What Hides in the Heights? The Case of the Iberian Endemism Bromus picoeuropeanus

Bromus picoeuropeanus is a recently described species belonging to a complex genus of grasses. It inhabits stony soils at heights ranging from 1600 to 2200 m in Picos de Europa (Cantabrian Mountains, northern Spain). This species is morphologically very similar to B. erectus, partially sharing its presumed distribution range. We aim to determine the relationship between these species and their altitudinal ranges in Picos de Europa and the Cantabrian Mountains by conducting phylogenetic analyses based on nuclear (ETS and ITS) and chloroplastic (trnL) markers. Phylogenetic trees were inferred by Maximum Likelihood and Bayesian Inference. Haplotype networks were estimated based on the plastid marker. Although the ITS topologies could not generate exclusive clades for these species, the ETS analyses generated highly supported B. picoeuropeanus exclusive clades, which included locations outside its altitudinal putative range. The ETS-ITS and ETS-ITS-trnL topologies generated B. picoeuropeanus exclusive clades, whereas the trnL-based trees and haplotype networks were unable to discriminate B. erectus and B. picoeuropeanus. This evidence suggests that B. picoeuropeanus is a separate species with a larger distribution than previously thought, opening new questions regarding the evolution of B. erectus and other similar species in European mountainous systems. However, more information is needed regarding B. picoeuropeanus susceptibility to temperature rises.


Introduction
Climate change as a consequence of anthropogenic activities is one of the processes driving the current Biological Diversity Crisis [1][2][3][4]). This has resulted, among other things, in high plant extinction rates [5], these extinctions being more significant in hotspots of biodiversity [6]. In this context, mountainous regions, one of the habitats comprising the most plant endemism, especially in Europe [7], have been reported to experience a faster warming process than other habitats [8,9]. These temperature rises may cause shifts in the range of distribution of mountainous species [10] which, in the case of the endemism of high mountain species, represent a double danger as they face not only a reduction in their potential range of distribution, but also new competitors from lower lands [11,12].
In this context of altitudinal segregation of members of the B. erectus complex, B. picoeuropeanus Acedo and Llamas (2019) has been recently described in the mountainous regions of the north of Spain [29] (see Figure 1). Morphologically, B. picoeuropeanus can be distinguished from B. erectus by the presence of well-developed rhizomes, its "loosely tufted" habit, its shorter height (no more than 40 cm) and its truncated or rounded ligule, among other features [29]. Bromus picoeuropeanus is endemic to the mountainous region of Picos de Europa in the Cantabrian Mountains (north Spain) and inhabits stony soils from 1600 to 2200 m a. s. l. [29]. On the other hand, B. erectus s. s. has been reported for the southern Cantabrian Mountains (in León and Palencia) at altitudes ranging from 1490 to 1520 m a. s. l. [13].
In the Iberian Peninsula, Acedo and Llamas [13,29] described 26 different Bromus species, 18 of which have been reported to occur in the mountainous systems of the north of Spain [33][34][35]. Among those 18, the Eurasian perennial tetraploid grass Bromus erectus Huds. (1762) sensu lato (s. l.) is a highly variable group of microspecies that inhabits mountainous areas of central Europe, the Atlantic and Mediterranean European Basins, including the British Islands as well as in Iran and Tibet [17,[36][37][38][39][40][41]. B. erectus s. l. has been reported to have evolved in at least two glacial refugia of Central Europe [40,42], something that could be related to the occurrence of many microspecies in its mountainous regions. For instance, B. erectus sensu stricto (s. s.) has been described by Bačič and Jogan [38] to inhabit lower lands up to an altitude of 600 m a. s. l. to experience an altitudinal segregation in some regions of the Slovenian Alps with another two closely related species of the "B. erectus group", i.e., B. transylvanicus Steud. (1854) and Bromus condensatus Hack. (1879), previously considered a subspecies or synonyms of B. erectus (i.e., B. erectus s. l.) [41]. Nevertheless, although B. erectus s. l. has been subject to morphological [43,44], cytological [45] and molecular studies [40,46], its microspecies remain relatively unknown [38].
