Molecular phylogenetics and new ( infra ) generic classification to alleviate polyphyly in tribe Hydrangeeae ( Cornales : Hydrangeaceae )

Tribe Hydrangeeae of Hydrangeaceae currently contains nine morphologically diverse genera, many of which are wellknown garden ornamentals. Previous studies have shown eight of these genera to be phylogenetically nested within Hydrangea, rendering the latter polyphyletic. To clarify the phylogeny of tribe Hydrangeeae, the present study sequenced four chloroplast regions and ITS for an extensive set of taxa, including the type for all nine genera involved. The resulting phylogenetic hypotheses corroborate the polyphyly of Hydrangea. Since polyphyletic taxa are deemed unacceptable by both sides in the ongoing debate concerning the adherence to strict monophyly in biological classifications, a new (infra)generic classification for tribe Hydrangeeae is proposed. In order to create a stable, evolutionary informative classification a broader circumscription of the genus Hydrangea is proposed, to include all eight satellite genera of the tribe. Such treatment is considered highly preferable to an alternative where Hydrangea is to be split into several morphologically potentially unidentifiable genera. To facilitate the acceptance of the new classification proposed here, and in order to create a classification with high information content, the familiar generic names were maintained as section names where possible.


INTRODUCTION
Over the past few decades, rapid advances in DNA technologies have brought about an increase in the use of phylogenetic hypotheses in taxonomy (e.g., phylogenetic systematics; Hennig, 1966).Indeed, the majority of contemporary taxonomic studies attempt to establish natural, genealogy-based classifications, guided by phylogenetic hypotheses.Therefore, a consensus seems to have arisen that common descent should play a major role in biological classification (Xiang X.G. & al., 2012).Disagreements, however, still exist with respect to the treatment of paraphyletic taxa, with two sides locked in ongoing debate (reviewed in : Hörandl & Stuessy, 2010;Schmidt-Lebuhn, 2012).On the one hand, the school of evolutionary systematics advocates a classification system with a high information content (Stuessy, 1987;Van Wyk, 2007;Hörandl, 2010;Mayr & Bock, 2002) and practicability (Brummit, 2002;Brickel & al., 2008), reflecting natural processes.In this philosophy, shared descent is viewed as an important character for grouping taxa, but an emphasis is placed on degrees of divergence and similarity between elements of a certain taxon (Hörandl & Stuessy, 2010).As a consequence, evolutionary systematists advocate the recognition of paraphyletic taxa, as these are argued to reflect similarity, high information content and practicability.The school of phylogenetic (or cladistic) systematics, on the other hand, proposes strict adherence to monophyletic (holophyletic) taxa, recognized by the presence of synapomorphic characters.This school argues that monophyletic groups are objective entities, considering all taxa above species level as human-devised, artificial constructs.Therefore, since paraphyletic taxa are based on a subjective idea of what is "divergent enough" (Schmidt-Lebuhn, 2012), these entities are rejected as artificial classes created to emphasize particular characters or divergence (Donoghue & Cantino, 1988;Ebach & al., 2006).Here, some of the prominent discussion points between both schools are illustrated with the taxonomy of Hydrangeaceae tribe Hydrangeeae.This group provides an interesting case study for solving complex classification problems due to the presence of (1) paraphyletic groups both at genus level and below, (2) a large polyphyletic Version of Record assemblage, and (3) important horticultural representatives with very distinct morphology.
A small but representative sampling of Hydrangeeae was included in studies addressing the evolutionary relationships within the Hydrangeaceae using both morphological (Hufford & al., 1997) and molecular (Soltis & al., 1995;Hufford & al., 2001) data.In addition to suffering from low statistical support, these studies resulted in different phylogenetic hypotheses.Sequencing a series of chloroplast regions for an extensive sampling of specimens, Samain & al. (2010) were able to identify two well-supported clades in tribe Hydrangeeae.A first clade, termed Hydrangea I, contained Cardi andra, Decumaria, Deinanthe, Pileostegia, Schizophragma and several representatives of Hydrangea.Relationships among these genera remained mainly unresolved.In the second major clade, termed Hydrangea II, Broussaisia and Dichroa were in a grade with two separate clades of Hydrangea representatives.Therefore, the results obtained by Samain & al. (2010) suggest that Hydrangea is a polyphyletic assemblage, with the remaining eight genera of Hydrangeeae phylogenetically nested within Hydrangea.Moreover, this study suggested that the infrageneric classification of Hydrangea proposed by McClintock (1957) is in need of revision.In a more recent study, Granados Mendoza & al. (2013) tested the utility of 13 plastid markers using a reduced sampling for resolving backbone relationships within tribe Hydrangeeae (Broussaisia not included).A highly supported phylogenetic hypothesis was recovered for Hydrangea I and II, offering better resolution within the first clade, and only leaving the position of H. arborescens L. unsupported.Furthermore, Hydrangea was once more recovered as a polyphyletic assemblage, corroborating the findings by Samain & al. (2010).
