Seminar in Plant Systematics and Evolution
Seminar in Plant Systematics and Evolution BOTANY 940
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How Not to Detect Design A review of William A Dembski s The Design Inference Eliminating Chance Through Small Probabilities Cambridge Cambridge University Press 1998 xvii 243 pg ISBN 0521623871 Branden Fitelson Christopher Stephens Elliott SoberTi Department of Philosophy University of Wisconsin Madison As every philosopher knows the design argument concludes that God exists from premisses that cite the adaptive complexity of organisms or the lawfulness and orderliness of the whole universe Since 1859 it has formed the intellectual heart of creationist opposition to the Darwinian hypothesis that organisms evolved their adaptive features by the mindless process of natural selection Although the design argument developed as a defense of theism the logic of the argument in fact encompasses a larger set of issues William Paley saw clearly that we sometimes have an excellent reason to postulate the existence of an intelligent designer Ifwe find a watch on the heath we reasonably infer that it was produced by an intelligent watchmaker This design argument makes perfect sense Why is it any different to claim that the eye was produced by an intelligent designer Both critics and defenders of the design argument need to understand what the ground rules are for inferring that an intelligent designer is the unseen cause of an observed effect Dembski s book is an attempt to clarify these ground rules He proposes a procedure for detecting design and discusses how it applies to a number of mundane and nontheological examples which more or less resemble Paley s watch Although the book takes no stand on whether creationism is more or less plausible than evolutionary theory Dembski s epistemology can be evaluated without knowing how he thinks it bears on this highly charged topic In what follows we will show that Dembski s account of design inference is deeply awed Sometimes he is too hard on hypotheses of intelligent design at other times he is too lenient Neither creationists nor evolutionists nor people who are trying to detect design in nontheological contexts should adopt Dembski s framework The Explanatory Filter Dembski s book provides a series of representations of how design inference works The exposition starts simple and grows increasingly complex However the basic pattem of analysis can be summarized as follows Dembski proposes an explanatory filter 37 which is a procedure for deciding how best to explain an observation E 1 There are three possible explanations of E Regularity Chance and Design They are mutually exclusive and collectively exhaustive The problem is to decide which of these explanations to accept 2 The Regularity hypothesis is more parsimonious than Chance and Chance is more parsimonious than Design To evaluate these alternatives begin with the most parsimonious possibility and move down the list until you reach an explanation you can accept 3 If E has a high probability you should accept Regularity otherwise reject Regularity and move down the list 4 If the Chance hypothesis assigns E a suf ciently low probability and E is speci ed then reject Chance and move down the list otherwise accept Chance 5 If you have rejected Regularity and Chance then you should accept Design as the explanation of E The entire book is an elaboration of the ideas that comprise the Explanatory Filter1 Notice that the lter is eliminativist with the Design hypothesis occupying a special position We have interpreted the Filter as sometimes recommending that you should accept Regularity or Chance This is supported for example by Dembski s remark 3 8 that if E happens to be an HP a high probability event we stop and attribute E to a regularity However some of the circumlocutions that Dembski uses suggest that he doesn39t think you should ever accept Regularity or Chance2 The most you should do is not reject them Under this alternative interpretation Dembski is saying that if you fail to reject Regularity you can believe any of the three hypotheses or remain agnostic about all three And if you reject Regularity but fail to reject Chance you can believe either Chance or Design or remain agnostic about them both Only if you have rejected Regularity and Chance must you accept one of the three namely Design Construed in this way a person who believes that every event is the result of Design has nothing to fear from the Explanatory Filter no evidence can ever dislodge that opinion This may be Dembski39s view but for the sake of charity we have described the Filter in terms of rejection and acceptance The Caputo Example Before discussing the lter in detail we want to describe Dembski s treatment of one of the main examples that he uses to motivate his analysis 919 162166 This is the case of Nicholas Caputo who was a member of the Democratic party in New Jersey Caputo s job was to determine whether Democrats or Republicans would be listed rst on the ballot The party listed rst in an election has an edge and this was common knowledge in Caputo s day Caputo had this job for 41 years and he was supposed to do it fairly Yet in 40 out of 41 elections he listed the Democrats rst Caputo claimed that each year he determined the order by drawing from an urn that gave Democrats and Republicans the same chance of winning In spite of his protestations Caputo was brought up on charges and the judges found against him They rejected his claim that the outcome was due to chance and were persuaded that he had rigged the results The ordering of names on the ballots was due to Caputo s intelligent design In this story the hypotheses of Chance and Intelligent Design are prominent But what of the first alternative that of Regularity Dembski l 1 says that this can be rejected because our background knowledge tells us that Caputo probably didn t innocently use a biased process For example we can rule out the possibility that Caputo with the most honest of intentions spun a roulette wheel in which 00 was labeled Republican and all the other numbers were labeled Democrat Apparently we know before we examine Caputo s 41 decisions that there are just two possibilities he did the equivalent of tossing a fair coin Chance or he intentionally gave the edge to his own party Design There is a straightforward reason for thinking that the observed outcomes favor Design over Chance If Caputo had allowed his political allegiance to guide his arrangement of ballots you d expect Democrats to be listed first on all or almost all of the ballots However if Caputo did the equivalent of tossing a fair coin the outcome he obtained would be very surprising This simple analysis also can be used to represent Paley s argument about the watch Sober 1993 The key concept is quot quot The quot39 quot39 J ofa 1 ti i is the probability it confers on the observations it is not the probability that the observations confer on the hypothesis The likelihood of H relative to E is PrE l H not PrH l E Chance and Design can be evaluated by comparing their likelihoods relative to the same set of observations We do not claim that likelihood is the whole story but surely it is relevant The reader will notice that the Filter does not use this simple likelihood analysis to help decide between Chance and Design The likelihood of Chance is considered but the likelihood of Design never is Instead the Chance hypothesis is evaluated for properties additional to its likelihood Dembski thinks it is possible to reject Chance and accept Design without asking what Design predicts Whether the Filter succeeds in showing that this possible is something we ll have to determine The Three Alternative Explanations Dembski defines the Regularity hypothesis in dilTerent ways Sometimes it is said to assert that the evidence E is noncontingent and is reducible to law 39 53 at other times it is taken to claim that E is a deterministic consequence of earlier conditions 65 l46n5 and at still other times it is supposed to say that E was highly probable given some earlier state of the world 38 The Chance Hypothesis is taken to assign to E a lower probability than the Regularity Hypothesis assigns 40 The Design Hypothesis is said to be the complement of the first two alternatives As a matter of stipulation the three hypotheses are mutually exclusive and collectively exhaustive 36 Dembski emphasizes that design need not involve intelligent agency 89 36 60 228 229 He regards design as a mark of intelligent agency intelligent agency can produce design but he seems to think that there could be other causes as well On the other hand Dembski says that the explanatory lter pinpoints how we recognize intelligent agency 66 and his section 24 is devoted to showing that design is reliably correlated with intelligent agency Dembski needs to supply an account of what he means by design and how it can be caused by something other than intelligent agency3 His vague remark 228229 that design is equivalent to information is not enough Dembski quotes Dretske 1981 with approval as deploying the concept of information that the design hypothesis uses However Dretske s notion of information is as Dembski points out the ShannonWeaver account which describes a probabilistic dependency between two events labeled source and receiver Hypotheses of mindless chance can be stated in terms of the ShannonWeaver concept Dembski 39 also says that the design hypothesis isn39t characterized by probability Understanding what regularity chance and design mean in Dembski s framework is made more difficult by some of his examples Dembski discusses ateacher who finds that the essays submitted by two students are nearly identical 46 One hypothesis is that the students produced their work independently a second hypothesis asserts that there was plagiarism Dembski treats the hypothesis of independent origination as a Chance hypothesis and the plagiarism hypothesis as an instance of Design Yet both describe the matching papers as issuing from intelligent agency as Dembski points out 47 Dembski says that context in uences how a hypothesis gets classified 46 How context induces the classification that Dembski suggests remains a mystery The same sort of interpretive problem attaches to Dembski s discussion of the Caputo example We think that all of the following hypotheses appeal to intelligent agency i Caputo decided to spin a roulette wheel on which 00 was labeled Republican and the other numbers were labeled Democrat ii Caputo decided to toss a fair coin iii Caputo decided to favor his own party Since all three hypotheses describe the ballot ordering as issuing from intelligent agency all apparently are instances of Design in Dembski s sense However Dembski says that they are examples respectively of Regularity Chance and Design The Parsimony Ordering Dembski says that Regularity is a more parsimonious hypothesis than Chance and that Chance is more parsimonious than Design 3 839 He defends this ordering as follows Note that explanations that appeal to regularity are indeed simplest for they admit no contingency claiming things always happen that way Explanations that appeal to chance add a level of complication for they admit contingency but one characterized by probability Most complicated are those explanations that appeal to design for they admit contingency but not one characterized by probability 39 Here Dembski seems to interpret Regularity to mean that E is nomologically necessary or that E is a deterministic consequence of initial conditions Still why does this show that Regularity is simpler than Chance And why is Chance simpler than Design Even if design hypotheses were not characterized by probability why would that count as a reason But in fact design hypotheses do in many instances confer probabilities on the observations The ordering of Democrats and Republicans on the ballots is highly probable given the hypothesis that Caputo rigged the ballots to favor his own party Dembski supplements this general argument for his parsimony ordering with two examples 39 Even if these examples were convincing 4 they would not establish the general point about the parsimony ordering It may be possible to replace Dembski s faulty argument for his parsimony ordering with a different argument that comes close to delivering what he wants Perhaps determinism can be shown to be more parsimonious than indeterminism Sober 1999a and perhaps explanations that appeal to mindless processes can be shown to be simpler than explanations that appeal to intelligent agency Sober 1998 But even if this can be done it is important to understand what this parsimony ordering means When scientists choose between competing curves the simplicity of the competitors matters but so does their fittodata You don t reject