In this context of altitudinal segregation of members of the B. erectus complex, B. picoeuropeanus Acedo and Llamas (2019) has been recently described in the mountainous regions of the north of Spain [29] (see Figure 1). Morphologically, B. picoeuropeanus can be distinguished from B. erectus by the presence of well-developed rhizomes, its "loosely tufted" habit, its shorter height (no more than 40 cm) and its truncated or rounded ligule, among other features [29]. Bromus picoeuropeanus is endemic to the mountainous region of Picos de Europa in the Cantabrian Mountains (north Spain) and inhabits stony soils from 1600 to 2200 m a. s. l. [29]. On the other hand, B. erectus s. s. has been reported for the southern Cantabrian Mountains (in León and Palencia) at altitudes ranging from 1490 to 1520 m a. s. l. [13].  [29]. The base map was obtained using marmap R Pakage [47] and edited using Inkscape [48].
Taking into account the evolutionary history of B. erectus s. l. and the fact that the Cantabrian Mountains have been reported to be a glacial refugium for other plant groups [49], the current evidence suggests that a similar segregation to that described by Bačič and Jogan (2001) [38] in the Alps could be found in Picos de Europa regarding B. erectus s. In the box, the blue dots represent the localities of B. picoeuropeanus reported by Acedo and Llamas [29]. The base map was obtained using marmap R Pakage [47] and edited using Inkscape [48].
Taking into account the evolutionary history of B. erectus s. l. and the fact that the Cantabrian Mountains have been reported to be a glacial refugium for other plant groups [49], the current evidence suggests that a similar segregation to that described by Bačič and Jogan (2001) [38] in the Alps could be found in Picos de Europa regarding B. erectus s. s. and B. picoeuropeanus. Nevertheless, since no phylogenetic study establishing the relationship has been conducted, we cannot rule out the possibility that B. picoeuropeanus, considered within the B. erectus complex by Acedo and Llamas [29], is an expression of the plasticity of B. erectus s. s. Therefore, we propose five hypotheses to test: (1) B. erectus s. s. and B. picoeuropeanus are two different species and B. erectus s. s. is found at up to 600 m of altitude, (2) B. erectus s. s. and B. picoeuropeanus are two different species and B. erectus s. s. is found at up to approximately 1600 m of altitude, (3) B. erectus s. s. and B. picoeuropeanus are two different species but there is no altitudinal segregation, (4) B. picoeuropeanus is another subspecies B. erectus s. s. that can be found at up to 2200 m of altitude and (5) B. picoeuropeanus is a different species from B. erectus s. s. and is the only one that inhabits the Picos de Europa (see Figure 2). In order to investigate the relationships between the Spanish populations of B. erectus s. s. and B. picoeuropeanus and their eventual different altitudinal distribution, we carried out molecular analyses based on both nuclear and plastidial markers.
s. and B. picoeuropeanus. Nevertheless, since no phylogenetic study establishing the relationship has been conducted, we cannot rule out the possibility that B. picoeuropeanus, considered within the B. erectus complex by Acedo and Llamas [29], is an expression of the plasticity of B. erectus s. s. Therefore, we propose five hypotheses to test: (1) B. erectus s. s. and B. picoeuropeanus are two different species and B. erectus s. s. is found at up to 600 m of altitude, (2) B. erectus s. s. and B. picoeuropeanus are two different species and B. erectus s. s. is found at up to approximately 1600 m of altitude, (3) B. erectus s. s. and B. picoeuropeanus are two different species but there is no altitudinal segregation, (4) B. picoeuropeanus is another subspecies B. erectus s. s. that can be found at up to 2200 m of altitude and (5) B. picoeuropeanus is a different species from B. erectus s. s. and is the only one that inhabits the Picos de Europa (see Figure 2). In order to investigate the relationships between the Spanish populations of B. erectus s. s. and B. picoeuropeanus and their eventual different altitudinal distribution, we carried out molecular analyses based on both nuclear and plastidial markers.  [38]; Hypothesis 2 also assumes the existence of two different species with the altitudinal range given by Acedo and Llamas [29] and Hudson [50] ; Hypothesis 3 assumes the existence of two distinct species that would co-occur in Picos de Europa; Hypothesis 4 assumes that B. picoeuropeanus would be a subspecies of B. erectus s. s. with the altitudinal range proposed by Acedo and Llamas [29] for B. picoeuropeanus; Hypothesis 5 assumes the existence of both species, B. picoeuropeanus being the only one found in Picos de Europa.

