Best Practices for Justifying Fossil Calibrations

Our ability to correlate biological evolution with climate change, geological evolution, and other historical patterns is essential to understanding the processes that shape biodiversity. Combining data from the fossil record with molecular phylogenetics represents an exciting synthetic approach to this challenge. The first molecular divergence dating analysis (Zuckerkandl and Pauling 1962) was based on a measure of the amino acid differences in the hemoglobin molecule, with replacement rates established (calibrated) using paleontological age estimates from textbooks (e.g., Dodson 1960). Since that time, the amount of molecular sequence data has increased dramatically, affording ever-greater opportunities to apply molecular divergence approaches to fundamental problems in evolutionary biology. To capitalize on these opportunities, increasingly sophisticated divergence dating methods have been, and continue to be, developed. In contrast, comparatively, little attention has been devoted to critically assessing the paleontological and associated geological data used in divergence dating analyses. The lack of rigorous protocols for assigning calibrations based on fossils raises serious questions about the credibility of divergence dating results (e.g., Shaul and Graur 2002; Brochu et al. 2004; Graur and Martin 2004; Hedges and Kumar 2004; Reisz and Müller 2004a, 2004b; Theodor 2004; van Tuinen and Hadly 2004a, 2004b; van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007; Parham and Irmis 2008; Ksepka 2009; Benton et al. 2009; Heads 2011). The assertion that incorrect calibrations will negatively influence divergence dating studies is not controversial. Attempts to identify incorrect calibrations through the use of a posteriori methods are available (e.g., Near and Sanderson 2004; Near et al. 2005; Rutschmann et al. 2007; Marshall 2008; Pyron 2010; Dornburg et al. 2011). We do not deny that a posteriori methods are a useful means of evaluating calibrations, but there can be no substitute for a priori assessment of the veracity of paleontological data. Incorrect calibrations, those based upon fossils that are phylogenetically misplaced or assigned incorrect ages, clearly introduce error into an analysis. Consequently, thorough and explicit justification of both phylogenetic and chronologic age assessments is necessary for all fossils used for calibration. Such explicit justifications will help to ensure that divergence dating studies are based on the best available data. Unfortunately, the majority of previously published calibrations lack explicit explanations and justifications of the age and phylogenetic position of the key fossils. In the absence of explicit justifications, it is difficult to distinguish between correct and incorrect calibrations, and it becomes difficult to reevaluate previous claims in light of new data. Paleontology is a dynamic science, with new data and perspectives constantly emerging as a result of new discoveries (see Kimura 2010 for a recent case where the age of the earliest known record of a clade was more than doubled). Calibrations based upon the best available evidence at a given time can become inappropriate as the discovery of new specimens, new phylogenetic analyses, and ongoing stratigraphic and geochronologic revisions refine our understanding of the fossil record. Our primary goals in this paper are to establish the best practices for justifying fossils used for the temporal calibration of molecular phylogenies. Our examples derive mainly, but not exclusively, from the vertebrate fossil record. We hope that our recommendations will lead to more credible calibrations and, as a result, more reliable divergence dates throughout the tree of life. A secondary goal is to help the community (researchers, editors, and reviewers) who might be unfamiliar with fossils to understand and overcome the challenges associated with using paleontological data. In order to accomplish these goals, we present a specimen-based protocol for selecting and documenting relevant fossils and discuss future directions for evaluating and utilizing phylogenetic and temporal data from the fossil record. We likewise encourage biologists relying on nonfossil calibrations for molecular divergence estimates (e.g., ages of island or mountain range formations, continental drift, and biomarkers) to develop their own set of rigorous guidelines so that their calibrations may also be evaluated in a systematic way. Most studies use a Bayesian framework for estimating divergence dates with probability curves between a minimum and a maximum bound to represent calibrations (time priors) (Thorne et al. 1998; Drummond et al. 2006; Yang 2006; Yang and Rannala 2006). An appropriately constructed fossil calibration uses the oldest assigned fossil of a taxon as the basis for its minimum age and then constructs these other parameters around it (Benton and Donoghue 2007; Donoghue and