Cistaceae) in the Canary Islands using genotyping-by-sequencing data. Annals of Botany 127: 597-611.) whose species were previously ascribed to H. sects. Argyrolepis or Lavandulaceum (Aparicio & al. 2017Aparicio A., Martín-Hernanz S., Parejo-Farnés C., Arroyo J., Lavergne S., Yesilyurt E.B., Zang M-L., Rubio E. & Albaladejo R.G. 2017. Phylogenetic reconstruction of the genus Helianthemum (Cistaceae) using plastid and nuclear DNA-sequences: systematic and evolutionary inferences. Taxon 66: 868-885. ).

Included taxa

Taxa not included in the phylogenetic analysis