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Turnip Time Travels: Age Estimates in Brassicaceae
Opinion
Turnip Time Travels: Age Estimates in Brassicaceae Andreas Franzke,1,* Marcus A. Koch,1,2 and Klaus Mummenhoff3 Results of research in life sciences acquire a deeper meaning if they can also be discussed in temporal contexts of evolution. Despite the importance of the mustard family (Brassicaceae) as a prominent angiosperm model family, a robust, generally accepted hypothesis for a family-wide temporal framework does not yet exist. The main cause for this situation is a poor fossil record of the family. We suggest that the few known fossils require a critical re-evaluation of phylogenetic and temporal assignments as a prerequisite for appropriate molecular dating analyses within the family. In addition, (palaeo)biogeographical calibrations, not explored so far in the family, should be integrated in a synthesis of various dating approaches, with each contributing their specific possibilities and limitations. What is Clade Age Estimation About? Age estimates based on molecular clock models are among one of the most important and fascinating applications in modern evolutionary biology. Early, somewhat simplistic approaches (e.g., relying on constant molecular clock models), have been replaced during the past two decades by highly elaborate methods and new concepts. Contemporary approaches for choosing suitable molecular clock models that accommodate different forms of evolutionary rate heterogeneity and methods for handling multilocus data sets and for different calibration techniques (see Glossary) are regularly reviewed (e.g., [1]). Due to advances in methods of estimation and recognition of the problems in measuring the absolute fit between evolutionary models and data, current approaches will be regarded in the near future as being overly simplistic. This also implies that dating analysis for a given group should always be interpreted with a healthy dose of historical criticism. For example, results of different studies might be difficult to compare as taxon sampling can have effects on molecular clock dating analysis and the manner in which palaeontological evidence is used for calibrating trees is often subjective (see [1] and references therein). Last, but not least, the result of molecular age estimates – in the same manner as a result of a phylogenetic reconstruction from a sample of gene loci – is ‘only’ one hypothesis regarding clade ages. Too often the jargon used in these respective publications sounds all too convincing, as the results of the dating analysis (ages based on molecular clock concepts) are presented as hard facts. This opinion article outlines our viewpoints on and critique of recent approaches used for age estimates within the mustard family. We believe our general thoughts on this to be relevant for a broad spectrum of the scientific community, as many Brassicaceae taxa now serve as model systems for numerous and diverse fields in plant sciences [2] (Boxes 1 and 2).
Trends The mustard family is one of the most important model plant families for virtually all areas of contemporary plant sciences. A reliable temporal framework for the evolutionary history of the family is essential as this unifies evolutionary hypotheses from various disciplines of plant research. The conflict of hypotheses on node ages within the family is often not appropriately perceived in the community. Previous dating methods relying solely on a few fossils attributed to the family and alternative approaches remain unsatisfactory.
1 Heidelberg Botanic Garden, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, D69120 Heidelberg, Germany 2 Department of Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, D-69120 Heidelberg, German 3 Biology Department, Botany, Osnabrück University, D-49069 Osnabrück, Germany
Current Problems of Age Estimates in the Brassicaceae Several recent Brassicaceae-focused studies aimed at providing age estimates for and within the family using relaxed molecular clock approaches, our own work included: Franzke et al. [3] estimated rather young ages (e.g., Brassicaceae crown group age of ca 15 My) and have
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http://dx.doi.org/10.1016/j.tplants.2016.01.024
*Correspondence: [email protected] (A. Franzke).
Box 1. An Introduction to Brassicales and Brassicaceae
Glossary
The order Brassicales comprises 16 families and about 4700 species, marked by the presence of specialised cells (myrosin cells) and the production of sulfur-containing metabolites known as glucosinolates (mustard oil glucosides). The Brassicaceae family or Cruciferae (mustards or crucifers) is the most species-rich member of the order Brassicales (ca 3700 species) and includes Arabidopsis thaliana as one of the most important model species in plant biology and numerous important crop plants such as cabbage (Brassica oleracea), canola (Brassica napus, Brassica rapa), and mustard (Sinapis alba, Brassica nigra). Moreover, the family comprises an increasing number of species that serve as study systems in many fields of plant science and evolutionary research. Brassicaceae are readily distinguished from other Brassicales families by a cruciform (cross-shaped) corolla, six stamens (the outer two shorter than the inner four), a capsule often with a septum, and a pungent, watery sap. However, the systematics and taxonomy of the family are very complex. Comprehensive molecular studies revealed 51 monophyletic tribes in four major lineages [8], with Aethionemeae as sister to the remaining Brassicaceae [57,58], and almost every character that has been used for classical taxonomy exhibits substantial homoplasy, especially those of fruits. Radiation of the family most probably started in the Irano–Turanian region. Most molecular datings indicate a pre-Miocene origin of the family and evidence from palaeobotany and palaeoecology favours a Miocene radiation [30] (but see [4] for older age estimates). Nevertheless, in both cases the radiation of Brassicaceae was largely ‘extrinsically’ driven by Miocene climate changes that created open and drier habitats and these new ecological niches became characteristically occupied by members of the family. Poorly resolved early cladogenesis argues for rapid colonisation of the newly formed arid and semiarid areas worldwide. The most important ‘intrinsic’ motor for the increase of this family of over 3700 extant species was suggested to be WGDs, which provided the genetic raw material for biological radiation and diversification [8,11,22,30,38].
Biogeography: study of the distribution of species and ecosystems in geographic space and through geological times. Calibration: converting genetic distances to absolute times, usually by means of fossils, geologic, or biogeographic evidence or nucleotide substitution rates. Clade: group of organisms (species, genera, etc.) derived from a common ancestor. Core Brassicaceae: all recent lineages except the sister tribe Aethionemeae. Crown group age: age of the clade that includes all recent taxa of a group. Ecological niche: the ecological role and space that an organism fills in an ecosystem. Geological ages: Miocene (23–5.3 Mya); Pleistocene (2.5–0.01 Mya); Quaternary (2.5–0 Mya). Homoplasy/homoplasious: nonhomologous similarities due to convergence or parallel evolution. By contrast, homologous similarity is inherited through common ancestry. Irano–Turanian region: one of the richest floristic areas of the Holarctic Kingdom in Southwest Asia, with most of its species diversity in the Iranian plateau, Anatolian plateau, and Central Asia. Model plant: reference species in furthering detailed understanding of mechanisms and processes in plants. Ideally, models are diploids, have few and small chromosomes, welldeveloped genetics, and rapid life cycles, are easily transformed, and have extensive sets of technical resources and databases curated by international resource centres. Molecular dating: estimating the divergence dates of two or more lineages by comparing their DNA or protein sequence data. Molecular systematics: use of the structure of molecules to gain information on evolutionary relationships. Monophyletic group: comprises a last common ancestor and all of its descendants. Node: in a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of the descendants. Radiation: increase in taxonomic diversity or morphological disparity.