Benton 2007). One key to improving the use of paleontological data is recognizing that this first step can be tied explicitly to one or a small set of museum specimens, creating a readily auditable chain of evidence. To minimize error and maximize clarity, all calibration data should be derived explicitly from specific fossil specimens. If links between calibration data and specimens cannot be made, then there are serious questions about the validity of the proposed time priors. In this respect, the fossil specimens used for calibrations represent a standard, much in the same way that a holotype specimen (or type series) is a taxonomic standard. In both cases, these specimens provide a necessary reference point for future inquiries. The explicit reporting of specimen data is just as crucial to the scientific integrity of a fossil calibration study as is making genetic sequences publicly available or reporting analytical methods. Thus, it is worthwhile to compile, reiterate, and expand on the caveats from previous studies that pertain to the construction and reporting of fossil calibrations (e.g., Graur and Martin 2004; Hedges and Kumar 2004; van Tuinen and Hadly 2004a, 2004b; Benton and Donoghue 2007; Donoghue and Benton 2007; Gandolfo et al. 2008; Parham and Irmis 2008; Benton et al. 2009; Ksepka 2009; Sanders et al. 2010) while providing a simple and explicit protocol (in checklist form) to address them. The checklist can be divided into justifying phylogenetic position and justifying age and In cases, the data to calibrations are in a but to be In to derived from is explicitly as for a rigorous and explicit approach is for justifying the use of paleontological and geological data for divergence The can be used to develop new calibrations and as a checklist for and justifying previously published calibrations based on fossils. If all are then a calibration can be of that all the relevant and data should be of specimens to the taxon should be An of the or an phylogenetic analysis that the should be on the of and molecular data should be The and stratigraphic the best of from the should be to a published age and of age should be a fossil used for calibration be based on a specimen that all the that it to be assigned to a taxonomic are from specimens, are to be from a divergence dating studies that use paleontological data for calibrations on from phylogenetic that are based on of specimens to a taxon In cases, the basis for a taxonomic can be as as documenting that the specimen was from the same or where other specimens previously Consequently, are a in et al. Parham fossil are not it is necessary to the and of specimens. may be to specimens from to a taxon there are or through phylogenetic analysis et al. 2004; et al. 2009; In where previously cannot be it is necessary to the calibration to a of specimens (e.g., and Parham or the from the calibration. Incorrect phylogenetic of fossil calibrations can introduce into divergence estimates Brochu van Tuinen and Hedges 2004; et al. dating studies on the paleontological for calibration but of the oldest of a have been in a phylogenetic analysis. Gandolfo et al. in incorrect and taxonomic to inappropriate fossil is a for that are a fossil in a taxon than the data can in the (e.g., and 2005; et al. 2010; Sanders et al. The that may use the same taxon to to biological the and may be the fossil record of is we the use of an approach to and phylogenetically specimens that are relevant for paleontological guidelines can also be to fossils (e.g., in the case that their are and evidence for the of a based on explicit and et al. 2008; et al. 2011). fossils are have phylogenetic the analytical on paleontological it is that evidence the taxonomic of relevant be explicitly A is the of to the oldest geological record of a based upon evidence. can be on provide evidence to are of or with specimens, it can be difficult to distinguish the fossil to the or the of the clade that it is used to the earliest will the of the of the and so assigning fossils to the or the of a clade of evolution that is not fossil specimens of may not be as lack one or more of the as a of or secondary Donoghue and 2009; et al. is for that are on the basis of molecular evidence but for is known (e.g., or et al. is also to in of that evolution their In those cases, the that might be of in the time of divergence from the may be difficult to the to the phylogenetic of specimen used for is not to a paper that the taxon or the of used in the phylogenetic of fossils it to The phylogenetic position of a fossil taxon can be specimens are a thorough of the paleontological is to that the recent study is claims about the oldest of a may as new data and are A of this is the case of the oldest the are fossils that to be the clade of of the of do not the In more recent analyses, have been the tree et al. and are to be on the of and where no evidence about a minimum for in phylogenetic position from about the of than from in study or