Results
The main features of the obtained alignments (displayed in Table 1) showed similar numbers of parsimonious-informative sites for ETS and ITS markers, the former having the higher number. On the other hand, the trnL plastid marker showed a lower number of parsimonious-informative sites. The five hypotheses of the occurrence of Bromus erectus s. s. and Bromus picoeuropeanus in Picos de Europa. Hypothesis 1 assumes that B. erectus s. s. and B. picoeuropeanus are two distinct species, being the altitudinal range of B. erectus s. s. described by Bačič and Jogan [38]; Hypothesis 2 also assumes the existence of two different species with the altitudinal range given by Acedo and Llamas [29] and Hudson [50]; Hypothesis 3 assumes the existence of two distinct species that would co-occur in Picos de Europa; Hypothesis 4 assumes that B. picoeuropeanus would be a subspecies of B. erectus s. s. with the altitudinal range proposed by Acedo and Llamas [29] for B. picoeuropeanus; Hypothesis 5 assumes the existence of both species, B. picoeuropeanus being the only one found in Picos de Europa.

Results
The main features of the obtained alignments (displayed in Table 1) showed similar numbers of parsimonious-informative sites for ETS and ITS markers, the former having the higher number. On the other hand, the trnL plastid marker showed a lower number of parsimonious-informative sites. Table 1. Main features of the different alignments used in the phylogenetic analysis and the haplotype network. The number of analyzed taxa, the conservative sites, the variable sites, and the parsimoniousinformative sites refers only to Bromus taxa. The number of sequences includes the outgroups, while the rest of the features does not include the outgroups. Variable sites are sites in which a minimum of two different nucleotides occur, while parsimonious-informative sites are those in which a minimum of two different nucleotides occur, two of which must have a minimum frequency of two. The ranges of length of the sequences and the alignment length were measured in pairs of bases (pb). The new ETS sequences of B. picoeuropeanus and B. erectus from individuals collected in the Cantabrian Mountains and the lowlands of Asturias (Br1-12) presented an indel of 119-121 bp ranging for position from 324 to 451 of the obtained alignment, identical in some cases or almost identical in others to the one presented by several B. erectus sequences, including the one collected in Picos de Europa (KJ632441). This insertion was also shared with other species such as B. sterilis L. (1753), B. riparius Rehmann (1872) or B. diandrus Roth (1787). Since the presence of this large indel in various species could influence the position of the Bromus samples and their inferred relationships, we performed an additional phylogenetic analysis on the ETS dataset excluding the indel region to determine the influence of this indel on the topology and the branch support ( Figure S1).