been quite rightly criticised [4] for relying on only a single secondary calibration (Box 3). Couvreur et al. [5] estimated a crown group age of the family of ca 37.5 My and was criticised [4] using only a single fossil constraint. The work of Beilstein et al. [4] is based on a molecular clock model calibrated with four fossils including a potentially overlooked Brassicaceae fossil, Thlaspi primaevum, as a minimum age constraint for the stem group age of Thlaspi (i.e., a rather terminal split in the Brassicaceae phylogeny between Thlaspi arvense and Alliaria petiolata). In this study, the crown node age of the Brassicaceae was dated at approximately 54 Mya and clade age estimates are two- to threefold older than previously calculated. More recent comprehensive nuclear transcriptome and multigene-locus-based studies provided independent and convergent evidence for a Brassicaceae crown group age close to the 37.5 My of [5]; however, not relying on the abovementioned Thlaspi fossil: 31.8 Mya [6] and 37.1 Mya [7]. Another recent Brassicaceae-focused whole-plastome-based analysis without the Thlaspi fossil calibration provided a Brassicaceae crown group age of 32.4 My [8]. These latter estimates on Brassicales-focused studies [5–8] are all in agreement with other recent angiosperm-wide studies (e.g., [9–12]). Interestingly, the most extreme and oldest Brassicaceae age estimates of [4] are preferentially cited, predominantly in context with ages for specific nodes within Brassicaceae (e.g., the split between Arabidopsis thaliana and Arabidopsis lyrata). In approximately one-third of these publications the authors cite age estimates from [4] only and draw major conclusions with no reference to alternative results, or used the results of this study to infer substitution rates for 18 nuclear and organelle markers frequently used for systematics and population genetics [13]. This includes many high-impact publications [14–25]. We believe that the outcome obtained by [4] is debatable at the very least as there are some major discrepancies between this study and the other abovementioned studies [5–12]. The general methodology for the phylogenetic analysis and molecular age estimates employed by [4] is without doubt state of the art. We appreciate their exemplary practice for a strict evaluation of the fossils used for calibration, accepting only well-documented vouchers fulfilling minimum requirements including a clear citation record, photographic evidence or accurate reproduction, and a fossil collection number. Consequently, due to the lack of missing primary literature, the authors excluded a fossil seed (and possibly part of the carpel) that was attributed to Rorippa [4], which had been used in prior studies for calibrating molecular clocks (e.g., [26,27]). However, the significantly older divergence times of [4] are also incongruent with other recent results: Bell et al. [9] estimated divergence times across the angiosperms (36 calibration points, 567 taxa, relaxed-clock model) and calculated the stem node age for the Brassicaceae at ca 32 Mya versus ca 65 Mya in [4] and ca 42 Mya versus ca 71 Mya in [4] for the Brassicaceae/Cleomaceae/Capparaceae clade. This
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Box 2. The Brassicaceae as a Model Plant Family Thirty years ago, Arabidopsis thaliana emerged as the model organism of choice for research in plant biology. No single species can address the diverse range of ecological and evolutionary questions of current interest, but the relatives of this model species provide experimental opportunities to use the information from and tools of A. thaliana to unravel the molecular mechanisms underlying important and putatively adaptive traits beyond A. thaliana and its shrunken genome [59]. Thus, in addition to the well-known model plants A. thaliana [60] and Brassica species [61], several other Brassicaceae taxa are currently used as study objects in modern plant biology [2]. Here we give a selection of these model species (for the phylogenetic position, see Figure 1 in [30]) and some key words to characterise some of the addressed research interests: Arabidopsis halleri, Noccaea caerulescens (heavy metal tolerance and hyperaccumulation [62]); Arabidopsis lyrata, Arabidopsis suecica (self-incompatibility and genome evolution [63]); Arabis alpina (perennial habit [64]); Cardamine hirsuta (leaf architecture [65]); Capsella sp. (self-incompatibility [66,67]); Capsella bursa-pastoris (flowering time, floral architecture [68]); Boechera sp. (apomixis [69] and plant–insect and plant–pathogen interactions [70]); Diplotaxis sp. (mating system changes [71]); Iberis sp. (flower symmetry [72]); Lepidium sp. (seed physiology [73] and fruit structure [74]); Eutrema, Thellungiella sp. (salt stress [75]); Brassicaceae sp. (WGDs, genome evolution, and diversification [8,22,38,52,76–78]); and Brassicales (coevolutionary interactions between plants and butterflies [6] and glucosinolate evolution [79]). To evaluate the evolutionary context of these characters there is great demand to classify the processes underlying these characters/traits in a geological time frame. This, however, requires proper calibration of molecular markers and phylogenies.
latter node was also very recently dated as being ca 44 My old [10] in the most comprehensive angiosperm-wide dating analysis so far, which relied on 137 fossil calibrations and several molecular markers. It should be noted, however, that Magallón et al. [10] presented this estimate incorrectly as the stem age of the Brassicaceae family s.str., although their analysis did not include representatives of Cleomaceae, the sister family to the Brassicaceae. The same node was dated as being even significantly younger (ca 34 Mya) in other recent comprehensive largescale dating analyses [11,12]. Also noteworthy are the unexpectedly high age estimates for splits within genera in [4] (e.g., Lepidium, ca 16 Mya), implying that many accepted Quarternary biogeographical scenarios for Brassicaceae taxa are highly questionable. This would also be true for the split between ecotypes of A. thaliana dated to 4.3 Mya in [4] versus Pleistocene splits in [28]. Arias et al. [29], following a similar approach, used fossil T. primaevum for dating nodes within the tribe Brassiceae. This approach resulted in a Pleistocene origin of Brassica cultivars (ca 90 000 ya), although it is well recognised that cultured plants did not originate (as a consequence of domestication) before the Holocene (11 500–2200 ya). The inferred comparatively high age estimates of [4] suggest, therefore, that the analysis may have been biased towards older age estimates, potentially due to the incorporation of the putative Thlaspi fossil dated to 30 Mya [4].