a simple curve and adopt a complex curve just by seeing how the simple curve fits the data and without asking how well the complex curve does so You need to ask how well both hypotheses fit the data Fitto data is important in curvefitting because it is a measure of likelihood curves that are closer to the data confer on the data a higher probability than curves that are more distant Dembski s parsimony ordering even if correct makes it puzzling why the Filter treats the likelihood of the Chance hypothesis as relevant but ignores the likelihoods of Regularity and Design Why Regularity is Rejected As just noted the Explanatory Filter evaluates Regularity and Chance in different ways The Chance hypothesis is evaluated in part by asking how probable it says the observations are However Regularity is not evaluated by asking how probable it says the observations are The filter starts with the question Is E a high probability event 3 8 This doesn t mean is E a high probability event according to the Regularity hypothesis Rather you evaluate the probability of E on its own Presumably if you observe that events like E occur frequently you should say that E has a high probability and so should conclude that E is due to Regularity If events like E rarely occur you should reject Regularity and move down the list5 However since a given event can be described in many ways any event can be made to appear common and any can be made to appear rare Dembski s procedure for evaluating Regularity hypotheses would make no sense if it were intended to apply to speci c hypotheses of that kind After all specific Regularity hypotheses e g Newtonian mechanics are often confirmed by events that happen rarely the return of a comet for example And specific Regularity hypotheses are often disconfirmed by events that happen frequently This suggests that what gets evaluated under the heading of Regularity are not specific hypotheses of that kind but the general claim that E is due to some regularity or other Understood in this way it makes more sense why the likelihood of the Regularity hypothesis plays no role in the Explanatory Filter The claim that E is due to some regularity or other by de nition says that E was highly probable given antecedent conditions It is important to recognize that the Explanatory Filter is enormously ambitious You don t just reject a given Regularity hypothesis you reject all possible Regularity explanations 53 And the same goes for Chance you reject the whole category the Filter sweeps the eld clear of all speci c Chance hypotheses 41 5253 We doubt that there is any general inferential procedure that can do what Dembski thinks the Filter accomplishes Of course you presumably can accept E is due to some regularity or other if you accept a speci c regularity hypothesis But suppose you have tested and rejected the various speci c regularity hypotheses that your background beliefs suggest Are you obliged to reject the claim that there exists a regularity hypothesis that explains E Surely it is clear that this does not follow The fact that the Filter allows you to accept or reject Regularity without attending to what speci c Regularity hypotheses predict has some peculiar consequences Suppose you have in mind just one speci c regularity hypothesis that is a candidate for explaining E you think that if E has a regularitystyle explanation this has got to be it If E is a rare type of event the Filter says to conclude that E is not due to Regularity This can happen even if the speci c hypothesis when conjoined with initial condition statements predicts E with perfect precision Symmetrically if E is a common kind of event the Filter says not to reject Regularity even if your lone speci c Regularity hypothesis deductively entails that E is false The Filter is too hard on Regularity and too lenient The Speci cation Condition To reject Chance the evidence E must be speci ed This involves four conditions CINDE TRACT DELIM and the description D that you use to delimit E must have a low probability on the Chance hypothesis We consider these in turn CINDE Dembski says several times that you can t reject a Chance hypothesis just because it says that what you observe was improbable If Jones wins a lottery you can t automatically conclude that there is something wrong with the hypothesis that the lottery was fair and that Jones bought just one of the 10000 tickets sold To reject Chance further conditions must be satis ed CINDE is one of them CINDE means conditional independence This is the requirement that PrEl H amp I PrE l H where H is the Chance hypothesis E is the observations and I is your background knowledge H must render E conditionally independent of I CINDE requires that H capture everything that your background beliefs say is probabilistically relevant to the occurrence of E CINDE is too lenient on Chance hypotheses it says that their violating CINDE suffices for them to be accepted or not rejected Suppose you want to explain why Smith has lung cancer E It is part of your background knowledge I that he smoked cigarettes for thirty years but you are considering the hypothesis H that Smith read the works of Ayn Rand and that this helped bring about his illness To investigate this question you do a statistical study and discover that smokers who read Rand have the same chance of lung cancer as smokers who do not This study allows you to draw a conclusion about Smith that PrE l HampI PrE l notH ampI Surely this equality is evidence against the claim that E is due to H However the lter says that you can t reject the causal claim because CINDE is false PrE l HampI PrE l H6 T RA CT and DELIM The ideas examined so far in the Filter are probabilistic The TRACT condition introduces concepts from a different branch of mathematics 7 the theory of computational complexity TRACT means tractability 7 to reject the Chance hypothesis it must be possible for you to use your background information to formulate a description D of features of the observations E To construct this description you needn t have any reason to think that it might be true For example you could satisfy TRACT by obtaining the description of E by brute force ithat is by producing descriptions of all the possible outcomes one of which happens to cover E 150 15 1 Whether you can produce a description depends on the language and computational framework used For example the evidence in the Caputo example can be thought of as a specific sequence of 40 Ds and 1 R TRACT would be satisfied if you have the ability to generate all ofthe following descriptions 0 Rs and 41 Ds 1 R and 40 Ds 2 Rs and 39 Ds 41 Rs and 0 Ds Whether you can produce these descriptions depends on the character of the language you use does it contain those symbols or others with the same meaning and on the computational procedures you use to generate descriptions does generating those descriptions require a small number of steps or too many for you to perform in your lifetime Because tractability depends on your choice of language and computational procedures we think that TRACT has no evidential significance at all Caputo s 41 decisions count against the hypothesis that he used a fair coin and in favor of the hypothesis that he cheated for reasons that have nothing to do with TRACT The relevant point is simply that PrEl Chance ltlt PrE 1 Design This fact is not relative to the choice of language or computational framework The DELIM condition as far as we can see adds nothing to TRACT A description D generated by one s background information delimits the evidence E just in case E entails D In the Caputo case TRACT and DELIM would be satisfied if you were able to write down all possible sequences of D s and R s that are 41 letters long They also would be satisfied by generating a series of weaker descriptions like the one just mentioned In fact just writing down a tautology satis es TRACT and DELIM 165 On the assumption that human beings are able to write down tautologies we conclude that these two conditions are always satis ed and so play no substantive role in the Filter D0 CINDE TRACT and DELIM Call the Chance Hypothesis into Question Dembski argues that CINDE TRACT and DELIM if true call the chance hypothesis H into question We quote his argument in its entirety The interrelation between CINDE and TRACT is important Because I is conditionally independent of E given H any knowledge S has about I ought to give S no knowledge about E so long as and this is the crucial assumption E occurred according to the chance hypothesis H Hence any pattern formulated on the basis of I ought not give S any knowledge about E either Yet the fact that it does in case D delimits E means that I is a er all giving S knowledge about E The assumption that E occurred according to the chance hypothesis H though not quite refuted is therefore called into question 147 Dembski then adds To actually refute this assumption and thereby eliminate chance S will have to do one more thing namely show that the probability PD l H that is the probability of the event described by the pattern D is small enough 147 We39ll address this claim about the impact of low probability later To reconstruct Dembski s argument we need to clarify how he understands the conjunction TRACT amp DELIM Dembski says that when TRACT and DELIM are satisfied your background beliefs I provide you with knowledge or information about E 143 147 In fact TRACT and DELIM have nothing to do with informational relevance understood as an evidential concept When I provides information about E it is natural to think that PrE l I PrE I provides information because taking it into account changes the probability you assign to E It is easy to see how TRACT amp DELIM can both be satisfied by brute force without this evidential condition s being satisfied Suppose you have no idea how Caputo might have obtained his sequence of D39s and Rs still you are able to generate the sequence of descriptions we mentioned before The fact that you can generate a description which delimits or even matches E does not ensure that your background knowledge provides evidence as to whether E will occur As noted generating a tautology satisfies both TRACT and DELIM but tautologies don t provide information about E Even though the conjunction TRACT amp DELIM should not be understood evidentially ie as asserting that PrE l I PrE we think this is how Dembski understands TRACT amp DELIM in the argument quoted This suggests the following reconstruction of Dembski39s argument 1 CINDE TRACT and DELIM are true of the chance hypothesis H and the agent S 2 If CINDE is true and S is warranted in accepting H ie that E is due to chance then S should assign PrE l I PrE 3 If TRACT and DELIM are true then S should not assign PrE l I PrE 4 Therefore S is not warranted in accepting H Thus reconstructed Dembski s argument is valid We grant premiss l for the sake of argument We39ve already explained why 3 is false So is premiss 2 it seems to rely on something like the following principle If S should assign PrElHampI p and S is warranted in accepting H then S should assign PrElI p If were true 2 would be true However is false For entails If S should assign PrHlH 10 and S is warranted in accepting H then S should assign PrH 10 Justifiably accepting H does not justify assigning H a probability of unity Bayesians warn against assigning probabilities of l and 0 to any proposition that you might want to consider revising later Dembski emphasizes that the Chance hypothesis is always subject to revision It is worth noting that a weaker version of 2 is true 2 If CINDE is true and S should assign PrHl then S should assign PrE l I PrE One then can reasonably conclude that 4 S should not assign PrH l However a fancy argument isn t needed to show that 4 is true Moreover the fact that 4 is true does nothing to undermine S39s confidence that the Chance hypothesis H is the true explanation of E provided that S has not stumbled into the brash conclusion that H is entirely certain We conclude that Dembski39s argument fails to call H into question It may be objected that our criticism of Dembski39s argument depends on our taking the conjunction TRACT amp DELIM to have probabilistic consequences We reply that this is a charitable reading of his argument If the conjunction does not have probabilistic consequences then the argument is a nonstarter How can purely nonprobabilistic conditions come into con ict with a purely probabilistic condition like CINDE Moreover since TRACT and DELIM sensu strictu are always true if the agent39s side information allows himher to generate a tautology how could these trivially satis ed conditions when coupled with CINDE possibly show that H is questionable The Improbability Threshold The Filter says that PrE l Chance must be sufficiently low if Chance is to be rejected How low