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

Molecular systematics requires the establishment of a robust phylogenetic framework including extensive geographical and taxonomic sampling. In this work, we proposed systematic changes in the genus Helianthemum based on phylogenetic trees obtained by both maximum likelihood and Bayesian analyses of GBS data. The implications of these phylogenetic results for the systematics of Helianthemum entail the establishment of a new subgenus and novel re-ascriptions of sections and species along with some nomenclatural novelties. The following new combinations are proposed: Helianthemum subg. Eriocarpum (Dunal) Martín-Hernanz, Velayos, Albaladejo & Aparicio; H. oelandicum subsp. conquense (Borja & Rivas Goday ex G.López) Martín-Hernanz, Velayos, Albaladejo & Aparicio; H. nummularium subsp. cantabricum (M.Laínz) Martín-Hernanz, Velayos, Albaladejo & Aparicio; H. nummularium subsp. tinetense (M.Mayor & Fern.Benito) Martín-Hernanz, Velayos, Albaladejo & Aparicio.

resolutive because they allow the discovery of thousands of loci even from non-model species (Fernández-Mazuecos & al. 2018).
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 2020), is a challenging case of study due to its complex taxonomy and broad geographical distribution Systematic implications from a robust phylogenetic reconstruction of the genus Helianthemum (Cistaceae) based on genotyping-bysequencing (GBS) data Sara MARTÍN-HERNANZ 1 , Mauricio VELAYOS 2 , Rafael G. ALBALADEJO 3 , Abelardo APARICIO 4* 2 Anales del Jardín Botánico de Madrid 78 (2): e113. https://doi.org/10.3989/ajbm.2601 (Janchen 1925;Quézel & Santa 1962;Proctor & Heywood 1968;Greuter & al. 1984;López-González 1992;Raynaud 1999). 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 , the other based on GBS data (Martín-Hernanz & al. 2019a). Previous phylogenetic analyses based on Sanger sequencing ) provided strong evidence for the generic boundaries in the family Cistaceae as stated by Janchen (1925), 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).  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. 2019a) (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  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 1836;Willkomm 1856;Grosser 1903;Janchen 1925;Quézel & Santa 1962;Proctor & Heywood 1968;López-González 1992) 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 1907;Proctor & Heywood 1968;Greuter & al. 1984). 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. 2019a), 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. 2014a(Soubani & al. , 2014bVolkova & al. 2016). 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. 2019a, 2019b. 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  and Martín-Hernanz & al. (2019a) 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  and Table S2 in Martín-Hernanz & al. 2019a). 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 3 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 2006) and Bayesian (ExaBayes 1.4.1; Aberer & al. 2014) analyses of a GBS assembly, applying the MaxResol configuration and the minimum 15% taxon coverage (Martín-Hernanz & al. 2019a). 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. 2019a). 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).

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. 2019a 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 1836;Willkomm 1856;Grosser 1903;Janchen 1925;Quézel & Santa 1962;Proctor & Heywood 1968;López-González 1992 3. Sect. Pseudomacularia Grosser, Engler, Pflanzenr. IV, 193: 62 (1903   The assignation of the endemic species from the Horn of Africa to this section is based on  and Martín-Hernanz & al. (2021).  Macularia (e.g., Grosser 1903;Janchen 1925;Proctor & Heywood 1968 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), ruling out the consideration of the separate genus Atlanthemum Raynaud (Raynaud 1987 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 con-sideration of H. marifolium and H. origanifolium (Lam.) Pers. as different species (e.g., Guinea 1954;Proctor & Heywood 1968;Greuter & al. 1984;Crespo & al. 2016) resulted firmly unsupported. Helianthemum oelandicum is an extremely polymorphic complex usually considered as a species' aggregate (Janchen 1907;Yuzepchuk 1974;Tzvelev 2006;Soubani 2010); 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 1992). 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.