Current Brassicales Fossil Situation We doubted earlier the attribution of T. primaevum to extant Thlaspi (for details see [30]). It is well known that fruit characters are highly homoplasious throughout the Brassicaceae family [30]; therefore, assignments of (such old) ‘Thlaspi’ fossil fruits to a distinct Brassicaceae taxon might be per se very questionable. Indeed, there is homoplasy in almost every morphological character in the crucifers [30]. Following this train of thought, one should also be sceptical when considering all described oldest macrofossils (fruits, seeds) that have been assigned to the genus level. Fossils assigned to Draba, Sinapis, Thlaspi, Cochlearia, and Clypeola are reported as being from the late Pliocene and the late Miocene of Germany, respectively (see references in [30]). The phylogenetic placements of these fossils, however, have never been confirmed. This is also true for fossils of Bunias from the Pleistocene of Russia and also the abovementioned Rorippa fossil from the Pliocene of Russia (see [4] and references therein). Fossil fruits from the late Miocene of Germany had been incorrectly determined as Draba and Lepidium, respectively, and also require further studies to determine their affinity (see [4] and references therein). These fossils are characterised by geological boundary intervals of temporal resolution. For example, the abovementioned Rorippa fossil is assigned to the Pliocene, reflecting a time span from 2.5 to 5 Mya. Therefore, in a molecular dating analysis,
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Relaxed molecular clock approach: analytical methods for molecular dating that relax the assumption of nucleotide substitution rate constancy among lineages. Stem group: organisms close to but outside a particular crown group that always lack features present at the base of the crown group to which they are attached. Systematics: study of evolutionary relationships between groups of organisms (species, genera, etc.). Taxon (plural, taxa): taxonomic rank at any level (e.g., species, genus, family, order, division). Taxonomy: description, identification, naming, and classification of organisms at various ranks. Tribe: a taxonomic category placed between a subfamily and a genus. Whole-genome duplication (WGD): an event creating an organism with extra copies of the entire genome (also called polyploidy). WGD events are of different ages and can be caused by hybridisation combining genomes of different species (allopolyploidy) or by the multiplication of the same genome.
Box 3. Dating Approaches Fossil Calibration Calibrations are usually based on fossil evidence, whereby a minimum constraint on the age of a clade is based on the timing of its earliest fossil representative. There has been extensive research into the proper use of fossil data for calibrating molecular trees and clocks and problems associated with the methodology (with uncertainty in fossil age and phylogenetic position representing the two greatest challenges); the reader is referred to [1,31,32,45] and the references therein. There is general agreement that the fossil record remains the most reliable source of information for the calibration of phylogenetic trees, although associated assumptions and potential bias must be taken into account. Secondary Calibration Secondary or indirect calibrations are node ages derived from previous analyses applied to an independent data set currently under study without reference to the original calibrations used to generate them [37]. Secondary calibration represents the most commonly applied age constraint after fossils, despite many problems associated with its use [37]. The primary problem with this approach is that sources of error generated by the first dating analysis become subsumed into new estimates, resulting in divergence dates of increasingly dubious reliability. Thus, the use of secondary calibration should be a last resort; the reader should consult [37] and the references therein for critical discussion of this method. Geological and (Palaeo)biogeographical Calibration Divergence of species can sometimes be attributed to geophysical isolating mechanisms or the appearance of new habitats (e.g., formation of islands, mountain systems, seaways, deserts, other geological events). This information can be used to calibrate phylogenetic trees and estimates of molecular rates once the timing of such an event is known in setting a maximum age at a node [46]. However, this procedure is prone to some distorting factors, including errors in the estimation of geological ages, the degree of association between geological events and genetic divergences, and the impacts of taxon sampling and lineage extinction [1]. Correlating the age of taxa with that of associated palaeogeographical events is probably one of the most promising methods, but the reader is referred to [1,45–47] and the references therein for details and critical evaluation of the methodology. Evidence from MA Lines In MA experiments the mutation rates of spontaneous mutations are studied in replicated inbred lines. The resulting rates, independent of any external calibration (e.g., fossils) could then potentially be used for age estimates. However, such experimentally estimated mutation rates could be an order of magnitude or more higher than substitution rates based on measurement over geological timeframes [45].
‘only’ the minimum age of the geological boundary should be used for node calibrations [31,32]. In addition, the Brassicaceae fossils (already) mentioned represent terminal taxa of the Brassicaceae. As it has been shown that calibrations at terminal nodes generally lead to rate and date estimates with higher error and lower precision [1], these fossils might be valuable for dating closely related terminal groups ‘only’. However, a critical re-evaluation of the taxonomic and temporal assignments of these fossils is urgently needed (Figure 1). Brassicaceae fossils for calibrating (deeper) nodes within the family are presently de facto not available. For larger-scale Brassicaceae phylogenies based on conservative-enough markers, it is putatively possible to include at least some external fossils of closely related families (Brassicales), which were also used in the abovementioned larger-scale dating analyses. However, the fossil record outside Brassicaceae (i.e., for the closely related families in the order Brassicales) is also scarce. At present this is as follows: (i) Turonian fossil flowers assigned to Dressiantha bicarpellata dated at ca 89 Mya [33], the oldest known putative Brassicales fossils [4]. As Dressiantha could be a stem representative or a member of the Brassicales crown group it should be used conservatively, calibrating the Brassicales stem age [10]. (ii) A leaf fossil from a Palaeocene formation (ca 61.7 Mya [34]) that was assigned to the genus Akania (Akaniaceae) but not formally assigned to a species (Akania sp.). This could be used as a minimum time constraint for the Akaniaceae stem group. (iii) Silicified wood from Neogene sediments (Late Karpatian, 17.0–16.3 Mya [35]) assigned to Capparidoxylon holleisii were clearly attributed to Capparaceae [35]. As this fossil wood is distinguished by only a very few anatomical characters from wood of the extant genus Capparis [35], it has been used to calibrate the crown age of the Capparaceae [4]. As stated above, we do not agree with the
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Figure 1. Correct Taxonomic Assignment of Fossils Is Crucial for Molecular Dating Analyses. We therefore suggest that the few known Brassicaceae fossils require critical re-evaluation. This cartoon illustrates our opinion that this is also true for Thlaspi primaevum, a fossil fruit that was recently used for molecular analysis and resulted in relatively old divergence times compared with several other published dating analyses. Cartoon drawn by Louis Werner.
attribution of fossil T. primaevum to extant Thlaspi. At present we would therefore suggest including T. primaevum only very conservatively to constrain the Brassicaceae stem/crown age and then to test the influence of this fossil on age estimates. A similar test (T. primaevum fossil included versus not included in dating analyses) was recently conducted by [7] and age estimates with T. primaevum were indeed higher. Interestingly, Magallón et al. [10] used T. primaevum as a constraint for the Arabidopsis–Brassica split, incorrectly designated as the crown node of Brassicaceae as representatives of the tribe Aethionemeae (sister to all remaining Brassicaceae) were not included. However, the authors used ca 23 My as a constraint, corresponding to the upper boundary of the Oligocene, instead of the currently established age of the according fossil flora being ca 32 My old [36]. The inclusion of Brassicales fossils (and corresponding sequences) is putatively valuable only for larger-scale phylogenies, as here only conservative markers can be applied, which allow uncritical alignments of sequences from Brassicaceae taxa and representatives of other Brassicales. This is also true for secondary calibrations (node ages from previous analyses) derived from the abovementioned large-scale analyses (e.g., [10]).