discovery of specimens. specimens, new of specimens, and phylogenetic lead to revisions in the phylogenetic of fossils. as the may and stratigraphic associated with fossil specimens, but relevant phylogenetic justifying the taxonomic of these specimens is rates of in and to be as as and the of taxonomic in as the to our specimen-based are useful for the oldest specimens to a given is necessary to the phylogenetic position of a specimen for calibration. In the best cases, fossil specimens that to be assigned to a with In these assigning fossils to is of the tree the fossil will the and as a calibration for all in it is In other cases, the position of a fossil is and is on the of a specific analysis. In to the position of a taxon given (see between of and molecular phylogenetic is a that has been (Benton et al. 2009; et al. 2010; et al. from and molecular can fossil calibrations in In cases, the of a fossil may become to about it can be used to If data of the of also may be to of evolution, the of fossils in a tree et al. 2005; et al. and molecular are in the phylogenetic position of a fossil cannot be to a a that a fossil taxon is justification for a fossil calibration. A fossil with can be assigned to a specific with of the the fossil will the and as a calibration for all it is phylogenetic from data can the position of fossil In the a fossil is to be to and the that the fossil A molecular study with a and making the of the fossil If the fossil is to then it If the fossil is to then it is a calibration for just one to can the of and of fossils. In the a fossil is in the to A molecular analysis the of the and In a the for the clade are in a way and so using the fossil to clade be problems of and molecular can be (e.g., Brochu and 2008; and 2008; Ksepka or through the use of a in and, the phylogenetic position of known from fossils (e.g., et al. and Parham 2006). those approaches and explicitly to the data from fossil specimens with the of molecular about the of molecular data. methods do not so a approach to based on or is In cases, it may be that the and molecular data are so that a evidence a molecular approach are for the position of an given the phylogenetic position of use of the oldest fossil specimens to has a probability of error into the analysis (see et al. et al. We using to divergence dating analyses. are to analysis is the of specimens used for calibrations be The with a fossil can be to a specific in a stratigraphic but on the data might be to a in a stratigraphic or a or or a specimens, those more than or those derived from the lack stratigraphic and data and so have for calibration fossil in can be assigned to its and to a stratigraphic that In the best cases, calibration data will be based upon fossils with and stratigraphic that can be assigned to a in a The with a fossil can be a stratigraphic framework will have a on estimates of its and in light of in revisions of and in (e.g., formations, and are the key used to correlate and the sequence in a have (e.g., and explicitly and fossil taxon has and geological that provide a basis for its The given is for on the can be a useful minimum calibration for specimens of are known and the of is the oldest specimens. is from the in the it is from the the it is from is of a stratigraphic for the the can be in the the can be assigned to the and a the of this and is to represent in the stratigraphic where et al. the is to on the basis of ages and methods based on the et al. specimen a minimum age of are of in of or represent of Most do not represent of may be might represent of with of the time range at do the between with geochronologic of the of a fossil to a a of the age of the fossil that can then be used to establish a age as is not a stratigraphic is or with the of new and new or and can lead to of the present at a (e.g., and The dynamic of the of for fossil specimens in order to the of stratigraphic and upon divergence dating calibrations and, divergence time dating ages, but do not use or The age of a fossil is the of for the geochronologic data for dates can be difficult to establish for a and much and so provide a more framework for reporting fossil The of fossil to ages a chain of through on the basis of geological and paleontological evidence (e.g., van Tuinen and Hadly Benton et al. 2009; 2011). for the majority of calibrations, this is not the used in are not The age of a fossil is not it is established through than through dating at the in the fossil was age for a fossil specimen is the best and can be through dating methods have dating an order of in the as a result of new of and methods (e.g., et al. 2004; 2006; et al. and ages that to (e.g., et al. of this ongoing it is to the basis upon the age is If the chain of is the of revisions will be its our for justifying the age of a calibration point is that the of from paleontological studies should reference or published that ages (e.g., and et al. et al. 2004; 2010; and on are constantly and can become