ETS
The phylogenetic analyses of the ETS dataset (see Figure 3A)     On the other hand, the phylogenetic analyses based on the plastid dataset (trnL) retrieved a topology in which independent phylogenetic analysis (see Figure 5A The topology of the ETS-ITS-trnL combined dataset (see Figure 6) presents a wellsupported B. picoeuropeanus

Discussion
The phylogenetic analyses based on ITS showed a topology in which the samples collected in the Cantabrian Mountains (Br2-12) were polyphyletic as they formed part of various clades or separate terminal branches of the same clade. However, the same analyses based on ETS generated an exclusive B. picoeuropeanus clade formed by all the individuals collected in the Cantabrian Mountains, including those outside the putative altitudinal range of this species as provided by Acedo and Llamas [29]. The combination of both nuclear markers (ETS-ITS) also showed a similar topology in which the Bromus of the Cantabrian mountains also formed an exclusive monophyletic clade separated and sister to that comprising B. erectus individuals. These results suggest that the Bromus collected in the Cantabrian Mountains would be a separate entity from B. erectus, thus discarding Hypothesis 4. The phylogenetic tree based on nuclear markers plus the plastid marker trnL further clarified these results, as the relationships of the obtained clades presented higher support values. On the other hand, the plastid marker was incapable of group taxa in species-exclusive clades. The trees and networks based on trnL presented large groups formed by many different species, with B. erectus and B. picoeuropeanus found in the section Bromopsis group. These results are similar to those of Nasiri et al. [51], who used a similar methodology to study the phylogenetic relationship within sect. Bromus and determined that neither the ITS nor the plastid markers were capable of discriminating at the species level, while the combination of ITS and ETS allowed them to be discriminated at species level.
The phylogenetic analyses strongly suggest that Bromus picoeuropeanus is molecularly distinct from B. erectus s. s., especially since the former did not form a subclade within a B. erectus s. s., exclusive clade from the ETS, ETS-ITS and ETS-ITS-trnL topologies. This phylogenetic position supports in part the hypothesis of Acedo and Llamas [29] who defined B. picoeuropeanus as an independent species based on morphological and ecological data. Therefore, the phylogenetic position results would support our Hypotheses 1, 2, 3 and 5, as all of them assume that both taxa are different species. Nevertheless, the altitudinal distribution of the sampled individuals neither supports the distribution or habitat proposed by Acedo and Llamas [29] by those two species in the study area (Hypothesis 2) nor the hypothesis of a sympatric distribution of the two species (Hypothesis 3). The presence of B. picoeuropeanus would not be restricted to Picos de Europa, as the samples collected in other locations of the Cantabrian Mountains also formed part of the B. picoeuropeanus clade, therefore indicating that B. picoeuropeanus would be a Cantabrian Mountains endemism rather than a Picos de Europa endemism.
Our results also shed some light on the altitudinal range of distribution of this endemism, since we found B. picoeuropeanus individuals occurring at altitudes ranging from at least 729 to 2200 m, a range wider than first supposed by Acedo and Llamas [29]. This indicates that the altitudinal occurrence might be wider at the lower altitudes than first thought by Acedo and Llamas [29], thus supporting our Hypothesis 5 regarding B. picoeuropeanus in Picos de Europa, namely that it is a different species from B. erectus s. s., the only one that inhabits the area. Interestingly, the samples from the south of the Cantabrian Mountains, where B. erectus s. s. has been reported to occur at height ranging from 1490 to 1520 m, Acedo and Llamas [13] was observed to belong to the B. picoeuropeanus clade. Although these sample were collected at lower and higher heights than that provided by Acedo and Llamas [13], these findings cast doubts regarding the actual distribution range of B. erectus s. s. in the south of the Cantabrian Mountains, as by the time this distribution was considered, B. picoeuropeanus had not yet been described. Biogeographically, we also determined that the two subclades within the B. picoeuropeanus subclade generated in the combined nuclear and plastid analyses separated B. picoeuropeanus individuals following bioclimes. On the one hand, we detect the highly supported subclade containing the B. picoeuropeanus individuals collected in Picos de Europa at heights ranging from 728 to 1653 m, which would belong to the lower altitudes of the orotemperate bioclime, while on the other hand, we have the other subclade containing samples found at subalpine regions of the orotemperate bioclime (at Picos de Europa from 1841 to 1928 m) plus the samples collected in the south of the Cantabrian Mountains, which belongs to the Orosubmediterranean (León sample) and to the supramediterranean bioclimes.
Another compelling result from our analyses is that B. picoeuropeanus belongs to a major clade that grouped together species from various sections with different ploidy levels, for instance B. diandrus, B. inermis and B. carinatus (8x), B. erectus and B. tectorum  (4x), B. catharticus (6x), B. sterilis and B. rubens (2x) [13,52,53]. This suggests that the ploidy level of B. picoeuropeanus should be investigated to provide a better understanding of its phylogenetic relationships.