is low enough Dembski s answer is that PrEn l Chance lt 12 where n is the number of times in the history of the universe that an event of kind E actually occurs 209 214 217 As mentioned earlier if Jones wins a lottery it does not follow that we should reject the hypothesis that the lottery was fair and that he bought just one of the 10000 tickets sold Dembski thinks the reason this is so is that lots of other lotteries have occurred If p is the probability of Jones winning the lottery if it is fair and he bought one of the 100000 tickets sold and if there are n such lotteries that ever occur then the relevant probability to consider is PrEn l Chance l lpquot If n is large enough this quantity can be greater than 12 even though p is very small As long as the probability exceeds 12 that Smith wins lottery L2 or Quackdoodle wins lottery L3 or or Snerdley wins lottery Ln given the hypothesis that each of these lotteries was fair and the individuals named each bought one of the 10000 tickets sold we shouldn t reject the Chance hypothesis about Jones Why is 12 the relevant threshold Dembski thinks this follows from the Likelihood Principle 190198 As noted earlier that principle states that if two hypotheses confer different probabilities on the same observations the one that entails the higher probability is the one that is better supported by those observations Dembski thinks this principle solves the following prediction problem If the Chance hypothesis predicts that either F or notF will be true but says that the latter is more probable then if you believe the Chance hypothesis and must predict whether F or notF will be true you should predict notF We agree that if a gun were put to your head that you should predict the option that the Chance hypothesis says is more probable if you believe the Chance hypothesis and this exhausts what you know that is relevant However this doesn t follow from the likelihood principle The likelihood principle tells you how to evaluate different hypotheses by seeing what probabilities they confer on the observations Dembski s prediction principle describes how you should choose between two predictions not on the basis of observations but on the basis of a theory you already accept the theory says that one prediction is more probable not that it is more likely Even though Dembski s prediction principle is right it does not entail that you should reject Chance if PrEn l Chance lt 12 and the other specification conditions are satis ed Dembski thinks that you face a probabilistic inconsistency 196 if you believe the Chance hypothesis and the Chance hypothesis leads you to predict notF rather than F but you then discover that E is true and that E is an instance of F However there is no inconsistency here of any kind Perfectly sensible hypotheses sometimes entail that notF is more probable than F they can remain perfectly sensible even if F has the audacity to occur An additional reason to think that there is no probabilistic inconsistency here is that H and notH can both confer an arbitrarily low probability on E In such cases Dembski must say that you are caught in a quotprobabilistic inconsistencyquot no matter what you accept Suppose you know that an urn contains either 10 green balls or 1 green balls perhaps you saw the urn being filled from one of two buckets you don t know which whose contents you examined Suppose you draw 10 balls from the um and find that 7 are green From a likelihood point of view the evidence favors the 10 hypothesis However Dembski would point out that the 10 hypothesis predicted that most of the balls in your sample would fail to be green Your observation contradicts this prediction Are you therefore forced to reject the 10 hypothesis If so you are forced to reject the 1 hypothesis on the same grounds But you know that one or the other hypothesis is true Dembski s talk of a probabilistic inconsistency suggests that he thinks that improbable events can t really occur a true theory would never lead you to make probabilistic predictions that fail to come true Dembski s criterion is simultaneously too hard on the Chance hypothesis and too lenient Suppose there is just one lottery in the whole history of the universe Then the Filter says you should reject the hypothesis that Jones bought one of 10000 tickets in a fair lottery just on the basis of observing that Jones won assuming that CINDE and the other conditions are satisfied But surely this is too strong a conclusion Shouldn t your acceptance or rejection of the Chance hypothesis depend on what alternative hypotheses you have available Why can t you continue to think that the lottery was fair when Jones wins it The fact that there is just one lottery in the history of the universe hardly seems relevant Dembski is too hard on Chance in this case To see that he also is too lenient let s assume that there have been many lotteries so that PrEn l Chance gt 12 The Filter now requires that you not reject Chance even if you have reason to consider seriously the Design hypothesis that the lottery was rigged by Jones cousin Nicholas Caputo We think you should embrace Design in this case but the Filter disagrees The aw in the Filter s handling of both these examples traces to the same source Dembski evaluates the Chance hypothesis without considering the likelihood of Design We have another objection to Dembski s answer to the question of how low PrEn l Chance must be to reject Chance How is one to decide which actual events count as the same with respect to what the Chance hypothesis asserts about E Consider again the case of Jones and his lottery Must the other events that are relevant to calculating En be lotteries Must exactly 10000 tickets have been sold Must the winners of the other lotteries have bought just one ticket Must they have the name Jones Dembski s En has no determinate meaning Dembski supplements his threshold of PrEn l Chance lt 12 with a separate calculation 209 He provides generous estimates of the number of particles in the universe 1080 of the duration of the universe 1025 seconds and of the number of changes per second that a particle can experience 1045 From these he computes that there is a maximum of 10150 speci ed events in the whole history of the universe The reason is that there can t be more agents than particles and there can t be more acts of specifying than changes in particle state7 Dembski thinks it follows that if the Chance hypothesis assigns to any event that occurs a probability lower than l21015 that you should reject the Chance hypothesis if CINDE and the other conditions are satis ed This is a fallacious inference The fact that there are no more than 10150 acts of specifying in the whole history of the universe tells you nothing about what the probabilities of those speci ed events are or should be thought to be Even if sentient creatures manage to write down only N inscriptions why can t those creatures develop a well con rmed theory that says that some actual events have probabilities that are less than l2N Conjunctive Disjunctive and Mixed Explanamla Suppose the Filter says to reject Regularity and that TRACT CINDE and the other conditions are satis ed so that accepting or rejecting the Chance hypothesis is said to depend on whether PrEn l Chance lt 12 Now suppose that the evidence E is the conjunction ElampE2ampamp Em It is possible for the conjunction to be suf ciently improbable on the Chance hypothesis that the Filter says to reject Chance but that each conjunct is suf ciently probable according to the Chance hypothesis that the Filter says that Chance should be accepted In this case the Filter concludes that Design explains the conjunction while Chance explains each conjunct For a second example suppose that E is the disjunction Elv E2 v v Em Suppose that the disjunction is suf ciently probable according to the Chance hypothesis so that the Filter says not to reject Chance but that each disjunct is suf ciently improbable that the Filter says to reject Chance The upshot is that the Filter says that each disjunct is due to Design though the disjunction is due to Chance For a third example suppose the Filter says that E1 is due to Chance and that E2 is due to Design What will the Filter conclude about the conjunction ElampE2 The Filter makes no room for mixed explanations it cannot say that the explanation of ElampE2 is simply the conjunction of the explanations of El and E2 Rejecting Chance as a Category Requires A Kind of Omniscience Although speci c chance hypotheses may confer de nite probabilities on the observations E this is not true of the generic hypothesis that E is due to some chance hypothesis or other Yet when Dembski talks of rejecting Chance he means rejecting the whole category not just the specific chance hypotheses one happens to formulate The Filter s treatment of Chance therefore applies only to agents who believe they have a complete list of the chance processes that might explain E As Dembski 41 says before we even begin to send E through the Explanatory Filter we need to know what probability distributions if any were operating to produce the event Dembski s epistemology never tells you to reject Chance if you do not believe you have considered all possible chance explanations Here Dembski is much too hard on Design Paley reasonably concluded that the watch he found is better explained by postulating a watchmaker than by the hypothesis of random physical processes This conclusion makes sense even if Paley admits his lack of omniscience about possible Chance hypotheses but it does not make sense according to the Filter What Paley did was compare a specific chance hypothesis and a specific design hypothesis without pretending that he thereby surveyed all possible chance hypotheses For this reason as well as for others we have mentioned friends of Design should shun the Filter not embrace it Concluding Comments We mentioned at the outset that Dembski does not say in his book how he thinks his epistemology resolves the debate between evolutionary theory and creationism8 Still it is abundantly clear that the overall shape of his epistemology re ects the main pattern of argument used in the intelligent design movement Accordingly it is no surprise that a leading member of this movement has praised Dembski s epistemology for clarifying the logic of design inference Behe 1996 pp 285286 Creationists frequently think they can establish the plausibility of what they believe merely by criticizing the alternatives Behe 1996 Plantinga 1993 1994 Phillip Johnson as quoted in Stafford 1997 p 22 This would make sense if two conditions were satisfied If those alternative theories had deductive consequences about what we observe one could demonstrate that those theories are false by showing that the predictions they entail are false If in addition the hypothesis of intelligent design were the only alternative to the theories thus refuted one could conclude that the design hypothesis is correct However neither condition obtains Darwinian theory makes probabilistic not deductive predictions And there is no reason to think that the only alternative to Darwinian theory is intelligent design When prediction is probabilistic a theory cannot be accepted or rejected just by seeing what it predicts Royall 1997 ch 3 The best you can do is compare theories with each other To test evolutionary theory against the hypothesis of intelligent design you must know what both hypotheses predict about observables Fitelson and Sober 1998 Sober 1999b The searchlight therefore must be focused on the design hypothesis itself What does it predict If defenders of the design hypothesis want their theory to be scientific they need to do the scientific work of formulating and testing the predictions that creationism makes Kitcher 1984 Pennock 1999 Dembski s Explanatory Filter encourages creationists to think that this responsibility can be evaded However the fact of the matter is that the responsibility must be faced References Behe M 1996 Darwin s BlackBox New York Free Press Dembski W 1998 Intelligent Design as a Theory of Information unpublished manuscript Reprinted electronically at the following web site httpwwwa1norgdocsdembski Dretske F 1981 Knowledge and the Flow ofInformation Cambridge MA MIT Press Fitelson B and Sober E 1998 Plantinga s Probability Arguments Against Evolutionary Naturalism Pacific Philosophical Quarterly 79 115 129 Kitcher P 1984 Abusing Science the Case Against Creationism Cambridge MA MIT Press Pennock R 1999 Tower ofBabel Cambridge MA MIT Press Plantinga A 1993 Warrant and Proper Function Oxford Oxford University Press 1994 Naturalism Defeated unpublished manuscript Royall R 1997 Statistical Evidence aLikelihood Paradigm London Chapman and Hall Sober E 1993 Philosophy ofBiology Boulder CO Westview Press 1998 Morgan s Canon In C Allen