In the present study, a comprehensive phylogeny of tribe Hydrangeeae is presented, sampling all major evolutionary clades retrieved in previous studies, using four plastid markers selected according to their phylogenetic informativeness (Granados Mendoza & al., 2013) and ITS.Using the resulting phylogenetic hypothesis, we address the polyphyletic nature of Hydrangea and evaluate the merits of creating a monophyletic Hydrangea.Finally, a new infrageneric classification is proposed, incorporating the inferred relationships among and within subclades Hydrangea I and II.Throughout the manuscript, all section names used are those of the here-proposed classification of Hydrangea s.l., the broad circumscription of Hydrangea, including the other eight genera of tribe Hydrangeeae.In contrast, Hydrangea s.str.refers to the previously recognized, polyphyletic Hydrangea, not including the eight satellite genera.

MATERIALS AND METHODS
Taxon sampling.-Taxa pertaining to all major clades and subclades recovered in Samain & al. (2010), all sections and subsections proposed in McClintock's (1957) infrageneric classification, as well as the eight allied genera Broussaisia, Cardiandra, Decumaria, Deinanthe, Dichroa, Pileostegia, Platycrater and Schizophragma were sampled.For all genera  S1. under study, a specimen representing the type was included.Two species of Loasaceae (Loasa tricolor Ker Gawl., Xylopodia klaprothioides Weigend) and two species of Hydrangeaceae tribe Philadelpheeae (Philadelphus mexicanus Schltdl., Philadelphus pekinensis Rupr.) were used as outgroups.Material used for DNA extraction consisted of silica-gel dried leaf tissue of wild collected accessions, while fresh leaves were used for material originating from botanical gardens.

Version of Record
Molecular methods and alignments.-Total genomic DNA was extracted from leaf tissue using a modified CTAB method (Doyle & Doyle, 1987).Four noncoding plastid regions, previously shown to be phylogenetically informative for tribe Hydrangeeae (Granados Mendoza & al., 2013), were utilized in this study.The rpl32-ndhF intergenic spacer (IGS), trnV-ndhC IGS, trnL-rpl32 IGS and the ndhA intron were sequenced for all accessions.Primer sequences and protocols for PCR amplification were taken from Granados Mendoza & al. (2013), with the exception of the amplification of the ndhA intron for the Asperae clade, which required the design of the additional primers ndhA-asp-F (GATTCGTTGAGACATAAATT) and ndhA-asp-R (GTACATGAGATTTTCACCT).These plastid markers are non-overlapping and distributed across the large and short single copies of the chloroplast genome (Granados Mendoza & al., 2013).In order to rule out incorrect conclusions based on incongruence between plastid and nuclear phylogenies, ITS was sequenced for a subset of taxa, representing all major clades found in the plastid analyses.Sequencing of this region was performed using primers ITS1 and ITS4 with PCR conditions as described in White & al. (1990).Raw sequences were edited in Sequencher v.5.0.1 (Gene Codes Corporation), and aligned with Muscle v.3.8.1 (Edgar, 2004).The obtained alignments were subsequently evaluated manually, excluding regions of uncertain homology such as mononucleotide repeats (for a list of excluded regions, see Electr.Suppl.: Table S2).Insertions and deletions (indels) were coded following the simple indel coding scheme of Simmons & Ochoterena (2000) available in SeqState v.1.4.1 (Müller, 2005).