Regarding the relationship of B. picoeuropeanus with B. erectus, they seem to be closely related as in the combined nuclear plastid analyses B. picoeuropeanus is a sister to the subclade comprising B. erectus (section Bromopsis), B. rubens (section Penicillius), B. diandrus and B. tectorum (section Genea). Nevertheless, our molecular evidence based on nuclear and plastid makers, separately and in combination, do not allow to determine the section in which B. picoeuropeanus should be placed, as either of the B. picoeuropeanus species exclusive clade belonged to clades formed by species from different sections, or members of the Bromopsis section were located in various clades. Hence, our evidence does not entirely support the proposal by Rico and Acedo [54] regarding the section Pnigma for B. picoeuropeanus. On the other hand, the described Bromus section Penicillus [29], which comprises B. madritensis, B. rubens and B. fasciculatus, is not supported as a monophyletic group by molecular evidence.
The ITS and ETS analyses portrayed B. erectus as a polyphyletic group, whereas the nuclear plastid analyses generated topologies in which three B. erectus samples generated a monophyletic clade in which the Bromus sample collected at lower altitudes (Br1), which corresponded to the description of B. erectus, was not included. This sample (Br1) belonged to the same subclade as B. erectus, although its relationship was closer to the species from section Genea B. diandrus, B. rubens and B. tectorum. The nuclear-combined analyses also indicated a closer relationship with B. armenus Boiss., B. adjaricus Sommier and Levier, B. riparius Rehmann, B. kopetdagensis Drobow and B. tomentellus Boiss. These differences in phylogenetic relationships could be explained by the fact that the combined nuclear analyses had more sequences than the combined analysis with trnL due to the lower availability of trnL sequences. Hence, although the latter analyses generated more reliable relationships, the former presented more reliable potential relationships for B. erectus samples. Therefore, our results cast doubts regarding the adscription of the individual Br1 as B. erectus s. s., since the morphological differentiation provided by Acedo and Llamas [29] included several B. erectus vouchers from different European herbaria. This means that our sample identified as B. erectus (Br1) could not be B. erectus s. s. This hypothesis would be in accordance with the position Br1 in the ETS-ITS and ETS-ITS-trnL topologies with respect to the rest of the individuals identified as B. erectus. In this sense, we cannot determine whether our sample Br1 is the one that does not belong to B. erectus s. s. or whether the other samples from previous studies are the ones that do not correspond, as no sample from the type locality has been sequenced yet (see Table S1). This situation has wider implications, affecting B. picoeuropeanus and other species morphologically similar to B. erectus as well. Therefore, our evidence, given the current absence of B. erectus type locality samples, only allow us to state that the individuals belonging to B. picoeuropeanus do not belong to the same taxon as the individuals known as B. erectus in our study area.
The altitudinal distribution of the taxon known as B. erectus in Picos de Europa and its surrounding areas seems to be more similar to that provided by Bačič and Jogan [38] for the B. erectus s. s. in the Alps, which considered 600 m its distributional limit, although our sample was collected in even lower lands (312 m). All this evidence suggests that a better understanding of the altitudinal distribution of B. erectus could be vital in understanding the evolutionary history of this species and its relationship with morphologically similar species, such as B. picoeuropeanus in the Cantabrian Mountains or B. transylvanicus and B. condensatus in the Alps [38]. This perspective seems interesting taking into account the fact that Rico and Acedo [54] estimate that there are around 30 endemic taxa that have been reported to have a similar relationship to that of B. picoeuropeanus and B. erectus in Europe and the Mediterranean basin. In the actual context, our knowledge of the B. erectus complex and the genus Bromus would benefit from wider studies focusing on (1) detecting the microspecies currently included in this complex, (2) determining their distributions and (3) understanding ecological and evolutionary processes involved in the formation of these species. These types of studies would also be of importance in detecting potential morphological adaptations in Bromus, which could clarify whether the species of B. erectus complex are phylogenetically related or whether their similar morphology is due to convergence.