and D Cummins eds The Evolution of Mind Oxford University Press pp 224242 1999a Physicalism from a Probabilistic Point of View Philosophical Studies forthcoming 1999b Testability Proceedings and Addresses of the American Philosophical Association forthcoming Stafford T 1997 The Making of a Revolution Christianity Today December 8 pp 1622 Notes Received April 1999 revised May 1999 T Send requests for reprints to Elliott Sober Philosophy Department University of Wisconsin Madison WI 53706 i We thank William Dembski and Philip Kitcher for comments on an earlier draft 1 Dembski 48 provides a deductively valid argument form in which E is due to design is the conclusion However Dembski s nal formulation of the design inference 2213 deploys an epistemic version of the argument whose conclusion is S is warranted in inferring that E is due to design One of the premisses of this latter argument contains two layers of epistemic operators it says that if certain epistemic assumptions are true then S is warranted in asserting that S is not warranted in inferring that E did not occur according to the chance hypothesis Dembski claims 223 that this convoluted epistemic argument is valid and defends this claim by referring the reader back to the quite different nonepistemic argument presented on p 48 This establishes nothing as to the validity of the official epistemic rendition of the design inference 2 For example he says that to retain chance a subject S must simply lack warrant for inferring that E did not occur according to the chance hypothesis H 220 3 Dembski 1998 apparently abandons the claim that design can occur without intelligent agency here he says that after regularity and chance are eliminated what remains is the hypothesis of an intelligent cause 4 In the first example Dembski 39 says that Newton s hypothesis that the stability of the solar system is due to God s intervention into natural regularities is less parsimonious than Laplace s hypothesis that the stability is due solely to regularity In the second he compares the hypothesis that a pair of dice is fair with the hypothesis that each is heavily weighted towards coming up 1 He claims that the latter provides the more parsimonious explanation of why snakeeyes occurred on a single roll We agree with Dembski s simplicity ordering in the first example the example illustrates the idea that a hypothesis that postulates two causes R and G is less parsimonious than a hypothesis that postulates R alone However this is not an example of Regularity versus Design but an example of RegularityampDesign versus Regularity alone in fact it is an example of two causes versus one and the parsimony ordering has nothing to do with the fact that one of those causes involves design In Dembski s second example the hypotheses differ in likelihood relative to the data cited however if parsimony is supposed to be a different consideration from fittodata it is questionable whether these hypotheses differ in parsimony 5 Dembski incorrectly applies his own procedure to the Caputo example when he says 1 1 that the regularity hypothesis should be rejected on the grounds that background knowledge makes it improbable that Caputo in all honesty used a biased device Here Dembski is describing the probability of Regularity not the probability of E 6 Strictly speaking CINDE requires that PrE l HampJ PrE l J for all J such that J can be generated by the side information I 145 Without going into details about what Dembski means by generating we note that this formulation of CINDE is logically stronger than the one discussed above This entails that it is even harder to reject chance hypotheses than we suggest in our cancer example 7 Note the materialistic character of Dembski s assumption here 8 Dembski has been more forthcoming about his views in other manuscripts The interested reader should consult the following web site httpwwwamorgdocsdembski Cryptic invasion by a nonnative genotype of the common reed Phragmites australis into North America Kristin Saltonstall Department of Ecology and Evolutionary Biology Yale University P O Box 208106 New Haven CT 0652078106 Edited by Barbara A Schaal Washington University St Louis MO and approved December 6 2001 received for review September 10 2001 Cryptic invasions are a largely unrecognized type of biological invasion that lead to underestimation of the total numbers and impacts of invaders because ofthe difficulty in detecting them The distribution and abundance of Phragmitesaustralis in North Amer ica has increased dramatically overthe past 1 50 years This research tests the hypothesis that a nonnative strain of Phragmites is responsible for the observed spread Two noncoding chloroplast DNA regions were sequenced for samples collected worldwide throughout the range of Phragmites Modern North American populations were compared with historical ones from herbarium collections Results indicate that an introduction has occurred and L i rLJri J W ii r44 to regions previously not known to have Phragmites Native types apparently have disappeared from New England and while still present may be threatened in other parts of North America iological invasions threaten species and ecosystems world wide An estimated 50000 exotic species have been introduced to the United States ofwhich 5000 are plant species that have escaped and now exist in natural environments Both the actual number of invaders and the impacts of these species may be underestimated because of the presence of cryptic invaders or species that cannot be easily classified as such invasions in marine ecosystems 476 and demonstrated both genetic and physiological differences be tween invading and native populations Given that cryptic invaders typically are unreco 39 d or are mistaken for native species knowledge of historical trends in geographic distribution and population genetic structure in cases of suspected introduc tions are of particular interest when trying to reconstruct the invasion history of a species In such cases museum or herbarium specimens are an invaluable resource for reconstructing popu lation history reed Phragmites australis Cav Trin ex Steudel hereafter referred to as Phragmites has a cosmopolitan dis tribution an is a undant in marsh communities and along the borders of lakes ponds and rivers It is a perennial grass that reproduces primarily through vegetative growth although dis persal by seeds may occur at low frequencies In North America the fossil record indicates that it has been present in the southwestern United States for at least 40000 years Paleo ecological investigations have shown it to have been present along both the Atlantic and Pacific coasts for several thousand years 8710 However over the last 150 years its distribution and relative abundance has increased dramatically particularly along the Atlantic coast Botanical records from the 1800s typically describe Phragmites as being rare or not common 11714 and a historical gap in its distribution was found in the southeastern states 15 By the early 1900s the species was considered more common and spreading 16 17 Today it exists in all of the mainland United States as well as throughout southem Canada and is considered an indicator of wetland disturbance It is also expanding into undisturbed sites particularly in inland areas To vwvw pnas orgcgidoi101073pnas 032477999 explain the spread of Phragmites it has been suggested that the rapid expansion could be the result of human activities causing habitat disturbances or stresses such as pollution changes in hydrologic regimes and increased soil salinity 18 Alterna tively nonnative genotypes of the species may have been introduced to North America sometime during the past 200 years 19721 although to date no studies have adequately supported this hypothesis This research asks the question of whether or not nonnative strains ofP australis exist in North America by using sequenc ing of two chloroplast DNA markers Although the rate of evolution of the chloroplast genome is relatively conservative variation has been found in chloroplast DNA at the intraspe cific level 22 It is maternally inherited in angiosperms and has been shown to be geographically structured in a diverse array of plant species 23 24 and therefore is an effective marker for use in the study of intraspecific phylogeography In this study modern samples ofPhrtzgmites collected across the continent were compared with historical specimens collected before 1910 to examine changes in the genetic structure ofthe North American population over the past 150 years Modern samples were also obtained worldwide for comparison and to determine the source of the introduction Materials and Methods Leaf tissues were collected from green Phragmites plants during the growing seasons of 199772001 by the author and collabora tors worldwide with a particular emphasis on obtaining samples from the presentday range of Phragmites in North America and Europe When available herbarium specimens also were ob tained to increase the number of samples from locations outside of North America Fresh specimens were dried by using silica gel and frozen on receipt in the laboratory Total DNA was ex tracted from 2 cm2 of fresh or dry leaf tissues using a 2 cetyltrimethylammonium bromide extraction protocol 25 Herbarium specimens were pretreated by scrubbing with 10 bleach to remove mounting glue placed under an UV light for 5 min to remove surface contaminants and extracted by using the same protocol All herbarium samples used in historical comparisons were collected before 1910 which is before the time period when references to expansion of Phragmites populations began to appear in the literature 16 Where possible modern samples also were collected at sites from which herbarium samples were obtained Sample collection locations herbarium accession numbers and haplotype designations for each sample are available on request This paper was supmmed directly Track II to the PNAS office Data deposition database acceSlon nus AY0163Z47AY016323 AY0163327AY016335 and AE4573827 AE457402 quotEsmail krislin sallonslall yale edu Th ph hls 39 H Jinparlbypg h a p This article must therefore be hereby marked quotadvertisementquot In accordance Wllh 18 u s c 1734 solely to Indlcale this fact PNAS l February 19 2002 l vol 99 l no 4 l 2445 2449 Q S n o n BIOLOGV networks were obtained with the software T05 29 using h algorithms of Templeton et 22 30 Haplotype diversity meae sures 31 analysis of molecular variance 32 and an exact test for population differentiation 33 were calculated by using ARLEQUIN z 000 34 All analyses were performed on the com bined data set Neumenearuapinygss haplotypes with fewer copies of the indel 2s Parsimony t e t samples er a igne the most likely h l as b on the available sequence and i in The are 26 variable characters of hi h 15 are indels four type Ia which are mononucleotide repeas Fig 1 Parslmony network ofPhragmltexmloroplasthaplotype diversity and nine type Ib which are deletions or duplications of 39 type 1 which are all other types substitutionsEleven aplotypes unique to North America haplotypes AaH s z and AA and are considered to be native to the continent These 11 are distinguished from all other haplotypes by five shared indels two type I o e two lotype represent one mutational dll fereme rolloWing odlng or lndel a of indels 35 and 11 are base lngle harader Unlabeled node lndKale lnferred step not round n the sampled populatlon Loop in the network are the result or homoplasle in the number or repeat in some lndel rne awestral haplotype orroot ortne ri a rllt u l land v Europe Leo and AwayAustralia l J L M o P Q UV and x have a Widesplead diSlllbullo 0quot multiple Co ll e ls half Aera K M R andv lotypes I and M with haplotype M being the most common type in North America Europe and Asia today Table 1 This type is most closely related to other haplotypes found in Twe quotOHCPdi g chloroplast regions were PCRramplified by Europe Asia and Africa Fig 1 It is also the predicted usmg the prlmer pans tmTUGU aquotacrnLUAA5 b 26 ancestral haplotype based on coalescent theory 36 and rbcLepscr 27 with annealing temperatures of 56 C With n orth America the exact test of population differena and 54 C respectively Smaller fragmerm were amplified in tiation indicates significant changes in aplotype frequencies the herbarium samples by using the primer pairs tmTUGU between the historical and modern samples P lt 0001 33 Fig aquotmTaR5 aTAGATTATTI TCCCAFCN I rUAA 2Thep L 39 L a widespread distribution b39ltranR 5CGGAGAAGATAGAATCATAGC VEELZF of the 11 native haplotypes across North America from New 5 aCGCAGCTTGTGAAATATGG45 L2R S LCGTATTTL England west to the Pacific coast Fig 2a Haplotype 1 which GATTCCATTATCGT and psuIZF S39VTGTCATAGAATV also is found in South America and Asia Fig 1 was distributed