these it for to A of this step in the protocol the of the age from the geological a minimum age the age of the fossil should be used the of the relevant time than the of a in the a fossil the of the to it is the age from an will the minimum from the age of it is to that the minimum age is one a and is to not on its the age of the minimum age should the age of the fossil the error associated with the geochronologic age Tuinen et al. 2004; Donoghue and Benton 2007; Benton and Donoghue 2007; Benton et al. age should be as a The assigning based on the age of the fossil has been (e.g., van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007). may to use in of or but The of a minimum age that the estimates for a fossil should be The justification for the might to a but paleontological data are established and that to introduce error into the analysis. In cases, of or the age of a fossil may not be a stratigraphic in cases, it is to much more and dates than are given a stratigraphic data may not be available in the the fossil specimens used for calibrations, and so it is necessary to evidence from fossil may not be to those data more than molecular but the specimen and ages in a may the the of dates In to the of the specimen-based we that about the of that or Such of calibrations in (e.g., Benton and Donoghue 2007; et al. 2007; Benton et al. it for to the justification the relevant and We should that through and analysis that the calibrations be or In order to the evolution of justifications, we that (or of should become a of calibration The justification of the phylogenetic position and age of a fossil is an first step to a in a divergence dating analysis. In to can be assigned time may not have this step the data from the fossil the minimum bound of a calibration The maximum bound and the of the are also based on the fossil but in a much more probability of the oldest known The of these other parameters from a protocol for them. the maximum is established as than all the oldest to a time the and for the of the are but no are the maximum an approach that into and phylogenetic has been proposed (e.g., Reisz and Müller Müller and Reisz 2005; Benton and Donoghue 2007; Donoghue and Benton 2007; Benton et al. approach is and from the fossil established Marshall who use this approach should provide justifying their so that can and, the of Benton and Donoghue and and the maximum should be and Most studies use a Bayesian framework for estimating divergence dates with probability curves between minimum and maximum In may be of the fossil but there is no way to parameters and and of little more than A of recent studies that these parameters are not et al. The of these are et al. 2010; et al. and et al. the that a is to that have a on results et al. et al. is a of molecular divergence dating The of methods for estimating maximum and probability curves should be a (see In order to the of our specimen-based we apply it to used calibrations in the vertebrate of the tree of the and the from A of the the of the paleontological data for these The of the specimen-based protocol to these results in new We also provide examples of our calibration as as maximum the the approach of Benton and Donoghue of ages for point calibrations, The data for this can be in The of Paleontology and holotype of et al. that the of (in the clade a of it in the clade also et al. a previously proposed calibration point for et al. can be in it the processes of the at the for the is the and the of the of the an is it a of the that is to the of the the of the the a is present at the of the of the for of of into the are on the and it the of of the et al. 2011). The of et al. is with molecular of that a (e.g., and 1998; Hedges and and et al. et al. 2005; et al. 2007; et al. 2009; et al. et al. et al. molecular data have for a clade and and et al. et al. or a clade and for these an where are the taxon to a is et al. et 2005; et al. The majority of recent molecular a et al. 2005; et al. 2007; et al. 2009; et al. 2009; et al. and of as or not the oldest calibration point or the phylogenetic of of the vertebrate fossils that are to be to other in and 2005; et al. is relevant is that the an and that is in age and and to other and 2007; and the also the taxon and a taxon in throughout et al. and 2005; and The data that these the and The is with ages, so the of the is to et al. The and is one of the fundamental calibrations for vertebrate studies (e.g., et al. 2007; et al. it as an calibration for both and molecular (e.g., et al. 2002; and 2006; 2007; and 2010) and is relevant to in (Benton and Donoghue 2007). used a secondary calibration for this has been and Martin 2004; Müller and Reisz on the to have not been used for calibration the earliest fossils from the and Reisz 2005; Benton and Donoghue 2007). Müller and Reisz proposed an age of for this based on the of the the of the is using recent age data et al. is phylogenetically as a of the (e.g., and 2006; 2007; and 2007; et