On the other hand, more information about B. picoeuropeanus is needed, since its mountain distribution indicates that this species is susceptible to experiencing range shifts as consequence of temperature rise as has already been reported in other Bromus species [55]. In the context of climate change, its capacity will depend on many factors, such as the genetic structure of the existing population [56]; therefore, future research efforts should focus on these conservational aspects.

Plant Material
A total of twelve individuals of B. erectus s. l. (i.e., B. erectus complex) were collected in Picos de Europa and other regions of the Cantabrian Mountains (see Figure 7A). Nine of those individuals were collected at different sites of Picos de Europa and their surrounding areas, following a clinal sampling scheme in which heights comprised altitudes ranging from 729 to 1928 m (see Figure 7B and Table 2), thus including the altitude ranges of B. erectus s. s. and B. picoeuropeanus described by Acedo and Llamas [29] for this area-from 1600 to 2200 m for B. picoeuropeanus and from 1490 to 1520 m for B. erectus s. s. Additionally, other two individuals of B. erectus complex, one fitting in the range of B. erectus s. s. and another fitting the range of B. picoeuropeanus as proposed by Acedo and Llamas [29], were sampled at locations in the Cantabrian Mountains outside the limits of Picos de Europa. Finally, an individual of B. erectus complex fitting the altitudinal range for B. erectus s. s. proposed by Bačič and Jogan [38]-up to 600 m a. s. l.-was sampled on the north side of the Cantabrian Mountains to serve as contrast from individuals identified as B. picoeuropeanus collected within the range of B. erectus s. s. sensu Acedo and Llamas [29].
This sampling design resulted in a total of six sites fitting within the altitudinal range of B. erectus s. s. sensu Acedo and Llamas [29], which also fitted its area of distribution of the north of Spain provided by Acedo and Llamas [29] and Rico and Acedo [54]: Br1, the only location fitting the distribution proposed by Bačič and Jogan [38], Br3 and Br9-12. The number of sites fitting the B. picoeuropeanus altitudinal range sensu Acedo and Llamas [29] was six (Br2 and Br4-Br8). All the collected individuals were later identified as either B. erectus s. s. or B. picoeuropeanus following the detailed comparative of Acedo and Llamas [29] (see Table 2).
Finally, three individuals belonging to B. diandrus Roth (1787), B. sterilis L. (1753) and B. rigidus Roth (1790), which form part of different sections from that of B. picoeuropeanus and B. erectus s. s., were identified following Rico and Acedo [54] and Smith [57] and collected (see Figure 7A). The collected material consisted of complete individuals, which were preserved in silica gel before the DNA extraction.  [29] have been highlighted in blue, while altitudes outside this range have been highlighted in gray. Llamas [29] have been highlighted in blue, while altitudes outside this range have been highlighted in gray.

DNA Extraction, Amplification and Sequencing
The DNA extraction was conducted using the NucleoSpin ® Plant II Columns (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) kit. The extracted DNA was stored at −20 • C. Three molecular markers which had proved useful in previous studies of Bromus were amplified: the two high-copy nuclear markers Internal Transcribed Spacer (ITS) and External Transcribed Spacer (ETS) and the plastid marker trnL. The regions 5.8S, ITS-1, and ITS-2 of the ribosomal nuclear maker ITS were amplified by PCR using the primers 17SE and 26SE [58]. The partial sequence 3 ETS of the intergenetic spacer (IGS) was amplified using the primers RETS-B4F [59] and 18S-R [60] following the recommended PCR conditions and cycles of Alonso et al. [59]. The exon of chloroplastic sequence trnL was amplified with the c and d pair of primers [61], following their proposed PCR conditions and the PCR cycle.
Obtained PCR products were sequenced at the DNA Synthesis and Sequencing Facility Macrogen (Amsterdam, The Netherlands).