AGGTGTCTCepsuI2R 5 aGATTAGAAGGATAGAAe along the Gulf Coast Haplotype M was found at foursites of the AGGC whichwere designed around the variable regions found 62 sampled New Haven CT Madison CT Camden NJ in the larger fragmens Doubleastranded PCR amplifications Chesapeake Beach g 20 In comparison whereas the were sequenced directly in both directions on an Applied native haplotypes and haplotype 1 remain throughout much of t e L two internal primers in t e chme region rpL23F 5t ofexpansion in the range of haplotypeMFig2c and d This GTAGTAGCTGTGAATAGC and rpL23R S VAGTCGATe pe has replaced native s in New England and expanded to GGCTATTCACAGC In total about 2000 bp were sequenced the southeast where thgmltes historically did not grow It is ri for each modern sample an 400 bp for herba um samples presently expanding to the west and becoming prevalent in the Because of the high incidence of large insertiondeletio Midwestern states mutations indels sequean were aligned by e e with SE7 Measures of h plotype diversity show a decline in diversity QUENCHER 1 Two mononucleotide repeat regions in the tmL between the historical population and the pr t 080 004 region which showed intrahaplotype ength variation were not vs 0 58 0 04 Further analysis of molecular variance shows used when 39 ishing hapl distingu otypes Before analysis all indels that amongrpopulation variation accouns for a larger proporr were coded as single characters to treat indels 39 L M 39 L L 39 39 L 39 39 39 L rather than multiple independent evens Where indels were the modern population 79 when compared with worldwide compose o several copies of a multipleasite insertion each populations P lt 0001 indicating that today the genetic copy was treated as a single unit and gaps were inserted in m L Am ri an 39 39 Table 1 Frequencies of Phragmifes haplotypes worldwide Totalno of quot0 Regional Geograpl39m region sample haplotype quot0 Haplotype l quot0 Haplotype M North Amerla after l96o l9s 3l 3 7 2 6l 5 North Amerla before l9lo 62 33 9 9 7 6 4 South Amerla ll 27 2 72 7 0 Europe Al 39 o 0 6l 0 AxleAustralla 27 55 6 ii i 33 3 Aera 9 33 9 0 ii i l lll 2446 l vvvvarlasorggldollol073pn35032477999 Saltonstall and on several islands in the southern Pacific The distribution of this type in North America appears to be the same as the Gulf Coast phenotype identified by Pellegrin and Hauber 20 based on isozyme hese data support their suggestion that the presence ofthis type in a wide variety ofhabitats across southern North America may be the result of the establishment of a single genetic lineage with broad ecological tolerances that has spread throughout the region However because of its prevalence in other parts of the world it is not possible to assign haplotype I to a category of native or introduced to North America although its distribution remained the same between the historic and modern samples Fig 2 a and 0 Given that its mostly closely related haplotype is found only in Asia Fig 1 it is possible that haplotype I originated there but it is not known when it arrived in North America The 11 native North American types show little change in their distribution between the historic and modem samples from the Midwest to the Pacific Coast Fig 2 a and 0 However the three native haplotypes that were found in the historical sample from southern and central New England were not detected in the modern sample despite resampling of all of the sites from which 19thcentury herbarium specimens were available Fig 3 Fur ther haplotype AA was restricted to this region in the historical samples and is not found in any of the modern samples Thus in addition to local changes in haplotype frequencies extinction of Phragmites lineages may have occurred over the past century Native haplotypes were found in only two sites along the Atlantic coast Allen MD and Chance VA in the modern sample in contrast to their widespread distribution throughout eastern North America in the historic sample Fig 2 a and c The lack of persistence of native types is surprising given the clonal nature of this species and suggests that haplotype M is highly compet itive and aggressive This is further evidenced by the rapid replacement of native lineages by the invasive one seen in marshes of Connecticut and Massachusetts by 1940 Fig 3 The rapid proliferation of haplotype M throughout the At lantic Coast could result from either an introduction of this type from elsewhere or a range expansion of an existing native type Because this haplotype was present in historical samples it is possible that humaninduced changes in the landscape or other unidentified causes gave it an advantage that allowed it to expand rapidly It is more likely that an introduction of Phrag mites has occurred because haplotype M shares none of the mutations that link the 11 native North American haplotypes it it is most closely related to EurAsian types Fig 1 and iii population structuring has declined significantly between the pre1910 and modem samples from North America This intro duction probably occurred sometime during the early part of the 19th century most likely at one or more coastal ports along the 1 Mack R N Simberloff D Lonsdale W M Evans H Clout M amp Bazzaz F A 2000 E4472 AppZ 10 6897710 Pimentel D Lach L Zuniga R amp Morrison D 2000 BiaSeienee 50 53765 Carlton J T 1996 Ee47247gy 77 165371655 Geller J B Walton E D Gorsholz E D amp Ruiz G M 1997 M472 E5472 6 9017906 Bastrop R Jurss K amp Strumbauer C 1998 M472 Bi472 Ev472 15 977103 6 McIvor L Maggs C A ProVan J amp Stanhope M J 2001 M472 E5472 10 9117919 Hansen R M 1978 Pa2e475i47247gy 4 3027319 Niering W A Warren R S amp Weymouth C G 1977 C47nneetiei4t Arhmetum 31122 22 2712 F SNN 5quot pad 9 Orson R 1999 BiaZ Inv 1 1497158 10 oman M amp Wells L 2000 Quat Res 54 2067217 11 Torrey J 1843 f2477a 47ftne State 47fNew Y477k Carroll and Cook Albany 12 Willis 0 R 1874 Cata247 i4e 47fP2ants G747wing Witn47i4t CuZtivati47n m the State 47f New Jersey J W Schermerhorn New York 13 MaCain J 1883 Cata247gi4e 47f Canadian P2ants Part 1 P472ypeta28 Dawson Brothers Montreal 2448 i vwvw pnas orgcgidoI101073pnas 032477999 Atlantic coast In the 1800s Phragmites was documented growing in places where ships ballast was dumped or used to fill marsh lands being converted to railroad and shipping hubs 38 Because Phragmites already grew in coastal marshes as a native component of the plant community and the introduced variety showed little or no morphological differences with native types the establishment of nonnative populations was not recognized After several decades of persisting in low densities rapid ex pansion of the type began and was probably facilitated by human dispersal by means of the widespread construction of railroads and major roadways across North America in the late 19th and early 20th centuries Given the aggressive patterns of spread seen over the past century it is likely that this expansion will continue to occur into western and northern parts of the continent The presence of native Phragmites lineages throughout these areas will only complicate efforts to control this spread It has been difficult for scientists to predict whether or not a species will become invasive upon entering a new habitat Detection of cryptic invasions is critical for quantifying both the numbers of invaders and their impacts For species with wide spread native distributions genetic diversity may play an impor tant role in their behavior when establishing at new sites Differences in physiological tolerance and behavior may give nonnative genotypes unforeseen advantages allowing them to proliferate and changing the genetic structure of the species This study presents compelling evidence of a cryptic invasion in a terrestrial plant species This invasion is on a scale comparable to if not greater than other known wetland invaders such as purple loosestrife Lythrum salictzritz and salt cedar Tamarix sp but appears to still be in a phase of expansion into new areas It is important to recognize that the structure and function of native terrestrial communities may be influenced by both cryptic and easily recognized invaders Thanks to J R Powell G Caccone D Skelly M Donoghue J Gleason J S Hall and two anonymous reviewers for comments and discussions J R Powell kindly provided the laboratory space and facilities for this work I thank the US National Herbarium Harvard University Her baria New England Botanical Society George Safford Torrey Herbar ium at the University of Connecticut Connecticut Botanical Society and Yale University Herbarium for providing herbarium samples This research was funded by an Environmental Protection Agency Science to Achieve Results graduate fellowship The Nature Conservancy Connect icut chapter The Long Island Sound Fund administered by the Con necticut Department of Environmental Protection through the sale of Long Island Sound license plates and contributions the New Jersey Public Service Electric and Gas Company and the US Fish and Wildlife Service through the Biological Control of Nonindigenous Plant Species Program at Cornell University and the National Oceanic and Atmospheric Administration Office of Sea Grant and Extramural Pro grams US Department of Commerce under Grant MERPSG 99721 14 Dame L L amp Collins F s 1888 F2477a anddZesex C47i4nty MA Middlesex Institute Malden MA 15 Hitchcock A S 1935 ManuaZ 47f the Grasses 47f the United States US Goverment Printing Office Washington DC Misc publication no 200 16 Graves C B Eames E H Bis C H Andrews L Harger E B amp Weatherby C A 1910 Bu22etin 47ftne C47nneetiei4t Ge47247giea2 and NaturaZ Histary Survey N47 14 Case Lockwood Brainard Hartford CT 17 Stone w 1911 1910 AnnuaZ Rep477t 47ftne New Jersey State Museum New Jersey State Museum Trenton 18 Marks M Lapin B amp Randall J 1994 NaturaZ Area I 142857294 5474 15176 20 Pellegrin D amp Hau 63 2417 9 21 Chambers R M Meyerson L A amp Saltonstall K 1999 Aquatic B47t 64 2617273 22 Soltis D E Soltis P s amp Milligan B 25 Doyle J J amp Dickson E E 1987 Tam 36 7157722 32 Excoffier L Smouse P amp Quattro J 1992 Genetics 131 4797491 26 Taberlet P Gielly L Pautou G amp Bouveg J 1991 P201722 M472 31472 17 33 Raymond M amp Rousseg F 1995 Ev472utz47n 49 128071283 110571109 34 Schneider 5 Roessli D amp Excoffier L 2000 ARLEQUIN A S47 wme f477 27 Saltonstall K 2001 M472 E5472 N472es 1 76778 P47pu2ati47n Genetics Data AmZysxs Genetics and Biometry Laboratory Univ 28 cGuireG DenhamM C ampBalding D J 2001 M472 Bx472Ev472 184817490 of Geneva Geneva version 20 29 Clement M Posada D amp Crandall K A 2000 M472 E5472 9 165771660 35 Golenberg E M Clegg M T Durbin M L Doebley J amp Ma D P 1993 30 Templeton A R Crandall K A amp Sing C F 1992 Genetics 132 M472 Phy247genet Ev472 2 52764 6197633 36 Castelloe J amp Templeton A R 1994 M472 Phy247genet EM 3 1027113 31 Nei M 1987 M472eeu2m Ev472utx47mry Genetics Columbia Univ Press New 3 Bahrman N amp Gorenflot R 1983 Rev GeneraZ B472 90 177 84 York 38 Burk I 1877 P7475 Aead Na22 Sex PhiZadethm 29 1057109 Saltonstall PNAS l February19 2002 1 vol 99 l no4 l 2449 POPU LATION BIOLOGV Molecular phylogenetic evidence for the independent evolutionary origin of an arthropod compound eye Todd H Oakley and Clifford W Cunningham Department of Biology Duke University Box 90325 Durham NC 2770870325 Edited by James W Valentine University of California Berkeley CA and approved November 30 2001 received for review September 122001 Eyes often take a central role in discussions of evolution with debate focused on how often such complex organs might have evolved One such debate is whether arthropod compound eyes arethe product of single or multiple origins Here we use molecular phylogeny to address this longstanding debate and find results favoring the multipleorigins hypothesis Our analyses of DNA sequences encoding rRNA unequivocally indicate that myo docopids the only Ostracoda Crustacea with compound eyes are nested phylogenetically within several groups that lack com pound eyes With our wellsupported phylogeny standard maximum likelihood ML character reconstruction methods sig nificantly reconstruct ancestral ostracods as lacking compound eyes We also introduce a likelihood sensitivity analysis and show that the singleorigin hypothesis is not significantly favored unless we assume a highly asymmetric model of evolution one favoring eye loss more than 301 over gain These results illustrate exactly why arthropod compound eye evolution has remained controver sial because one of two seemingly very unlikely evolutionary histories must be true Either compound eyes with detailed simi larities evolved multiple times in different arthropod groups or compound eyes have been lost in a seemingly inordinate number of arthropod lineages he number of times eyes originated during evolution is often debated including within anthropods 173 Many biologists