al. 2011). its age is is from the of the in this has been to the using vertebrate and and this is with et al. and age data and Unfortunately, vertebrate is in (e.g., et al. 2009; Irmis et al. so the age of is not an age for this it a minimum age of divergence for the of et al. studies have this calibration (e.g., et al. Benton and Donoghue proposed an age of for this based on the of the from the of is a for the it has been in a phylogenetic and there is evidence that it is a of phylogenetically the and et al. from the of a that is the oldest of the of phylogenetically this fossil is no than also problems in the age of is vertebrate (see et al. et al. from the but these the same problems as other (see and are no than from the of was first as a of and with this phylogenetic of the specimen et al. that it to the age of a minimum for the for the maximum age for is difficult recent fossil discoveries have the age of divergence for this fossil evidence that the earliest based on the that a of have to the et al. but this not the that of will be to have a the of the with present in and et al. 2011). this (e.g., the the with We that the age of the oldest of the but to the a for a maximum it is the oldest fossil and all fossils. is clearly as a and and is to the and The is ages to that we do not of the is we that a maximum for be studies inappropriate calibrations that have error into divergence dating (e.g., Graur and Martin 2004; Gandolfo et al. 2008; Ksepka 2009; Sanders et al. In order to the of the specimen-based protocol can identify inappropriate calibrations, we examples and In the the published minimum age cannot be with specimen-based evidence so we a much minimum age in the the published minimum age cannot be with specimen evidence. In we cannot identify specimen that will all the of the protocol for that and so that future do not is not that these be but then it not be the fossil data calibrations is clearly to data into analysis. The checklist is an first step to other incorrect calibrations and more reliable time priors. calibrations are more to be of will molecular divergence dates more and provide in A specimen-based protocol will attention on between the fossil record and published calibration making it for to identify and correct and refine calibrations as new data to the reporting of data in (e.g., our is a crucial first In to providing this we identify as the for more methods for selecting parameters of time maximum and probability and the associated with from In both cases, can be in our checklist protocol will help identify the oldest fossil of a that can a time with an minimum fossils the time of the represent (e.g., Marshall Benton and Benton and Donoghue 2007). The probability of the oldest fossil is the other Bayesian calibration parameters to these parameters estimates of that to the of a et al. for of fossil and to provide of in the fossil The amount of to rigorous paleontological for is To studies have of fossil with of record at small taxonomic or Benton et al. 2004; and 2007; and for the of time priors. approach was to Bayesian and 2010; and 2010) on dates that be as Bayesian for divergence dating but we do not of studies that have this of time based on the temporal of and then used as time for divergence dating et al. 2011). The and of these and other methods to time parameters should be a for the divergence dating the of will be the of relevant data. genetic sequences is not the but the will more from in order to and the data from the fossil record. A the problems we address is the associated with from the first step of the specimen-based specimen and justifying is a for a molecular a for their Such challenges can be through or a study that has the are not but also introduce and these data be of time or at more The step is to that the and of paleontological calibration data to the way that molecular sequence data are on The is an for this as the based on Benton and Donoghue We can a of that et al. that is to other of biological data as the and the of We encourage and to a more providing data that the in and to to provide these data to their have to calibration data for divergence If paleontological data can be to their position in this it will result in more and the to to be explicitly with molecular will encourage the of phylogenetic for of data and for future (e.g., differences in rates of and molecular evolution, between and The recommendations to explicitly ages will and with The for more to maximum dates should the of methods for the fossil record. on the fossils will all the of to the we can a new community of to develop a more and rigorous approach to the study of evolution and the of life. can be in the data was the and of the of the of and a and The and and from the is the to the was through an from the of and are for their and was at a in in The of at the of is for this of with and the fossil record. We for to use the of from the for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.