Phylogenetic Analysis
The obtained sequences were visualized and manually edited in Geneious Prime v. 1.3 [62]. The bases and polymorphism were coded following the International Union of Pure and Applied Chemistry (IUPAC). SNPs were considered "true" if they occurred at the same site in both reverse and forward amplicons and the lower peak reached at least a third of the height of the higher one.
Since previous molecular studies have revealed that the section Bropmosis, in which B. picoeuropeanus was classified by Rico and Acedo [54], is polyphyletic e.g., [14,51], sequences from different Bromus sections generated in previous studies were included in the analyses. The newly generated sequences together with Bromus sequences from previous studies available at GenBank (see Table S1) were used to generate several datasets: the ITS dataset, ETS dataset, trnL dataset, nuclear dataset, and the combined dataset. The nuclear dataset consisted of the concatenation of ETS and ITS sequences, while the combined dataset was formed by the concatenation of all three markers. We only included Bromus species from which sequences of all three makers were available; these included the type of the genus  [59,63].
The sequences were aligned in MUSCLE [64] using the online server EMBL-EBI [65] and the alignment was manually reviewed and edited in Geneious Prime v.1.3 [66] The nucleotide substitution model of each dataset was estimated in JModelTest 2 v.1.10 [67] by the default setting of the corrected Akaike Information Criterion (AIC) [68]. In the case of the ITS and ETS datasets, the inferred substitution model was the symmetrical substitution model with gamma distribution (SYM + G) [69], while the substitution model estimated for the chloroplastic sequence trnL (UAA) was the Hasegawa-Kishino-Yano substitution model with gamma distribution (HKY + G) [70].
The phylogenetic relationships of the samples were inferred by two different phylogenetic methods: Maximum Likelihood (ML) and Bayesian Inference (BI). The ML analysis was conducted in the IQ-TREE web service [71,72]. For this analysis, the initial tree was estimated by Neighbor-Joining (NJ) and the posterior full-tree rearrangement operations were performed by Neighbor Interchange (NNI). The branch support values were statistically inferred by 10,000 bootstrap (BS) replications [73][74][75]. The BI inference using MrBayes [76] was performed by 6 Monte Carlo Markov Chains (MCMC) (1 cold chain and 5 hot chains) for 10,000,000 generations and a 0.25 of burnin fraction. This burnin fraction was visualized using Tracer v1.7.1 [77]. The branch support was statistically inferred by posterior probability (PP).
The three phylogenetic analyses were performed on each maker (ETS, ITS, and trnL (UAA)) and combined datasets (the nuclear ETS-ITS and the plastid plus nuclear markers). In the cases where the sequences were concatenated, the ML and BI analyses were performed using a partitioned analysis and applying the corresponding model. Since a large indel was found in the ETS dataset, we performed and compared analyses with and without the insertion, finding similar topologies with similar support values. For this reason, we used the ETS dataset containing the large indel to perform the phylogenetic analyses.
We also inferred the phylogenetic relationships through the generation of phylogenetic networks based on the plastid haplotype diversity, hence we generated a second trnL dataset (trnLB) which only included taxa belonging to Bromus. The best-fitting substitution model for this dataset was HKY + G [70]. On the one hand, we constructed a Neighbournet in SplitsTree 4.16.2 [78], estimating branch support by performing 10,000 bootstrap repeti-tions. On the other hand, we also generated a gene genealogy by Templeton, Crandall and Sing (TCS) cladistics methods [79] in PopART 1.7 [80].

Author Contributions:
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by C.G.-T., H.S.N. and E.C. and by J.A.F.P. until the moment of his death. The first draft of the manuscript was written by C.G.-T., under the supervision and following the corrections of H.S.N. and E.C. All authors have read and agreed to the published version of the manuscript.
Funding: Claudia González-Toral had the financial support of the Government of Asturias (2002166-Ayudas del programa "Severo Ochoa" para la investigación y docencia). This research was partially supported by the research programme (PAPI-19-GR-2016-0010) funded by the University of Oviedo.

Data Availability Statement:
The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 16 March 2023). The reference number of each sequence are specified in Table 2.