Phylogenetic analysis.-The most appropriate model for nucleotide evolution was selected with the Akaike information criterion (AIC) in jModelTest v.2.1.3 (Darriba & al., 2012).This procedure selected the TVM + G model for all regions except for the trnL-rpl32 IGS, for which GTR + G was preferred.Bayesian inference analysis was run in MrBayes v. 3.2.1 (Ronquist & al., 2012), for each of the four plastid regions and ITS separately, a concatenated matrix containing all four plastid regions, and a concatenated matrix combining the plastid regions with ITS.The concatenated dataset was generated to examine the impact of the information in the ITS dataset on the phylogenetic relationships recovered, and only attempted since there were no supported (posterior probability > 0.95) incongruences.For each of the above-mentioned alignments, two analyses were run; one with and one without indels coded.All analyses were run using the GTR + G model, since the TVM model is not implemented in MrBayes.The analyses of the concatenated matrices were run with partitions for each region, unlinking model parameters for each partition.The Markov Chain Monte Carlo (MCMC) was run using four simultaneous runs with four chains each, for a total of five million generations, sampling trees every 100 generations.Parameter sampling was checked in Tracer v.1.6(Rambaut & al., 2014) to ensure stationarity for each run.Discarding the first 12,500 trees as burn-in, the remaining trees were used to calculate the posterior probabilities (PP) of clades using the majority-rule consensus.The Cyber infrastructure for Phylogenetic Research (Cipres Science gateway; http://www.phylo.org;Miller & al., 2010) was used to run all Bayesian analyses.A maximum likelihood analysis in RAxML v. 7.2.8 (Stamatakis & al., 2005) was performed on both concatenated datasets (plastid and plastid + ITS) without indel coding, using the GTRGAMMA model for sequence evolution, with the dataset partitioned according to marker regions, and 1000 rapid bootstrap replicates (Stamatakis & al., 2008).
Phylogenetic hypothesis testing.-Bayesian phylogenetic inference did not resolve the evolutionary position of three taxa: Broussaisia arguta Gaudich., Hydrangea arborescens and H. quercifolia W.Bartram.Therefore, all possible resolutions of the unsupported branches in the phylogenetic hypothesis were statistically compared using Bayesian inference and the combined plastid dataset with indels coded.The marginal likelihoods for each possible resolution were calculated using the stepping stone algorithm (Xie & al., 2011), as implemented in MrBayes v. 3.2.2 (Ronquist & al., 2012).For each hypothesis under study, a phylogenetic tree with all major clades constrained to match the phylogenetic hypothesis was used as a prior, in accordance with the preferred approach of Bergsten & al. (2013).The stepping stone algorithm was run for 10 million generations over 50 steps, with the first step as burn-in for four independent runs.The marginal likelihoods for each hypothesis were then compared using Bayes Factors (Kass & Raftery, 1995).
Estimating phylogenetic informativeness.-The online application PhyDesign (López-Giráldez & Townsend, 2011) was used to calculate the net phylogenetic informativeness (Townsend, 2007) for each marker used in this study.This calculation used an ultrametric tree generated from the combined plastid and ITS dataset without indel coding.Substitution rates were estimated in HyPhy v.2.2.1 (Pond & al., 2005).Phylogenetic informativeness profiles for each individual region were compared to the reference ultrametric tree.Maximum net phylogenetic informativeness (PImax) was documented for each separate region, in order to determine the point in time at which each region is phylogenetically most informative.

Version of Record
Phylogenetic inference.-In the plastid combined analysis (concatenated chloroplast nucleotide dataset, including indel data; Fig. 2), Hydrangea sect.Dichroa is sister to a grade of the monophyletic sect.Macrophyllae, sect.Hirtae and sect.Chinenses.Hydrangea sect.Stylosae is recovered as sister to this entire assemblage, completing a clade congruent with Hydrangea II without Broussaisia arguta.This latter taxon is sister to a strongly supported clade (PP: 1) coinciding with Hydrangea I.This sister relationship, however, remains weakly supported (PP: 0.61).Within Hydrangea I, H. arborescens and H. quercifolia are grouped in a weakly supported clade (PP: 0.52), and are sister to the rest of Hydrangea I.In this major clade, sect.Pileostegia is sister to a clade containing the monophyletic sections Schizophragma and Decumaria, while sect.Heteromallae is sister to this entire assemblage (PP: 0.7).Hydrangea sect.Cardiandra is recovered as monophyletic and in a sister relationship with a monophyletic sect.Deinanthe, while this assemblage is sister to the clade comprising sections Heteromallae, Schizophragma, Decumaria and Pileo stegia.All these sections are in turn sister to a clade containing sects.Asperae, Cornidia, Calyptranthe and Platycrater arguta.The last is phylogenetically nested within sect.Asperae, which Fig. 2. The 50% majority-rule consensus tree based on the combined plastid dataset with indels coded, posterior probabilities obtained from Bayesian inference indicated on the respective branches when below 1. Section names according to the new infrageneric classification presented here.Hydrangea angustipetala* = H.angustipetala "f.macrosepala".in turn is sister (PP: 1) to a clade (PP: 1) containing the two highly supported monophyletic sister sections Cornidia and Calyptranthe.Analysis of the indel-coded concatenated dataset including the ITS region recovered a similar phylogenetic hypothesis, the only topological difference being the position of Broussaisia arguta.This taxon is sister to a well-supported clade (PP: 1) consisting of sect.Chinenses, sect.Hirtae, sect.Macrophyllae, sect.Dichroa and sect.Stylosae.Furthermore, support for the deeper nodes is reduced by adding ITS to the analysis (Fig. 3).