argue that arthropod compound eyes are the product of a single origin because detailed similarities exist among the eyes of diverse groups For example genes involved in eye development such as Fax6 and sine oculis appear functionally conserved across phyla and may also be conserved within Arthropoda In addition a highly stereotyped number and arrangement of cells develop in a similar manner to form the individual eye facets of different arthropod groups 478 Finally neural circuitry of the optic lobe is highly conserved in many arthropods Despite the many similarities some scientists suggest that compound eyes may result from multiple origins Nilsson 10 argues that the different biophysical properties of some eyes make homology unlikely Others postulate based on phyloge netic arguments drawn from taxonomy that compound eyes may have multiple origins 11 12 We have taken advantage of the power of molecular systematics and the recent advances in methods for analyzing character evolution to study the question of compound eye evolution in a phylogenetic framework ere we use these tools and examine the phylogeny of the Ostracoda Crustacea to test the hypothesis that one ostracod group independently evolved compound eyes with respect to all other arthropods 11 12 The Ostracoda are a diverse and ancient group of bivalved crustaceans with a superb fossil record dating back at least 500 million years 13 Taxonomically ostracods are often divided into three major groups Podocopa Palaeocopa and Myodocopa 13 The Myodocopa are further divided into the Halocyprida and Myodocopida Although most Podocopa and Myodocopida have a noninlageforming and anterodorsally located eye called the median eye the Myo docopida myodocopids are the only ostracods that also have a pair of lateral compound eyes Our molecular phylogeny clearly indicates that myodocopids are monophyletic and are nested 1426 1430 1 PNAS i February52002 1 vol 99 i no 3 within several groups lacking compound eyes Based on this phylogeny methods of character reconstruction significantly favor the independent origin of myodocopid compound eyes constituting the strongest phylogenetic evidence to date for multiple origins of arthropod eyes If this is not an independent origin and compound eyes were actually lost many times then this is a case where commonly used methods of historical inference are positively and significantly misleading Methods Taxa Our analysis contains representatives of the major groups of Ostracoda 13 with the possible exception of the Platyco pida which lack both median and compound eyes and are often placed within the Podocopa 14 We sampled all five taxonomic families of Myodocopida the only ostracods with compound eyes to test for monophyly As outgroups we chose two maxillopods crustaceans thought to be close relatives of ostracods eg ref 5 39 e most maxillopods both chosen outgroups have a median eye However only one of these outgroups also has compound eyes Argulus sp Branchyura the other does not Tigriopus Copep oda This outgroup choice is conservative with respect to the independent compound eyes hypothesis because most maxillo pods lack compound eyes 5 and the inclusion of additional outgroups lacking compound eyes could strengthen but is un likely to weaken our conclusion of independent origins Collec tion details for all taxa are available from T 0 Sequences and Phylogenetic Analysis We used standard primers and methods to obtain a complete sequence for DNA encoding 18S rRNA rDNA and a partial 28S rDNA sequence including the ddff eemm and vx regions 15 16 We aligned sequences with CLUSTALX 17 and removed ambiguously aligned regions although the same maximum likelihood ML tree topology was obtained when including all data results not shown We determined the bestfit model of molecular evolution to be TamuraiNei 18 gamma invariant sites by using MODEL TEST 19 We then fixed parameters to their ML estimates transversions 1 AiG CiT 41785 gamma shape 06894 proportion of invariant sites 03228 for a ML heuristic search and for 00 ML bootstrap pseudoreplicates in PAUPquot 20 We estimated relative branch lengths by using the ML tree and assuming a molecular clock Ancestral State Reconstruction Taxa were scored for presence absence of compound eyes and separately for presenceabsence This paper was submitted directly Track II to the PNAS office A m M Data deposition database accesion nos AF3632947AF363360 and Evolution University of Chicago 1101 East 57th Street Chicago IL 60637 Email39 thomidway uchicago e u Th ph thls 39 H Jinpartbypg h a p This article must therefore be hereby marked quotadvertisementquot in accordance Wlth 18 u s c 1734 soier to indicate this fact vwvw pnas orgcgidoi101073pnas 032483599 30 7 VU prper Bounds of Loss Parameter24 t m Independent Origin Favored E 20 quot6 E ru n 10 Model Asymmetry Favoring Loss loss rategain rate Flg z A sensitiVity analysis omparlng llkellhood models Wlth dlfferlng amounts of asmmetry in rate of ompound eye evolution The anlS repree 0 nodes flxed to abseme The xaxlsvalue refer to the amount of asymmetry o 20 3o favor ng loss of ompound yes reasonable models beaue loss of eyes is R t 6 probably more llkely that gain For example l on the x aXlS is an equal rate a e 7 339quot model A 30 on the anl l a model where loss 30 gtlt galn e 30 B Fig 3 leellhoodsurfaeforatwoeparametermodel orostratod ompound of the Myodocopida 100 boootrap Fig 1 Furthermore a X2 distribution w the loss parameterfltsthe data lgnlflantlyworehanthe ML monophyletic Myodocopida is nested within Podocopa Palaeoe w a a L mi c a L lack compound eye Fig 1 ML methods of ancestralestate reconstruction significantly support the independent origin of s a s I39BThe394rdo t i t favor eye loss over gain Fig 2 The assumed model of Dome before our data favor the singleeorigin hypothesis and must kewed over 301 before that result is statistically significant Fig 2 We found that the 95 1 for the loss parameter has a large range Fig 3 presumably because this phylogeny has relatively few taxa and estimated rates ofeye evolution are therefore based on little data Larger and roa er taxonsampli cou dprovide tighter estimates of rate parameters Despite the imprecision of rate estimates the absence of compound eyes is still favored a value significantly greater than the rate estimated from the data which is 0 In contrast to compound eyes some ambiguity exists in the s s t a r ostracods and outgroups is supported by ML reconstructions that used relative branch lengths estimated from DNA Fig 1A This result is different from reconstruction based on h h r at of which favor a loss of median eyes within ostracods coupled with a regain in the myodocopids The difference in parsimony and ML reconstruction for median eyes clearly illustrates a difference in the assumptions of these methods Unlike parsia mony ML considers branch lengths as a proxy for time when t h nr a separate the three ostracods that lack median eyes from their relati e w w L l v l L t t likely L t t branches resulting in the difference between parsimony and ML reconstruction This interpretation is further supported by the 1423 l wwwpnascrgsgidcilolo73pna5032483599 remnnru lon or all nodes basal Io myodowpld rav e remnstrualon are not illustrated lndlvlduallythese remne structions are not atlsmally SlgnlfKanti all have similar proportions of llkellhood favorlng ompound eye abseme about 0 53 fact on w 39branch lengths concurs with parsimony reconstruction of median eyes ur se uence data are equivocal with respect to ostracod the ML tree does not support monophyly but how strong support for any alternative tree The best tree consistent with ostracod monophyly also pound eyes Other relationships indicated by our data are consistent with previous views of ostracod phylogeny based on morphology with the possible exceptions of paraphyletic Haloe cyprids and Philomedidae Myodocopida 25 Our phylogee indicating that hirsute flaps are probably reduced compound eyes rather than an intermediate step between absence and presence 11 ssion ur very well supported molecular phylogeny unequivocally indicates that the only ostracods with compound eyes are altogether These resulm indicate one of two possibilities either arthropod com ound eyes have originated more t an once or compound eyes were actually lost in several ostracod lineages ds of character reconstruction strongly favor the independent origin hypothesis 1f compound eyes did not evolve independently in my 7 docopids then these commonly used methods of historical inference are significantly and positively misleading Here we s k c A t f39 nodes ancestral to myodocopids by using ML reconstruction a method that is often conservative eg ref 22 A case in point Oakley and Cunningham for ambiguity is ostracod median eyes where ML ancestral state reconstruction was equivocal at three nodes Fig 1A and nificant results were obtained when assuming either equal rates of eye gainloss or rates estimated by maximum likelihood Assumptions about branch lengths also did not change the conclusion of an independent origin of compound eyes Despite these apparently unambiguous results for compound eyes an important consideration is that eyesilike other com plex charactersiare pro a y more easily lost than gained which can cause character reconstruction methods to be mis leading 26728 We explored this possibility in two different ways First we showed that compound eye absence is favored at all five nodes basal to myodocopids even when using avalue for the rate of compound eye loss that is significantly higher than the ML estimate from the available data Fig 3 Second we devised a likelihood sensitivity analysis to estimate the extent that loss would have to be favored over gain before the independent origin hypothesis was no longer supported Herein lies a central problem in character reconstruction analysis there is no way to know what value of a sensitivity analysis is large enough to be considered significant 28 29 For example even though 30 is a seemingly very large number perhaps eyes really are lost over 30 times as often as gained during evolution From a perspective of Bayesian statistics this problem can be stated as a difficulty in choosing an appropriate prior distribution for a parameter that estimates directional bias 22 30 These prior probabilities will almost certainly vary among different charac ters and analyses at different taxonomic levels Therefore additional data that corroborate unambiguous character recon struction results or clarify ambiguous results are particularly valuable 28 31 For the cases in question further evidence could be ob tained by examining molecular development eye morphology or neural circuitry Of these only eye morphology has been examined previously in ostracods and these data are consis tent with median eye homology and an independent origin of compound eyes The ostracod median eye is typical of maxil lopods in having a tripartite structure and diagnostic tapetal cells 32 With the exception of Mystacocarida and Tantulo carida which completely lack eyes other maxillopo ds have this diagnostic median eye consistent with a conclusion of median eye homology in maxillopods and ostracods Unlike median eyes ostracod compound eyes are unique among arthropods as we might expect if those eyes had an independent evolu tionary origin Ostracod compound eye facets ommatidia each have six photoreceptive cells retinular or R cells and two lens cells crystalline cone cells 33 34 In contrast the ommatidia ofmany diverse groups of arthropods including the maxillopodArgulus an outgroup in our phylog 1 SalvinirPlaWen L V amp Mayr E 1977 On the Evalutwn aanatareeeptars and Eyes Plenum New York Gehring w J amp Ikeo K 1999 Trends Genet 15 3717377 Gould s J 1994 Nat Hist 103 12 10720 Paulus H F 1979 in Artnmpad Pnylageny ed Gupta A P Van Nostrand Reinhold New York pp 2997383 Brusca R C amp Brusca G J 1990 Invertebrates Sinauer Sunderland MA Melzer R R Diersch R Nicastro D amp Smola U 1997 Naturwxssenr senaften 34 5427544 M lzer R R Michalke c amp Smola U 2000 Naturwissensenaften 37 3087311 Dohle W 2001 Ann Sac Ent Fr 37 857103 Osorio D amp Bacon J P 1994 BwEssays 16 4197424 Nilsson D E 1989 in Facets afoswn eds Stavenga D G amp Hardie R C Springer Berlin pp 30773 Parker A R 1995 Prue R Sac Landan B 262 3497355 Fryer G 1996 Brat J Linn SM 58 1755 Cohen A C Martin J W amp Kornicker L S 1998 Letnaia 31 2517265 i 999 9 5quot 9 HHH wmr Oakley and Cunnlngham eny have eight Rcells and four conecells 4 6 This co mon arrangement of cells has been one of the main arguments for the singleorigin hypothesis of arthropod com pound eyes 4 6 35 The name tetraconata has even been suggested for a CrustaceanHexapod clade because of the abundance of species with four cone cells per facet 8 We consider the deviation of ostracods from the common 84 cell