Alternatives to Primary Fossil Dating Approaches Secondary calibrations are derived from node age estimates from previous studies applied to an independent data set without reference to the original calibrations used to generate them. Therefore, uncertainty in the original analyses (e.g., due to non-critical assessment of fossil evidence) could lead to compounded errors. As reliable (oldest) fossils for primary calibrations within the Brassicaceae are not available at present, secondary calibration, successively applied from more basal nodes to more terminal nodes within the family, is one of the remaining options for dating estimates within the crucifers. Potential bias associated with the original study should then be taken into account and reported [37]. Before discussing geologic and (palaeo)biogeographical calibrations, where we see some unexploited potential for calibrating more terminal nodes within the family, we wish to address two other approaches in the context of age estimates in the family. A key finding is that all Brassicaceae species share a common whole-genome duplication (At-/ WGD) event in their history [11,38]. Thus, the age of the At-/ WGD should coincide with the age of the Brassicaceae and, therefore, should represent an alternative independent approach of estimating the age of the Brassicaceae. Analogically, this should be true for at least four additional independent lineage-specific WGDs within the family (e.g., the Br-/ WGD characterising the tribe Brassiceae). However, inferring the exact timing of WGDs is not straightforward, as the general
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problems described above also apply for dating WGDs; for example, rate heterogeneity within and among lineages and rate variation among genes, even at synonymous sites [39,40]. However, the most important point in this respect is the fact that the substitution rates used to finally scale molecular divergence into time were also calibrated, directly or indirectly, with fossils; moreover – to the best of our knowledge – in Brassicaceae studies always under the assumption of a strict molecular clock. Another fascinating approach for divergence time estimates could be based on (general) mutation rates inferred from mutation accumulation (MA) experiments (Box 3). Such an experiment was performed for A. thaliana and the time of divergence between A. thaliana and A. lyrata was calculated as being ca 18 Mya [41]. This result is in general agreement with the (T. primaevum) fossil-based high age estimate for the same split (13 Mya) of [4], which was consequently regarded as independent confirmation of the MA dating approach [42]. However, the MA-based dating approach for this particular split was based on the assumption of a generation time of 1 year. This is true for extant A. thaliana. However, closely related Arabidopsis taxa including A. lyrata are biennials, mostly even perennials [43]. As annuality and selfing are commonly associated [44], the lineage of A. thaliana might be annual since only very recent times (Middle Pleistocene, ca 0.44–1 Mya), when transition to selfing in A. thaliana occurred (see [43] and references therein). This would imply that the MA-based dating approach for the A. lyrata–A. thaliana split would be at least (unbelievably) 36 My old. Moreover, as mutation rates from MA experiments typically exceed long-term substitution rates at least by an order of magnitude (see [45] and references therein), this particular split would ‘realistically’ be 72 My old. In conclusion, much more research is needed to clarify how the mutation rate observed in one species can be assigned to related species to base age estimates on results of MA experiments.
Outstanding Questions Is it possible to improve the reliability of phylogenetic and temporal assignment of known Brassicaceae fossils for molecular clock calibrations within the family? We suggest exploiting the synergy of transdisciplinary research efforts of palaeobotanists and Brassicaceae (molecular) systematists. Can (palaeo)biogeographical molecular clock calibration approaches, new for the Brassicaceae, improve the temporal framework of the family? Recent approaches in this developing field also correlate demographic events with geological calibrations. Is it possible to unify hypotheses from various dating approaches for a familywide temporal framework? This might also deepen our understanding of how MA experiment-derived spontaneous mutation rates correlate with long-term substitution rates.
Concluding Remarks: Biogeography as Part of the Solution? Besides results based on critical re-evaluated Brassicaceae fossils and the stepwise secondary calibration approaches mentioned above to infer age estimates within the family, biogeographical calibrations could be an alternative option for mutual comparison between the two methods (see Outstanding Questions). As in all other calibrating methods, this type of external calibration approach carries numerous risks [1,45–48] that we do not want to address here in detail; nevertheless, it would seem to be a promising approach [49]. Here we see unexploited potential in biogeographical calibrations for the Brassicaceae family as – to our knowledge – this approach has not so far been applied. We therefore suggest that geological or palaeoclimate events in the Neogene–Quarternary should be tested to calibrate terminal nodes in Brassicaceae phylogenies; for example, the onset of the Messinian Salinity Crisis and the origin of the Aegean Islands (Ricotia [50]), the formation of Socotra (endemic Brassicaceae [51]), the emergence of the Sahara (South African endemic Brassicaceae [27,52], the uplift of the Southern Alps in New Zealand (Pachycladon [53]), the uplift of the Himalayas and the Tibetan plateau (tribe Arabidae [54]), the longitudinal range split and genetic differentiation (Eurasian steppe plant Clausia aprica [55]), the origin of the Hawaiian islands (Hawaiian endemic Lepidium [27]), or the recolonisation of formerly glaciated areas such as the Arctic or High Alpine regions during Pleistocene glaciations (e.g., Arabis alpina [56]). This approach is no panacea but might be a starting point for a more holistic view on the evolutionary history of the family, where, for example, a Miocene age estimate for typical ‘known’ Quaternary biogeographical patterns should provoke some scepticism. Acknowledgments € Nora Hohmann, Nicolai Nürk, José Ignacio Lucas-Lledó, Diego Salariato, Barıs¸ Ozüdog ˘ ru, Lydia Gramzow, Günter Theißen, and Steven Manchester are acknowledged for discussion, Graham Muir and Lucille Schmieding for proofreading, and Louis Werner for drawing the cartoon. The authors thank two anonymous reviewers and Fabien Condamine for constructive advice on an earlier version of the manuscript. A.F., M.A.K., and K.M. were funded by the German Research Foundation (DFG) (grants MU 1137/7-1/2, MU 1137/9-1, and KO 2302/13-1).