Including the data from the simple indel coding scheme generally improved clade support in the Bayesian analysis for the separate regions.Topology was not affected by inclusion of these characters, except for the position of Broussaisia arguta in the analysis of the rpl32-ndhF IGS and the concatenated dataset (Electr.Suppl.: Figs.S1, S2).For the rpl32-ndhF region, B. arguta was sister to the Hydrangea II clade with weak support (PP: 0.82) when only nucleotide data were analyzed (not shown), while this relationship was not recovered when indel data were added to the analysis (Electr.Suppl.: Figs.S2-S6).A parallel pattern for this taxon occurred in the combined plastid analysis, with B. arguta sister to Hydrangea II for the nucleotide data (PP: 0.80; Electr.Suppl.: Fig. S1), and sister to Hydrangea I (PP: 0.61) when indel data were included in the analysis (Fig. 2).Bayesian analysis of the datasets combining plastid and ITS data recovered B. arguta as sister to Hydrangea II (PP: 0.90, not shown) when indels were not coded, while this relationship was not supported when indels were coded (PP: 0.67, Fig. 3).
Analyses of separate regions did not yield well-supported conflicts.The position of H. arborescens and H. quercifolia remains unresolved in all single-gene trees and the combined analyses.However, these taxa are recovered as part of a wellsupported clade with the representatives of Hydrangea I in the combined analyses (with and without indel data, Figs. 2, 3 and Electr.Suppl.: Fig. S1) and the single gene trees for rpl32-ndhF IGS and trnV-ndhC IGS (Electr.Suppl.: Figs.S2, S3).Phylogenetic hypotheses resulting from the ML analyses did not show any supported topological differences with those generated with Bayesian inference (Electr.Suppl.: Fig. S7).
Hypothesis testing.-Comparing the marginal likelihoods obtained from the stepping stone algorithm for each of the nine hypotheses (Fig. 4) showed four hypotheses (M3-M6) to be strongly preferred over the alternatives (Table 2).Models placing Broussaisia arguta sister to the rest of Hydrangea II are preferred over alternative models with the same configuration for H. arborescens and H. quercifolia.Between models sharing the same placement of B. arguta (Fig. 4A-C, D-F and G-I), the model placing H. quercifolia sister to the rest of Hydrangea I shows the highest marginal likelihood.Bayes Factor analysis only shows this difference to be strongly supported for model M3 over M2 and M1, and for M9 over M8 and M7.
Phylogenetic informativeness.-The phylogenetic informativeness profiles of all sequenced regions are plotted below   the ultrametric tree based on the concatenated dataset with ITS and plastid regions, without indel coding in Fig. 3.The profile for the ITS region reaches a clear maximum at time 0.35, which is prior to the divergence of tribe Hydrangeeae at time 0.43, and sharply declines towards more ancient times.The plastid regions show lower, flatter profiles, steadily increasing in informativeness towards deeper nodes.Of the plastid regions, the rpl32-ndhF IGS reaches the highest informativeness, followed by trnV-ndhC, trnL-rpl32 intergenic spacers and finally the ndhA intron, respectively.