pattern important evidence that is consistent with the independent origin hypothesis yet we recognize that other explanations are possible One possibility is that there was a change in cell numbers during the evolution of homologous compound eyes a plausible hypothesis because mutations in single genes like sevenless reduce Rcell number from eight to seven in Drosophila ommatidia 36 37 In addition ostracods are not the only arthropods that vary from the 84 pattern Despite possible alternative explanations phylogeny and facet morphology are both consistent with the independent origin of compound eyes in ostracods The possible independent origin of an arthropod compound eye is particularly interesting in light of recent controversy surrounding claims for the homology of all metazoan eyes based on highly conserved developmental genes like Fax6 38 This controversy originally focused on definitions of homology 39 40 but more recently two opposing evolutionary models have emerged separate from disagreements about homology defini tions One model is that prototype photoreceptive structures and associated developmental genes evolved once and have been elaborated along independent lines A second model is that photoreceptive structures evolved multiple times and each time coopted homologous genes for use in those structures 41 The key difference between the models is whether a photoreceptive an was resent in all common ancestors or not The data presented here suggest that compound eyes were not present in ostracod ancestors If independently derived eyes of ostracods use the same developmental genes as other eyes then the cooption model above might be favored suggesting that a structurally nonhomologous eye coopted homologous eye de velopment genes during evolution Investigating the genes in volved in ostracod eye development will provide additional insight into these matters We thank E Oakley K Abe and members of his laboratory K Swanson T Jellenick K Tanoue M o t wr an m m ers of his laboratory especially A Parker collecting B K p p m H D H D m a D 1 E gt m m D Q N 4 D 1 H 9 S 1 00 H 1 m 1 i D H m D a 1 0 ct Explorer s Club uke niversity and a atio eron autics and Sp ace Administration Graduate Student Researchers Program fellowship 14 Cohen A C 1982 in Synapsxs and Classi catmn 0f wang Organisms ed Parker S P McGraerill New York pp 1817202 15 Hillis D M amp Dixon M T 1991 Q Rev Brat 66 4117453 rman S N Nicol S Elliott N G amp McMinn A 2000 M02 Pnylagenet Eva 17 26736 17 Higgins D G Bleasby A J amp Fuchs R 1992 Camput Appz Eraser 3 897191 18 Tamura K amp Nei M 1993 M02 Brat Eval 10 5127526 19 Posada D amp Crandall K W 1998 wanfarmatxes 14 8177818 20 Swofford D L 1999 PAUPquot Phylogenetic Analysis Using Parsimony and other methods Sinauer Associates Sunderland MA Version 40 Pagel M D 1994 Pm R 505 Landan B 255 37745 Schluter D Price T Mooers A D amp Ludwig D 1997 Evalutmn Lawrence Kans 51 169971711 Pagel M D 1999 Syst 3m 48 6127622 Ree R amp Donoghue M 1999 Syst BM 48 6337641 Kornicker L S 1976 Smxtnsanxan Cantr Z4702 219 1782 26 Omland K E 1993327214th Lawrence Kans 51 163671646 as IONIC u w PNAS i FebruaryS 2002 1 vol 99 i no3 i 1429 EVOLUTION Sggg www 92M 1430 l Cunningham C W Omland K O amp Oakley T H 1998 Trends Ech E12272 13 3617366 Oakley T H amp Cunningham C W 2000 Evalunzm Lawrean Kam 54 3977405 1 Omland K E 1999 Syst Big 48 604761 Schultz T R amp Churchill G A 1999 Syst Bid 48 6517664 Hoekstra H E amp Edwards s V 2000 Pm R 5272227112127 3131272 52 267 Elofsson1 1992 Am 227272 73 3697372 Andersson A 1979 PhD thesis University of Lund Lund Sweeden HuVard A L 1990 Acta Z2702 71 2177224 vwvw pnas orgcgldoI101073pnas 032483599 9292 9a m 4 paw HOW Paulus H F 2000 I Z0272 Syst E12272 Res 38 1897208 Harris W A Stark W S amp Walker J A 1976 I Physwl Landcn 256 4157439 Hafen E Basler K Edstroem J E amp Rubin G M 1987 Science 236 5576 Quiring R Walldorf U Kloter U amp Gehring W J 1994 Science 265 78 7789 Canatella D 1997 Syst 3272 46 3667369 Abouheif E 1997 Trends Ech 13m 12 4057408 Ni 42 lsson D E 1996 Curr 31272 6 397 Oakley and Cunnlngham Botany 940 Review 1 Parsimony Likelihood Principle Ingredients of molecular evolution models Substitution rate matrix Distribution of rates Bootstrap Divergence times Next week Bayesian methods Testing monophyly Parsimony TAATAAT TAAAA TAT T T T T TATAA TAAATAATTTTT a TAATAAATTAAT gtlt b d ab cd L11 ac bd L14 ad I be L12 QOU39SD Bootstrap with Parsimony 12 volunteers choose a number between 1 and 12 Get a new alignment and a new MP tree Repeat Likelihood principle Probability model can be used to make predictions C a X gt many b d Data probability model gt likelihood probability of the data under the model measures the fit of the model to the data and few 7 338 38353 wwwa awn Maximum Likelihood l ML tree find the tree and parameters that maximize the likelihood Tree ablcd acbd adlbc score log likelihood parameters 3034 a0b5535c55d0 3102 a0c790b55d35 3102 a0d3527b27c76 Bootstrap with Maximum Likelihood 12 volunteers choose a number between 1 and 12 Get a new alignment and a new ML tree Repeat Ingredients of molecular evolution models Topology I Branch lengths in number of substitutions per site Usually unconstrained no clock Substitution rate matrix A 1 33 33 33 1 20 60 20 85 40 35 10 C 33 1 33 33 20 1 20 60 15 6O 35 10 G 33 33 1 33 60 20 1 20 15 40 65 10 T 33 33 33 1 20 60 20 1 15 40 35 90 JC K80 k3 F81 unequal base freq Ingredients of molecular evolution models Common models reversible The root doesn t matter Base freq tiltv ratio param JC equal 1 0 K80 equal free 1 F81 free 1 3 HKY85 free free 4 F84 free free 4 TN93 free two titv ratios 5 are free GTR free All rate ratios 8 are free Ingredients of molecular evolution models Distribution of rates across sites 1 Some sites are slow some are fast a Model each site chooses a rate r at random from some distribution i M r0 2 r1 r1 2 r0 8 Ingredients of molecular evolution models Distribution of rates across sites a Model Gamma distribution of rates continuous and discretized for calculations Ingredients of molecular evolution models Distribution of rates across lineages a Covarion models hidden onoff process for absencepresence of constraint 13 parameters iWwW Ingredients of molecular evolution models Partitions in the data a W 2nol and 3rOI codon positions a several genes are available a or several exons and introns a or nuclear mt DNA Allow blocks to have their own models andor parameters Estimation of Divergence Times Times and rates are counfounded Sites can a evolve for 1 million years at 510397 substyearsite a or for 10000 years at 510395 substyearsite the alignement will look the same 05 substsite on average Difficult problem a fossil calibrations usually few and uncertain a rates vary a lot Origin of a complex key innovation in an obligate insect plant mutualism Olle Pellmyr and Harald W Krennt Department of Biology Vanderbilt University Box 1812 Station B Nashville TN 37235 and Department of Evolutionary Biology Institute of Zoology University of Vienna Althanstrasse 14 A710 0Vienna Austria Edited by May R Berenbaum University of Illinois at UrbanarChampaign Urbana IL and approved January 30 2002 received for review 1 November 2 200 Evolutionary key innovations give organisms access to new eco logical resources and cause rapid sometimes spectacular adaptive radiation Thewell known obligate pollination mutualism between yuccas and yucca moths is a major model system for studies of coevolution and it relies on the key innovation in the moths of complex tentacles used for pollen collecting and active pollination These structures lack apparent homology in other insects making them a rare example ofa novel limb We performed anatomical and LL 39 39 439 39 39 quotoiiginand quot quot a remarkably simple mechanism Morphological analyses of the tentacles and adjacent mouthparts in pollinators and closely re lated taxa showed that the tentacle appears abruptly in female pollinating yucca moths Several morphological synapomorphies cles to propose a possible developmental genetic basis for the trait The Function of the Tentacles The pollinating yucca moth genera Tegeticultz and Parategeticultz constitute a monophyletic group within the Prodoxidae Fig 1 Jointly they contain at least 25 extant species 5 two of which are derived nonpollinating Tegeticultz species that oviposit into yucca fruit created by coexisting pollinator species 16 The sister group Prodoxus coexists with the pollinators on yuccas but feed as larvae on plant parts other than the seeds Their radiation was thus directly facilitated by the pollinator radiation Together these genera constitute a major adaptive radiation on yuccas with a species diversity more than 20fold that of their sister group the between the galeae which constitute the characteristic Iepidop teran proboscis and 39 39 ihat 39 39 39 quot quickly 39 g r forthe galea at an apical growth bud on the first segment of the maxillary palp Behavioral data indicate that tentacle and proboscis movements are controlled by a shared hydraulic extension mechanism thus no new mechanism was needed for tentacle function Known devel opmental paths from other insects can explain the origin of this sexspecific key innovation in a few steps us bligate mutualisms between plants and pollinators provide some of the most apparent examples of coevolution 1 2 Alongrecognized association ofthis kind between yucca moths Prodoxidae and yucca plants Agavaceae has become an important model in understanding how obligate mutualisms coevolve 37 6 In this association established at least 40 million years ago 7 yuccas are pollinated exclusively by yucca moths whose larvae in turn consume some of the developing yucca seeds This has been an evolutionarily and ecologically highly successful association with some 30745 yucca species 8 9 being important vegetation components throughout much of the North American deserts and semiarid regions 10 Prior analyses of the coevolution between yucca moths and yuccas have shown that the transition from antagonism to mutualism primarily involved quantitative changes in already existing traits rather t an evo utionary novelties The one exception is the evolution of elaborate tentacular mouthparts in the yucca moths used for handling pollen with great precision These tentacles are an evolutionary key innovation both in the sense that it is a truly novel trait that evolved quickly 7 Fig 1 and that it is linked to an adaptive radiation 11713 Under standing how this trait evolved then is central to understanding the coevolutionary history of diversification and changing inter actions between yuccas and yucca moths Although reported when the relationship was first described over a century ago 14 15 no analyses have been performed of tentacle anatomy or homolo Here we present anatomical data from phylogenetically piv otal moth species indicating that t A 39 trait for the mutualism has a surprisingly simple origin We also use trait expression in the pollinators their nonpollinating sister group and derived species that have secondarily lost the tenta 5498 5502 I PNAS I April 162002 I vol 99 I no 8 seedparasitic Mesepioltz whose larvae feed on plants in the Nolinaceae 17 Female yucca moths possess unique tentacles on their mouth parts that are used to actively pollinate host flowers where they oviposit The female moth gathers the glutinous pollen of yucca flowers by scraping it off the anthers with her tentacles The pollen is immediately compacted by using tentacles and some times the forelegs as well and placed as a solid batch on the concave posterioventral surface of the head Fig 2 The pollen mass may approach 10000 grains and weigh up to 10 of the moth body mass 18 Prolific pollen coating maintains batch cohesion and the tentacles are not involved in its retention After pollen gathering the moth seeks out flowering yucca plants where she oviposits into Tegeticultz or near Paratege ticultz pistils As oviposition is completed the female flexes her tentacles and uses the apical portion to remove a small pollen load from her batch She walks to the floral stigma and very deliberately places the pollen on it In all but one host species the stigmatic papillae line the interior ofthe hollow style and the moth packs in the pollen with 10720 repeated bobbing motions in the course of 3710 sec Movie 1 which is available as supporting information on the PNAS web site wwwpnasorg In the single exception the host Hesperoyucctz whipplei has a capshaped stigma and the moth pollinates by using the same dragging behavior on the stigma as is used for pollen collection on the anthers Materials and Methods Male and female moths of six species were included in the study Fig 1 Two outgroup taxa were used to determine the basal condition Nemophortz degeerelltz Adelidae is a basal member of the superfamily