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Turnip Time Travels: Age Estimates in Brassicaceae Andreas Franzke,1,* Marcus A. Koch,1,2 and Klaus Mummenhoff3 Results of research in life sciences acquire a deeper meaning if they can also be discussed in temporal contexts of evolution. Despite the importance of the mustard family (Brassicaceae) as a prominent angiosperm model family, a robust, generally accepted hypothesis for a family-wide temporal framework does not yet exist. The main cause for this situation is a poor fossil record of the family. We suggest that the few known fossils require a critical re-evaluation of phylogenetic and temporal assignments as a prerequisite for appropriate molecular dating analyses within the family. In addition, (palaeo)biogeographical calibrations, not explored so far in the family, should be integrated in a synthesis of various dating approaches, with each contributing their specific possibilities and limitations. What is Clade Age Estimation About? Age estimates based on molecular clock models are among one of the most important and fascinating applications in modern evolutionary biology. Early, somewhat simplistic approaches (e.g., relying on constant molecular clock models), have been replaced during the past two decades by highly elaborate methods and new concepts. Contemporary approaches for choosing suitable molecular clock models that accommodate different forms of evolutionary rate heterogeneity and methods for handling multilocus data sets and for different calibration techniques (see Glossary) are regularly reviewed (e.g., [1]). Due to advances in methods of estimation and recognition of the problems in measuring the absolute fit between evolutionary models and data, current approaches will be regarded in the near future as being overly simplistic. This also implies that dating analysis for a given group should always be interpreted with a healthy dose of historical criticism. For example, results of different studies might be difficult to compare as taxon sampling can have effects on molecular clock dating analysis and the manner in which palaeontological evidence is used for calibrating trees is often subjective (see [1] and references therein). Last, but not least, the result of molecular age estimates – in the same manner as a result of a phylogenetic reconstruction from a sample of gene loci – is ‘only’ one hypothesis regarding clade ages. Too often the jargon used in these respective publications sounds all too convincing, as the results of the dating analysis (ages based on molecular clock concepts) are presented as hard facts. This opinion article outlines our viewpoints on and critique of recent approaches used for age estimates within the mustard family. We believe our general thoughts on this to be relevant for a broad spectrum of the scientific community, as many Brassicaceae taxa now serve as model systems for numerous and diverse fields in plant sciences [2] (Boxes 1 and 2).
Trends The mustard family is one of the most important model plant families for virtually all areas of contemporary plant sciences. A reliable temporal framework for the evolutionary history of the family is essential as this unifies evolutionary hypotheses from various disciplines of plant research. The conflict of hypotheses on node ages within the family is often not appropriately perceived in the community. Previous dating methods relying solely on a few fossils attributed to the family and alternative approaches remain unsatisfactory.
1 Heidelberg Botanic Garden, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, D69120 Heidelberg, Germany 2 Department of Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, D-69120 Heidelberg, German 3 Biology Department, Botany, Osnabrück University, D-49069 Osnabrück, Germany
Current Problems of Age Estimates in the Brassicaceae Several recent Brassicaceae-focused studies aimed at providing age estimates for and within the family using relaxed molecular clock approaches, our own work included: Franzke et al. [3] estimated rather young ages (e.g., Brassicaceae crown group age of ca 15 My) and have
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http://dx.doi.org/10.1016/j.tplants.2016.01.024
*Correspondence: [email protected] (A. Franzke).
Box 1. An Introduction to Brassicales and Brassicaceae
Glossary
The order Brassicales comprises 16 families and about 4700 species, marked by the presence of specialised cells (myrosin cells) and the production of sulfur-containing metabolites known as glucosinolates (mustard oil glucosides). The Brassicaceae family or Cruciferae (mustards or crucifers) is the most species-rich member of the order Brassicales (ca 3700 species) and includes Arabidopsis thaliana as one of the most important model species in plant biology and numerous important crop plants such as cabbage (Brassica oleracea), canola (Brassica napus, Brassica rapa), and mustard (Sinapis alba, Brassica nigra). Moreover, the family comprises an increasing number of species that serve as study systems in many fields of plant science and evolutionary research. Brassicaceae are readily distinguished from other Brassicales families by a cruciform (cross-shaped) corolla, six stamens (the outer two shorter than the inner four), a capsule often with a septum, and a pungent, watery sap. However, the systematics and taxonomy of the family are very complex. Comprehensive molecular studies revealed 51 monophyletic tribes in four major lineages [8], with Aethionemeae as sister to the remaining Brassicaceae [57,58], and almost every character that has been used for classical taxonomy exhibits substantial homoplasy, especially those of fruits. Radiation of the family most probably started in the Irano–Turanian region. Most molecular datings indicate a pre-Miocene origin of the family and evidence from palaeobotany and palaeoecology favours a Miocene radiation [30] (but see [4] for older age estimates). Nevertheless, in both cases the radiation of Brassicaceae was largely ‘extrinsically’ driven by Miocene climate changes that created open and drier habitats and these new ecological niches became characteristically occupied by members of the family. Poorly resolved early cladogenesis argues for rapid colonisation of the newly formed arid and semiarid areas worldwide. The most important ‘intrinsic’ motor for the increase of this family of over 3700 extant species was suggested to be WGDs, which provided the genetic raw material for biological radiation and diversification [8,11,22,30,38].
Biogeography: study of the distribution of species and ecosystems in geographic space and through geological times. Calibration: converting genetic distances to absolute times, usually by means of fossils, geologic, or biogeographic evidence or nucleotide substitution rates. Clade: group of organisms (species, genera, etc.) derived from a common ancestor. Core Brassicaceae: all recent lineages except the sister tribe Aethionemeae. Crown group age: age of the clade that includes all recent taxa of a group. Ecological niche: the ecological role and space that an organism fills in an ecosystem. Geological ages: Miocene (23–5.3 Mya); Pleistocene (2.5–0.01 Mya); Quaternary (2.5–0 Mya). Homoplasy/homoplasious: nonhomologous similarities due to convergence or parallel evolution. By contrast, homologous similarity is inherited through common ancestry. Irano–Turanian region: one of the richest floristic areas of the Holarctic Kingdom in Southwest Asia, with most of its species diversity in the Iranian plateau, Anatolian plateau, and Central Asia. Model plant: reference species in furthering detailed understanding of mechanisms and processes in plants. Ideally, models are diploids, have few and small chromosomes, welldeveloped genetics, and rapid life cycles, are easily transformed, and have extensive sets of technical resources and databases curated by international resource centres. Molecular dating: estimating the divergence dates of two or more lineages by comparing their DNA or protein sequence data. Molecular systematics: use of the structure of molecules to gain information on evolutionary relationships. Monophyletic group: comprises a last common ancestor and all of its descendants. Node: in a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of the descendants. Radiation: increase in taxonomic diversity or morphological disparity.