Generic relationships, congruences and conflicts in tribe
Hydrangeeae.-This study presents the most comprehensive phylogenetic hypothesis for tribe Hydrangeeae to date.Single gene trees for the ITS region (Electr.Suppl.: Fig. S6) showed the same major clades as the chloroplast markers.Resolution for the deeper nodes remained much lower than in the combined plastid analysis.Furthermore, inclusion of ITS into the concatenated analysis drastically reduced support for evolutionary relationships among large clades (sections) within Hydrangea I (Fig. 3).The inclusion of the ITS data therefore introduced noise into the dataset, as can be deduced from the phylogenetic informativeness profile in Fig. 3.The maximum phylogenetic informativeness of ITS is reached more recently (t = 0.35) than the divergence of the major clades in Hydrangea I.This region was therefore fairly uninformative for resolving evolutionary relationships prior to this time, as more recent changes in its sequence might obscure signals that have arisen within the time interval of the divergence of these major Hydrangea I lineages (Townsend, 2007).The more uniform informativeness profiles of the plastid markers, the better suited they are for resolving deeper nodes in tribe Hydrangeeae.Consequently, the new classification presented here is discussed using the phylogenetic tree based on the concatenated chloroplast regions (Fig. 2), as this is the most complete dataset, with best support for relationships among sections.In this phylogenetic hypothesis, the morphologically diverse genera Broussaisia, Cardiandra, Decumaria, Deinanthe, Dichroa, Pileostegia, Platycrater and Schizophragma were recovered as monophyletic, but nested within the larger polyphyletic Hydrangea (Fig. 2).These findings were in general agreement with earlier studies (Samain & al., 2010;Granados Mendoza & al., 2013).A combined analysis of 13 chloroplast regions by Granados Mendoza & al. (2013) recovered H. quercifolia in a grade with H. arborescens and a clade containing sect.Asperae (plus Platycrater) as sister to the sister sections Calyptranthe and Cornidia.The short branch subtending H. arborescens, however, remained unsupported in Granados Mendoza & al. (2013).In the present study, phylogenetic placement of H. arborescens and H. quercifolia was only partly resolved (with low support) for the combined plastid dataset with indels coded and both analyses of the rpl32-ndhF IGS (Electr.Suppl.: Fig. S2).Furthermore, the Bayesian test of phylogenetic hypotheses did not prefer one configuration of these taxa over alternative configurations.The reason for this absence of resolution is the presence of deep, short branches connecting the two North American taxa to the rest of the tribe, combined with long branches subtending these monophyletic species.Resolving such short branches positioned deep in a phylogeny is considered a difficult endeavour (Townsend & Leuenberger, 2011), and requires multiple genes of high phylogenetic signal and demonstrated absence of incongruence (Salichos & Rokas, 2013), or loci highly informative on that specific time scale (Townsend, 2007).Moreover, resolving the position of H. arborescens is of pivotal importance as this taxon is the type of Hydrangea.
A second conflict between the present and previous studies was the position of the Hawaiian endemic Broussaisia arguta.The phylogenetic hypothesis generated by Samain & al. (2010) placed this taxon sister to Hydrangea II with high support (bootstrap: 96, PP: 0.98).The current study, however, recovered a weakly supported sister relationship (PP: 0.61, Fig. 2) between B. arguta and Hydrangea I in the plastid concatenated analysis incorporating indel data, while B. arguta was sister to Hydrangea II (PP: 0.80) when indels were not coded (Electr.Suppl.: Fig. S1).When ITS was added to the concatenated Bayes factors calculated with the stepping stone algorithm for comparison of the nine alternative phylogenetic hypotheses (M1-M9) presented in Fig. 4. Values > 3 but <10 signify strong support for H1 over H2, values >10 signify strong support for H1 over H2, in which H1 is the model in the first column, H2 the model in the top row (Jeffreys, 1961).