Incurvarioidea which includes Prodoxidae Prodoxus decipiens represents the pollinator sister group thus eing a close relative of the common ancestor of the tentacle bearing moths of Tegeticultz and Parategeticultz A clade of three This paper was submitted directly Track II to the PNAS office M a r i Th h hls 39 H Jinpartbypg h a c This article must therefore be hereby marked quotadvertisementquot In accordance Wllh 18 u s c 1734 soier to Indlcale hls fact vwvw pnas orgcgidoi101073pnas 072588699 Nemophora degeereIa decrplens o Prodoxus Fig 1 sister or lln elephant39peIa yuccasella cassandra Inlermedla Tegelicula Paraleg tentacles lost tentacles present pollination yuccas colonized in ml the is proportional to tlme and the bottom of l The lnternode e three genera n n r nil h overlap thus the uniform tentade seen in all pollinator moth must have evolved very unKly Data are from ref Sand 7 and o P and M Baka zarrtara unpublished data Tegerrcnzc species was used including the pollinators Tegcrrcnzc yucccsezzc and and the l i tnr ccsscudm 5 thus providing information on loss of the tentar cles A single representative of the smaller pollinator genus Pumiegetrculu Parategetrculu elephumpellu was included to cover the phylogenetic range of species with tentacles Sample data are provided in Table 1 39ve mot of all Tcgcrrcnlc Pmdzmm and Ncmcplrm same 24 h in alcoholic Bouins fixative and then electron microscopy Internal anatomy was exa serial semithin sections of specimens embedded in epoxy resin T as e size In T E E a z E linear scale from 0 to 10 where 0 was no trace and length on a 10 represented the longest rudiment which was onerhalf of the lethinquot 39mld 39 each sex were used for the com ariso us in tentacles palps and proboscis during 39 39 drinking were observed to e n anrsm a hare be tentacles and other par Fourt en f l T yuccnsezzc and T ccsscudm gathered in the field were placed individually in glass vialswith a L L 39 late dusk we observed mout par ove no through a x10 us under red narrowrbandwidth illumination peak 626 nm that did not affect moth behavior on the flowers Results 39entatle Anatomy The tentacle emerges frontally on the first maxillary palp segment and is found only in females of the Pellmyr and Krenn pollinating species of Tegerrcnzc and Pmrcgcrrcnzc Fig 3 t n s i No traces of a tentacle were found in either Pmdzmm or Nemcplrm No variation in tentacle morphology was found among the three analyzed pollinator species The nonsegmented tentacle is 24 023 mmrlong u 10 in T yucccsczzc about 1 pm incrosssection at midpoint and ittaperstowar the tip During rest the ten der the ad hen female does not carry pollen F pollen load when one is present Fig 2 The surface Fig 3c is annulated ensely covere with microtrichia an as numerr ous trichoid sensilla with a hooked tip These sensilla are located e the apex The most prominent internal feature of the tentacle of pollir nators Fig 4 is longitudinal musculature consisting of several a r ennolan eral walls although some fibers have an oblique dorsolateral course Fig AB These muscles permit recoiling and possibly 4 39 39 tentacle They are quite distinct from muscles attached to the base of the second max illa palp segment in all studied species where they serve as flexors and extenso Ot er features of the tentacle include a thickened dorsal epidermis and cuticle nerves and a single longitudinal trachea Fig 4 are present in both sexes Fig AB and there is no significant difference in length between them r test P gt 012 Micro trichiation and a reduced number of hooked sensilla are still present Fig 5 A reduced number of muscles are present sexually monomorphic in the nonpollinators but dimorphic in their pollinating ancestor PNAS l Aplll 16 2002 l vol 99 i no 8 l 5499 EVOLUHON Fig 4 A internal anatomy of the tentatle of female T yuttasea in ihi x n rt dorsal epidermis and tutitle arrowheads a nerve n and a trathea tr 8 Cross section through tentatles left and right sections and prooostis the irir xii i r w nerve and trathea tr L39 L L 18 an 2192 that the genetic template fox th ea is fundamental pxoducing the tentacle as well They include the xaxe condition of a nonaxticulate and coilable axthxopod limb similaxly are l musculatuxe doxsally thickened epidexmis and t I L basal maxillaxy paxt the stipes tube which has been functionally intexpxeted to be a hemolymph pump in the highex glossate Lepidoptexa 19 20 The behaviox data indicate that the hydxaulic extension mechanism is shaxed as well as tentacle xt 39 39 xoboscis extension and mover segmene The lessex mover pxoboscis is extended fox dxinking is expected fox two xeasons Fixst the laxgexvolume of the tentacle 39 39 L p 39 39 pxoboscis extension Second when the moth caxxies a pollen load the tentacle 39 alxeady paxtly uncoiled without intexnal liquid pxese suxe as it wxaps axound the pollen and influx will cause only fuxthex extension as it xeaches beyond the uncoiling Modifications in the tentacle fxom the galea axe modest and a galea and the numbex of ventxolatexal muscle fibeis 39 e bristlealike sensilla axe clustexed in the ventxal n xge e gained the distinctive texminal hook They aid in handling the pollen and seem to evolve eas39 pollenacollecting insece similax hookedatip sensilla have 39 al asll 39 thanSObee species that collect pollen fxom flowexs whose anthexs axe concealed in naxxow tubes 21724 In bees the hooked haiis re s r a paxticulax species uses fox pollenagathering Finally a medial PeHmyr and Krenn mourn Flg s A Tentatle rudiment t on first maXillary palp segment moi of female T intermedia seton maXillary palp segment mp2 With stales min trotrithiated probostis p B Tentade rudiment of female T intermedia in from flattened side of the galea that foxms the centxal food canal of the pxoboscis is ost Genet it is stxi sis ol the Iantacle Fxom a morphological peispective ng that such a complex stxuctuxe as the tentacle has oths The 39 p t pl t fox the shaxed specializations between the tentacle and galea is that a shaxed developmental is x x t e r 26 this maxillaxy appendage is highly modified in most Lepie doptexa The most basal clades Micxoptexigoidea Agathiphaa goidea and Hetexobathmioidea have a minute pxotxusion but the clade Glossata which const39tutes about 999 of all den scxibed Lepidoptexa is chaxactexized by elonga nected galeae that foxm the distinctive pxoboscis 2 Whe as the eaxliest pxoboscides lacked intxinsic musculatuxe fox tight coi ing this txait evolved well befoxe the oxigin of the Pxodoxidae 28730 T a moths have a xelatively shoxt o oscis consisting of loosely connected galeae they axe splayed apaxt on wet suxfaces of yucca floweis allowing liquids such as watex an nectax to be imbibed by capillaxy foxce 31 lnfoxmation about galea developmental genetics is limited but me x t x t t n homeodomain txanscxiption factox chaxactexistic of and xee quixed fox the development of distal limb stxuctuxes 32734 in 39 f39 t a Galea development is usually sexually monomoxphic including in the yucca moths In contxast functional tentacles axe sexually dimoxphic in pole linating yucca moths but sexually monomoxphic paxtial expresr sion of maxillaxy tentacle xudimene in nonpollinating T mm PNAS l Apiil 162002 l vol 99 i no 8 l 5501 EVOLUIION media is evidence that the developmental template is present in both sexes of pollinators but repressed in ma es A simple explanation for the observed patterns holds that the basic tentacle is an appendage using the genetic template for the galea D11 is expressed in all maxillary palp segments 35 36 and known to integrate homeotic and other pathways to cause sexual dimorphism in Drosophila 37 A simple genetic basis is con sistent with the rapid reversal to sexual monomorphism in the derived nonpollinators but could also be explained by a more complex trait with at least one majoreffect gene In contrast the variable degree of rudiment expression in nonpollinators rang ing from a blunt point to a short tentacle with some intrinsic musculature indicates slow functional loss through mutations in different parts of the tentacle template This hypothesis is consistent with life history reconstruction of the nonpollinating yucca moths which suggests 1 ss of pollination behavior was secondary to a shift from ovipositing in flowers to ovipositing in fruit 5 making the structures and behaviors associated with pollination redundant and subject to gradual loss through ab sence of purifying selection In conclusion the morphological data strongly indicate that the unique tentacles of female yucca moths originated through expression in a novel site of the genetic template for the 1 Herre E A 1999 in Levels afSelecti an in Evalutwn ed Keller L Princeton Univ Press Princeton pp 2097237 Thompson J N 1999 Science 284 211672118 Bogler D J Neff J L amp Simpson B B 1995 Prac Natl Acad Sci USA 92 686476867 Pellmyr 0 Thompson J N Brown J amp Harrison R G 1996Arn Nat 148 8277847 paw s 5quot Pellmyr O amp leebenerack J 2000 Am Nat 156 5627576 Bronstein J L 2001 Ecal Lett 4 2777287 Pellmyr O amp Leebenerack J 1999 Prac Natl Acad Sci USA 96 917879183 Reveal J L 1977 in Interrnauntain Flara eds Cronquist A Holmgren H Holmgren N H Reveal J L amp Holmgren P K New York Botanical Garden Press New York Vol 6 pp Clary K H 1997 PhD thesis Univ of Texas Austin TX Brown D E ed 1994 Biatic Camrnunities Sauthwestern United States and Narthwestern MEXMJ Univ of Utah Press Salt Lake City Simpson G G 1953 The Mdjz FedlMYES avaaluti an Columbia Univ Press New York B amp Hauser D L 1995 Hist Bial 10 1517173 13 Schluter D 2000 The Ecalagy aan39apti ve Radiatian Oxford Univ Press xford b b l prooa ax i N Riley c v 1872 Nature Landan 6 444 Riley C V 1873 Mssauri St Entarnal Annu Rep 5 1507160 Pellmyr 0 Leebenerack J amp Huth c J 1996 Nature Landan 330 557156 Frack D C 1982 MS thesis California State Polytechnic University Pomona CA Pellmyr o 1997 Ecalagy 78165571660 b b b l 9m i n i on 5502 i vwvw pnas orgcgidoI101073pnas 072588699 elongated galeae of Lepidoptera Prior work on the interaction has suggested that the tentacles are the only truly novel mor phological trait in these moths and the present findings show that acquisition of this complex trait and its mechanism of movement may have been evolutionarily simple Meanwhile the behavioral component of pollination is likely derived from a obing behavior for nectar in floral tubes in more basal prodoxids 4 and on the wet stigmas of yuccas thus a passive less efficient pollination mechanism may have preceded the origin of the tentacles and active pollination in the yucca moths Given the significance of these organisms in studies of coevolutionary processes coupled with the rarity of evolution of new limbs it should be important to test the explicit predictions about patterns of gene expression that derive from this ypot esis about tentacle origin We dedicate this paper to the memory of Ebbe Schmidt Nielsen who contributed greatly to our understanding of basal evolution of Lepidop e thank Kari Segraves and Manuel BalcazarLara for help gathering specimens Ursula Hannappel and Alexander Pernstich for the preparation of the semithin sections and Jim Kane for guidance on bee sensilla Florida State Parks provided permission to gather samples in Torreya State Park Behavior experiments were performed at Archbold Biological Station This work was supported by the National Geographic Society the National Science Foundation and the Austria Science Fund P 13944 19 Banziger H 1971 Mtt Schweiz Entarnal Ges 43 2257239 20 Krenn H w 1990 Zaarnarphalagy 110 1057114 21 Miiller A 1995 Entarnal Gener 20 43757 22 Alves dos Santos I amp Wittmann D 2000 Plant Syst Evil 223 1277137 23 Michener C D 2000 The Bees 0f the W0er Johns Hopkins Univ Press Baltimore 24 Thorp R W 2000 Plant Syst Eval 222 211 25 Snodgrass R E 1935 Principles afInsect Marphalagy McGraerill New York 26 Chapman R F 1998 The Insects Structure and Functian Cambridge Univ Press Cambridge UK 27 Kristensen N P amp Skalski A w 1998 in Lepidaptera Maths and Butter ies Z Handbmk afZaalagy ed Kristensen N P de Gruyter Berlin pp 7725 Kristensen N P 1968 Ent Medd 36 2397293 Nielsen E S amp Kristensen N P 1996 Invert Tawn 10 119971302 e n H n N P 2000 Zaal Anz 239 1797196 Pellmyr o 1999 Syst Entarnal 24 2437271 Cohen S Bronner M Kuttner F Jurgens G amp Jackle H 1989 Nature Landan 333432 34 33 Panganiban G Irvine S M Lowe C Roehl H Corley L S Sherbon B Grenier J Fallon J F Kimble J Walker M et al 1997 Prac Natl Acad Sci USA 94 516275166 34 Abzhanov A amp Kaufman T 2000 Dev Bial 227 6737689 35 Popadic A Panganiban G Rusch D Shear W A amp Kaufman T C 1998 Dev Genes Eval 208 1427150 36 Scholtz G Mittmann B amp Gerberding M 1998 Int J Dev Bial 42 8017810 37 Kopp A Duncan 1 amp Carroll S B 2000 Nature Landan 408 5537559 Pellmyr and Krenn
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