been quite rightly criticised [4] for relying on only a single secondary calibration (Box 3). Couvreur et al. [5] estimated a crown group age of the family of ca 37.5 My and was criticised [4] using only a single fossil constraint. The work of Beilstein et al. [4] is based on a molecular clock model calibrated with four fossils including a potentially overlooked Brassicaceae fossil, Thlaspi primaevum, as a minimum age constraint for the stem group age of Thlaspi (i.e., a rather terminal split in the Brassicaceae phylogeny between Thlaspi arvense and Alliaria petiolata). In this study, the crown node age of the Brassicaceae was dated at approximately 54 Mya and clade age estimates are two- to threefold older than previously calculated. More recent comprehensive nuclear transcriptome and multigene-locus-based studies provided independent and convergent evidence for a Brassicaceae crown group age close to the 37.5 My of [5]; however, not relying on the abovementioned Thlaspi fossil: 31.8 Mya [6] and 37.1 Mya [7]. Another recent Brassicaceae-focused whole-plastome-based analysis without the Thlaspi fossil calibration provided a Brassicaceae crown group age of 32.4 My [8]. These latter estimates on Brassicales-focused studies [5–8] are all in agreement with other recent angiosperm-wide studies (e.g., [9–12]). Interestingly, the most extreme and oldest Brassicaceae age estimates of [4] are preferentially cited, predominantly in context with ages for specific nodes within Brassicaceae (e.g., the split between Arabidopsis thaliana and Arabidopsis lyrata). In approximately one-third of these publications the authors cite age estimates from [4] only and draw major conclusions with no reference to alternative results, or used the results of this study to infer substitution rates for 18 nuclear and organelle markers frequently used for systematics and population genetics [13]. This includes many high-impact publications [14–25]. We believe that the outcome obtained by [4] is debatable at the very least as there are some major discrepancies between this study and the other abovementioned studies [5–12]. The general methodology for the phylogenetic analysis and molecular age estimates employed by [4] is without doubt state of the art. We appreciate their exemplary practice for a strict evaluation of the fossils used for calibration, accepting only well-documented vouchers fulfilling minimum requirements including a clear citation record, photographic evidence or accurate reproduction, and a fossil collection number. Consequently, due to the lack of missing primary literature, the authors excluded a fossil seed (and possibly part of the carpel) that was attributed to Rorippa [4], which had been used in prior studies for calibrating molecular clocks (e.g., [26,27]). However, the significantly older divergence times of [4] are also incongruent with other recent results: Bell et al. [9] estimated divergence times across the angiosperms (36 calibration points, 567 taxa, relaxed-clock model) and calculated the stem node age for the Brassicaceae at ca 32 Mya versus ca 65 Mya in [4] and ca 42 Mya versus ca 71 Mya in [4] for the Brassicaceae/Cleomaceae/Capparaceae clade. This
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Box 2. The Brassicaceae as a Model Plant Family Thirty years ago, Arabidopsis thaliana emerged as the model organism of choice for research in plant biology. No single species can address the diverse range of ecological and evolutionary questions of current interest, but the relatives of this model species provide experimental opportunities to use the information from and tools of A. thaliana to unravel the molecular mechanisms underlying important and putatively adaptive traits beyond A. thaliana and its shrunken genome [59]. Thus, in addition to the well-known model plants A. thaliana [60] and Brassica species [61], several other Brassicaceae taxa are currently used as study objects in modern plant biology [2]. Here we give a selection of these model species (for the phylogenetic position, see Figure 1 in [30]) and some key words to characterise some of the addressed research interests: Arabidopsis halleri, Noccaea caerulescens (heavy metal tolerance and hyperaccumulation [62]); Arabidopsis lyrata, Arabidopsis suecica (self-incompatibility and genome evolution [63]); Arabis alpina (perennial habit [64]); Cardamine hirsuta (leaf architecture [65]); Capsella sp. (self-incompatibility [66,67]); Capsella bursa-pastoris (flowering time, floral architecture [68]); Boechera sp. (apomixis [69] and plant–insect and plant–pathogen interactions [70]); Diplotaxis sp. (mating system changes [71]); Iberis sp. (flower symmetry [72]); Lepidium sp. (seed physiology [73] and fruit structure [74]); Eutrema, Thellungiella sp. (salt stress [75]); Brassicaceae sp. (WGDs, genome evolution, and diversification [8,22,38,52,76–78]); and Brassicales (coevolutionary interactions between plants and butterflies [6] and glucosinolate evolution [79]). To evaluate the evolutionary context of these characters there is great demand to classify the processes underlying these characters/traits in a geological time frame. This, however, requires proper calibration of molecular markers and phylogenies.
latter node was also very recently dated as being ca 44 My old [10] in the most comprehensive angiosperm-wide dating analysis so far, which relied on 137 fossil calibrations and several molecular markers. It should be noted, however, that Magallón et al. [10] presented this estimate incorrectly as the stem age of the Brassicaceae family s.str., although their analysis did not include representatives of Cleomaceae, the sister family to the Brassicaceae. The same node was dated as being even significantly younger (ca 34 Mya) in other recent comprehensive largescale dating analyses [11,12]. Also noteworthy are the unexpectedly high age estimates for splits within genera in [4] (e.g., Lepidium, ca 16 Mya), implying that many accepted Quarternary biogeographical scenarios for Brassicaceae taxa are highly questionable. This would also be true for the split between ecotypes of A. thaliana dated to 4.3 Mya in [4] versus Pleistocene splits in [28]. Arias et al. [29], following a similar approach, used fossil T. primaevum for dating nodes within the tribe Brassiceae. This approach resulted in a Pleistocene origin of Brassica cultivars (ca 90 000 ya), although it is well recognised that cultured plants did not originate (as a consequence of domestication) before the Holocene (11 500–2200 ya). The inferred comparatively high age estimates of [4] suggest, therefore, that the analysis may have been biased towards older age estimates, potentially due to the incorporation of the putative Thlaspi fossil dated to 30 Mya [4].