Version of Record dataset, B. arguta was recovered as sister to Hydrangea II whether or not indel data were included, although higher support was achieved with the inclusion of indel data (PP: 0.90; Fig. 3).Comparison of marginal likelihoods for the different positions of B. arguta (Fig. 2; Table 2) preferred the sister relationship with Hydrangea II over the alternative positions, which is congruent with the results shown in Samain & al. (2010).The contrasting position of B. arguta in the phylogenetic analysis of the concatenated data with indels coded might therefore be heavily influenced by the presence of large indels within the trnV-ndhC IGS.The long branches subtending this species might indicate an accelerated rate of molecular change, obscuring the evolutionary relationships of Broussaisia.A similar pattern was recovered in the Cornales family Hydrostachyaceae (Xiang & al., 1998;Xiang Q.Y., 1999;Fan & Xiang, 2003;Xiang X.G. & al., 2012), where the difficulties of reconstructing relationships in this group were suggested to be caused by an acceleration of evolution in molecular and morphological characters.Shifts into novel environments, followed by selection, increased mutation rates and genetic drift were suggested as likely to have caused this accelerated accumulation of variation.
Similarly, the long branches subtending B. arguta, as well as its deviating molecular sequences might be caused by its isolated geographic location, as the only member of tribe Hydrangeeae endemic to the Hawaiian Islands.
From a polyphyletic Hydrangea s.str.to a monophyletic Hydrangea s.l.-Unraveling the polyphyletic nature of Hydrangea is a necessity, as neither of the large schools of systematics accepts polyphyletic taxa (Hörandl & Stuessy, 2010;Schmidt-Lebuhn, 2012).Phylogenetic hypotheses resulting from the present study suggest two possible resolutions: (1) creating new genera to accommodate monophyletic groups of Hydrangea not directly related to the type H. arborescens, retaining the eight satellite genera as separate entities, or, (2) including the eight satellite genera into Hydrangea, creating a broadly circumscribed, monophyletic Hydrangea s.l.The first approach would entail splitting Hydrangea, with the description of minimally seven new genera, of which two would be monotypic.Furthermore, splitting Hydrangea s.str.would result in morphologically very similar taxa which would be very difficult to distinguish.Several degrees of splitting can be proposed, depending on the acceptance of monotypic and paraphyletic genera.For example, in order to retain the genus Platycrater, McClintock's subsect.Asperae would have to be split into three genera, two of them monotypic.The second approach entails the creation of a large genus Hydrangea, containing all species of the eight satellite genera, among which several taxa would require new specific epithets.Furthermore, the newly created Hydrangea s.l.would display wide variation in morphology, losing the practicability of classifying morphologically aberrant taxa as separate (satellite) genera.
It is argued here that a splitting approach, creating several new genera, would complicate Hydrangeeae taxonomy, resulting either in a large amount of monotypic genera or multiple morphologically very variable, and hence potentially unrecognizable, taxa.Furthermore, small changes in relationships between clades potentially recovered in future studies may possibly require new changes in number and configuration of genera.Therefore, a broad circumscription of Hydrangea to include Broussaisia, Cardiandra, Decumaria, Deinanthe, Dichroa, Pileostegia, Platycrater and Schizophragma would best serve the science of taxonomy, in creating a stable classification.
We do recognize the point made by evolutionary systematists that a classification should carry information about similarities between its constituents.Therefore, a new infrageneric classification is proposed, which is expected to facilitate the acceptance of the taxonomical changes in horticulture.By circumscribing the previous satellite genera as distinct sections, these entities remain recognizable for the broader public, with already well-known names, albeit at a different taxonomic level.A new infrageneric classification of Hydrangea, including new sections and combinations.-The eight satellite genera of Hydrangea are recognized as distinct sections, with the exception of Platycrater, which is placed in sect.Asperae in order to avoid the creation of a polyphyletic Asperae.The subsections in the classification of McClintock (1957) are raised to section level.Assignment of all currently recognized Hydrangeeae species names to their respective section is provided in Electr.Suppl.: Table S1

Fig. 4 .
Fig. 4. Phylogenetic hypothesis used for Bayesian hypothesis testing.A, The full tree corresponding to model M1, monophyly of all sections was constrained, as were all depicted nodes.B-I, alternative hypotheses, clade A and B are constrained as depicted in Fig. 4A, positions of Broussaisia, H. quercifolia and H. arborescens differ between models (B: model M2, C: model M3, D: model M4, E: model M5, F: model M6, G: model M7, H: model M8, I: model M9).

Table 1 .
Genera in tribe Hydrangeeae, with number of published names and broad distribution, prior to merging the satellite genera into Hydrangea.Hydrangea s.l. in bold.Table with all currently recognized species names in the Electr.Suppl.: Table

Table 2 .
Comparison of the nine different hypotheses presented in Fig.4using Bayes factors.