Current Brassicales Fossil Situation We doubted earlier the attribution of T. primaevum to extant Thlaspi (for details see [30]). It is well known that fruit characters are highly homoplasious throughout the Brassicaceae family [30]; therefore, assignments of (such old) ‘Thlaspi’ fossil fruits to a distinct Brassicaceae taxon might be per se very questionable. Indeed, there is homoplasy in almost every morphological character in the crucifers [30]. Following this train of thought, one should also be sceptical when considering all described oldest macrofossils (fruits, seeds) that have been assigned to the genus level. Fossils assigned to Draba, Sinapis, Thlaspi, Cochlearia, and Clypeola are reported as being from the late Pliocene and the late Miocene of Germany, respectively (see references in [30]). The phylogenetic placements of these fossils, however, have never been confirmed. This is also true for fossils of Bunias from the Pleistocene of Russia and also the abovementioned Rorippa fossil from the Pliocene of Russia (see [4] and references therein). Fossil fruits from the late Miocene of Germany had been incorrectly determined as Draba and Lepidium, respectively, and also require further studies to determine their affinity (see [4] and references therein). These fossils are characterised by geological boundary intervals of temporal resolution. For example, the abovementioned Rorippa fossil is assigned to the Pliocene, reflecting a time span from 2.5 to 5 Mya. Therefore, in a molecular dating analysis,
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Relaxed molecular clock approach: analytical methods for molecular dating that relax the assumption of nucleotide substitution rate constancy among lineages. Stem group: organisms close to but outside a particular crown group that always lack features present at the base of the crown group to which they are attached. Systematics: study of evolutionary relationships between groups of organisms (species, genera, etc.). Taxon (plural, taxa): taxonomic rank at any level (e.g., species, genus, family, order, division). Taxonomy: description, identification, naming, and classification of organisms at various ranks. Tribe: a taxonomic category placed between a subfamily and a genus. Whole-genome duplication (WGD): an event creating an organism with extra copies of the entire genome (also called polyploidy). WGD events are of different ages and can be caused by hybridisation combining genomes of different species (allopolyploidy) or by the multiplication of the same genome.
Box 3. Dating Approaches Fossil Calibration Calibrations are usually based on fossil evidence, whereby a minimum constraint on the age of a clade is based on the timing of its earliest fossil representative. There has been extensive research into the proper use of fossil data for calibrating molecular trees and clocks and problems associated with the methodology (with uncertainty in fossil age and phylogenetic position representing the two greatest challenges); the reader is referred to [1,31,32,45] and the references therein. There is general agreement that the fossil record remains the most reliable source of information for the calibration of phylogenetic trees, although associated assumptions and potential bias must be taken into account. Secondary Calibration Secondary or indirect calibrations are node ages derived from previous analyses applied to an independent data set currently under study without reference to the original calibrations used to generate them [37]. Secondary calibration represents the most commonly applied age constraint after fossils, despite many problems associated with its use [37]. The primary problem with this approach is that sources of error generated by the first dating analysis become subsumed into new estimates, resulting in divergence dates of increasingly dubious reliability. Thus, the use of secondary calibration should be a last resort; the reader should consult [37] and the references therein for critical discussion of this method. Geological and (Palaeo)biogeographical Calibration Divergence of species can sometimes be attributed to geophysical isolating mechanisms or the appearance of new habitats (e.g., formation of islands, mountain systems, seaways, deserts, other geological events). This information can be used to calibrate phylogenetic trees and estimates of molecular rates once the timing of such an event is known in setting a maximum age at a node [46]. However, this procedure is prone to some distorting factors, including errors in the estimation of geological ages, the degree of association between geological events and genetic divergences, and the impacts of taxon sampling and lineage extinction [1]. Correlating the age of taxa with that of associated palaeogeographical events is probably one of the most promising methods, but the reader is referred to [1,45–47] and the references therein for details and critical evaluation of the methodology. Evidence from MA Lines In MA experiments the mutation rates of spontaneous mutations are studied in replicated inbred lines. The resulting rates, independent of any external calibration (e.g., fossils) could then potentially be used for age estimates. However, such experimentally estimated mutation rates could be an order of magnitude or more higher than substitution rates based on measurement over geological timeframes [45].
‘only’ the minimum age of the geological boundary should be used for node calibrations [31,32]. In addition, the Brassicaceae fossils (already) mentioned represent terminal taxa of the Brassicaceae. As it has been shown that calibrations at terminal nodes generally lead to rate and date estimates with higher error and lower precision [1], these fossils might be valuable for dating closely related terminal groups ‘only’. However, a critical re-evaluation of the taxonomic and temporal assignments of these fossils is urgently needed (Figure 1). Brassicaceae fossils for calibrating (deeper) nodes within the family are presently de facto not available. For larger-scale Brassicaceae phylogenies based on conservative-enough markers, it is putatively possible to include at least some external fossils of closely related families (Brassicales), which were also used in the abovementioned larger-scale dating analyses. However, the fossil record outside Brassicaceae (i.e., for the closely related families in the order Brassicales) is also scarce. At present this is as follows: (i) Turonian fossil flowers assigned to Dressiantha bicarpellata dated at ca 89 Mya [33], the oldest known putative Brassicales fossils [4]. As Dressiantha could be a stem representative or a member of the Brassicales crown group it should be used conservatively, calibrating the Brassicales stem age [10]. (ii) A leaf fossil from a Palaeocene formation (ca 61.7 Mya [34]) that was assigned to the genus Akania (Akaniaceae) but not formally assigned to a species (Akania sp.). This could be used as a minimum time constraint for the Akaniaceae stem group. (iii) Silicified wood from Neogene sediments (Late Karpatian, 17.0–16.3 Mya [35]) assigned to Capparidoxylon holleisii were clearly attributed to Capparaceae [35]. As this fossil wood is distinguished by only a very few anatomical characters from wood of the extant genus Capparis [35], it has been used to calibrate the crown age of the Capparaceae [4]. As stated above, we do not agree with the
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Figure 1. Correct Taxonomic Assignment of Fossils Is Crucial for Molecular Dating Analyses. We therefore suggest that the few known Brassicaceae fossils require critical re-evaluation. This cartoon illustrates our opinion that this is also true for Thlaspi primaevum, a fossil fruit that was recently used for molecular analysis and resulted in relatively old divergence times compared with several other published dating analyses. Cartoon drawn by Louis Werner.
attribution of fossil T. primaevum to extant Thlaspi. At present we would therefore suggest including T. primaevum only very conservatively to constrain the Brassicaceae stem/crown age and then to test the influence of this fossil on age estimates. A similar test (T. primaevum fossil included versus not included in dating analyses) was recently conducted by [7] and age estimates with T. primaevum were indeed higher. Interestingly, Magallón et al. [10] used T. primaevum as a constraint for the Arabidopsis–Brassica split, incorrectly designated as the crown node of Brassicaceae as representatives of the tribe Aethionemeae (sister to all remaining Brassicaceae) were not included. However, the authors used ca 23 My as a constraint, corresponding to the upper boundary of the Oligocene, instead of the currently established age of the according fossil flora being ca 32 My old [36]. The inclusion of Brassicales fossils (and corresponding sequences) is putatively valuable only for larger-scale phylogenies, as here only conservative markers can be applied, which allow uncritical alignments of sequences from Brassicaceae taxa and representatives of other Brassicales. This is also true for secondary calibrations (node ages from previous analyses) derived from the abovementioned large-scale analyses (e.g., [10]).
Alternatives to Primary Fossil Dating Approaches Secondary calibrations are derived from node age estimates from previous studies applied to an independent data set without reference to the original calibrations used to generate them. Therefore, uncertainty in the original analyses (e.g., due to non-critical assessment of fossil evidence) could lead to compounded errors. As reliable (oldest) fossils for primary calibrations within the Brassicaceae are not available at present, secondary calibration, successively applied from more basal nodes to more terminal nodes within the family, is one of the remaining options for dating estimates within the crucifers. Potential bias associated with the original study should then be taken into account and reported [37]. Before discussing geologic and (palaeo)biogeographical calibrations, where we see some unexploited potential for calibrating more terminal nodes within the family, we wish to address two other approaches in the context of age estimates in the family. A key finding is that all Brassicaceae species share a common whole-genome duplication (At-/ WGD) event in their history [11,38]. Thus, the age of the At-/ WGD should coincide with the age of the Brassicaceae and, therefore, should represent an alternative independent approach of estimating the age of the Brassicaceae. Analogically, this should be true for at least four additional independent lineage-specific WGDs within the family (e.g., the Br-/ WGD characterising the tribe Brassiceae). However, inferring the exact timing of WGDs is not straightforward, as the general
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problems described above also apply for dating WGDs; for example, rate heterogeneity within and among lineages and rate variation among genes, even at synonymous sites [39,40]. However, the most important point in this respect is the fact that the substitution rates used to finally scale molecular divergence into time were also calibrated, directly or indirectly, with fossils; moreover – to the best of our knowledge – in Brassicaceae studies always under the assumption of a strict molecular clock. Another fascinating approach for divergence time estimates could be based on (general) mutation rates inferred from mutation accumulation (MA) experiments (Box 3). Such an experiment was performed for A. thaliana and the time of divergence between A. thaliana and A. lyrata was calculated as being ca 18 Mya [41]. This result is in general agreement with the (T. primaevum) fossil-based high age estimate for the same split (13 Mya) of [4], which was consequently regarded as independent confirmation of the MA dating approach [42]. However, the MA-based dating approach for this particular split was based on the assumption of a generation time of 1 year. This is true for extant A. thaliana. However, closely related Arabidopsis taxa including A. lyrata are biennials, mostly even perennials [43]. As annuality and selfing are commonly associated [44], the lineage of A. thaliana might be annual since only very recent times (Middle Pleistocene, ca 0.44–1 Mya), when transition to selfing in A. thaliana occurred (see [43] and references therein). This would imply that the MA-based dating approach for the A. lyrata–A. thaliana split would be at least (unbelievably) 36 My old. Moreover, as mutation rates from MA experiments typically exceed long-term substitution rates at least by an order of magnitude (see [45] and references therein), this particular split would ‘realistically’ be 72 My old. In conclusion, much more research is needed to clarify how the mutation rate observed in one species can be assigned to related species to base age estimates on results of MA experiments.
Outstanding Questions Is it possible to improve the reliability of phylogenetic and temporal assignment of known Brassicaceae fossils for molecular clock calibrations within the family? We suggest exploiting the synergy of transdisciplinary research efforts of palaeobotanists and Brassicaceae (molecular) systematists. Can (palaeo)biogeographical molecular clock calibration approaches, new for the Brassicaceae, improve the temporal framework of the family? Recent approaches in this developing field also correlate demographic events with geological calibrations. Is it possible to unify hypotheses from various dating approaches for a familywide temporal framework? This might also deepen our understanding of how MA experiment-derived spontaneous mutation rates correlate with long-term substitution rates.
Concluding Remarks: Biogeography as Part of the Solution? Besides results based on critical re-evaluated Brassicaceae fossils and the stepwise secondary calibration approaches mentioned above to infer age estimates within the family, biogeographical calibrations could be an alternative option for mutual comparison between the two methods (see Outstanding Questions). As in all other calibrating methods, this type of external calibration approach carries numerous risks [1,45–48] that we do not want to address here in detail; nevertheless, it would seem to be a promising approach [49]. Here we see unexploited potential in biogeographical calibrations for the Brassicaceae family as – to our knowledge – this approach has not so far been applied. We therefore suggest that geological or palaeoclimate events in the Neogene–Quarternary should be tested to calibrate terminal nodes in Brassicaceae phylogenies; for example, the onset of the Messinian Salinity Crisis and the origin of the Aegean Islands (Ricotia [50]), the formation of Socotra (endemic Brassicaceae [51]), the emergence of the Sahara (South African endemic Brassicaceae [27,52], the uplift of the Southern Alps in New Zealand (Pachycladon [53]), the uplift of the Himalayas and the Tibetan plateau (tribe Arabidae [54]), the longitudinal range split and genetic differentiation (Eurasian steppe plant Clausia aprica [55]), the origin of the Hawaiian islands (Hawaiian endemic Lepidium [27]), or the recolonisation of formerly glaciated areas such as the Arctic or High Alpine regions during Pleistocene glaciations (e.g., Arabis alpina [56]). This approach is no panacea but might be a starting point for a more holistic view on the evolutionary history of the family, where, for example, a Miocene age estimate for typical ‘known’ Quaternary biogeographical patterns should provoke some scepticism. Acknowledgments € Nora Hohmann, Nicolai Nürk, José Ignacio Lucas-Lledó, Diego Salariato, Barıs¸ Ozüdog ˘ ru, Lydia Gramzow, Günter Theißen, and Steven Manchester are acknowledged for discussion, Graham Muir and Lucille Schmieding for proofreading, and Louis Werner for drawing the cartoon. The authors thank two anonymous reviewers and Fabien Condamine for constructive advice on an earlier version of the manuscript. A.F., M.A.K., and K.M. were funded by the German Research Foundation (DFG) (grants MU 1137/7-1/2, MU 1137/9-1, and KO 2302/13-1).
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