CHAPTER 17 Floral characters and diversification

Kathleen M. Kay, Claudia Voelckel, Ji Y. Yang, Kristina M. Hufford, Debora D. Kaska, and Scott A. Hodges Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA, USA

Outline The burgeoning of phylogenetic information during the past 15 years has focused much interest on whether specific features of clades enhance or hinder the evolution of species diversity. In the angiosperms many of the traits thought to affect clade diversity are floral in nature, because of their association with reproduction and thus species isolation. Therefore, we briefly review mechanisms by which floral traits can affect diversification. We then consider the possible influences of four specific traits by comparing the species diversity of a clade possessing a trait with that of its sister clade that lacks the trait. Clearly, this approach requires correct identification of sister groups, so that changes in phylogenetic reconstruction can have profound effects on these analyses. Here we use a recent supertree analysis of the angiosperms, which includes nearly all described families, along with other phylogenies to reexamine a number of floral traits thought to affect diversification rates. In addition, because many of the previous analyses employed a statistical test that has since been shown to be misleading, we use a suite of signed-rank tests to assess associations with diversification. We find statistical support for the positive effect of animal pollination and floral nectar spurs and a negative effect of dioecious sexual system on diversification, as proposed previously. However, our results for the effect of bilaterally symmetric flowers on species diversity are equivocal. We discuss several factors that will aid in future analyses and the need for both more detailed phylogenetic analyses and more studies on floral biology.

17.1 Introduction occurred concurrently with their radiation and rise The angiosperms are the most abundant and to ecological dominance. exhibit an diverse group of on Earth today. Since their amazing variety of sizes, shapes, colours, first appearance in the fossil record during the arrangements, scents, rewards, and sexual sys- early Cretaceous (ca. 130 Ma, Crane et al. 2004), tems, from the tiny self-fertilizing flower of Arabi- they have colonized almost every habitat on the dopsis thaliana to the intricate flowers of Ophrys planet, and now number approximately 260,000 orchids, which mimic a female mate for an extant species (Soltis and Soltis 2004). These myr- unsuspecting male wasp, to the enormous putrid iad species vary impressively in morphology, life inflorescence of the corpse flower, Amorphophallus history, chemistry, and reproductive biology. titanium. Especially striking is the floral diversification, Which factors promote angiosperm diversifica- which fossils show began among early angios- tion, especially the role of floral traits and sexual perms (Friis et al. 2000), and therefore must have systems, is an enduring question and its resolution

311 312 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S is a central goal of evolutionary biology. 1995; Hodges 1997a, b) or relied largely on Darwin puzzled over the apparent sudden angiosperm phylogenies that lacked representa- appearance of the angiosperms in the fossil record, tives of many families and were constructed clearly finding it a challenge to his view of mostly with plastid-gene sequences (Dodd et al. ‘‘extremely gradual evolution’’ and forever label- 1999; Heilbuth 2000; Sargent 2004). Clearly, incor- led the phenomenon with his famous quotation as rect statistical techniques can cause misinterpreta- ‘‘an abominable mystery’’ (Darwin 1903). Knowl- tions about diversification hypotheses. The limited edge of the timing and pace of angiosperm diver- taxon sampling in phylogenies can both lead to sification has progressed considerably with both errors in inferring sister-group relationships and the discovery of fossil flowers (e.g., Dilcher and the timing of the origin of a trait of interest and Crane 1984; Crane et al. 1995; Gandolfo et al. 1998) reduce the sample of replicate origins of a key trait. and molecular-based phylogenies (e.g., Qiu et al. Finally, because plastids do not undergo recom- 2000; Zanis et al. 2002). These findings have bination, plastid genes are inherited essentially as revealed an increasingly detailed view of patterns a single locus, so that sequences of different genes of floral diversification. provide limited independent phylogenetic infor- During the past 10 years or so, phylogenies have mation. Consequently, phylogenetic information been used extensively to assess whether species from plastid genes should be combined with data diversity occurs non-randomly among clades from other loci for a robust phylogeny based on (Sims and McConway 2003) and whether parti- multiple independent lines of evidence. These cular traits may be responsible for these patterns. problems all call for a reanalysis of the role of Although studies have reported that changes in floral traits in angiosperm diversification. angiosperm diversity correlate with several traits, Here we reanalyse four purported floral corre- including the rates of molecular evolution (Barra- lates of angiosperm diversity—animal pollination, clough et al. 1996; Barraclough and Savolainen floral nectar spurs, bilateral symmetry, and the 2001), latex and resin canals (Farrell et al. 1991), dioecious sexual system—using both more com- herbaceous growth habit (Dodd et al. 1999), and plete phylogenetic analyses and appropriate sta- climbing habit (Gianoli 2004), floral traits have tistical tests. We use a recently constructed and been implicated most commonly. This apparent nearly comprehensive supertree of angiosperm evolutionary importance of floral traits is perhaps families derived from 46 source (Davies et al. not surprising, because a correlation between the 2004), along with other phylogenies at lower rise and diversification of angiosperms and the taxonomic levels, and circumscriptions diversification of pollinating insects has long been consistent with APGII (2003). For each character recognized (Crepet 2000). As much of this volume we identify phylogenetically independent con- attests, aspects of both sexual system and floral trasts and compare the species richness of the sis- morphology can affect how a plant reproduces and ter clades composing each contrast. We discuss our with which other plants it mates. Thus, these traits findings in light of hypotheses for how these traits are natural subjects for investigating their effects affect diversification, and suggest avenues for on species diversity. future work to clarify the mechanisms responsible Comparative studies have identified several for any correlations. floral characters that affect rates of angiosperm diversification, including animal pollination (Dodd 17.2 How might floral traits affect et al. 1999), floral nectar spurs (Hodges and Arnold diversification? 1995; Hodges 1997a, b), bilateral symmetry (Sargent 2004), and a dioecious sexual system For a trait to affect diversification rates, it must (Heilbuth 2000) (Plate 7). However, most previous influence the probability of speciation, , analyses of the effects of floral traits on species or both. Speciation involves the evolution of diversity either used now-discredited statistical reproductive isolation and is generally initiated by methods (Dodd et al. 1999; Hodges and Arnold geographical isolation (Coyne and Orr 2004). F L O R A L C H A R A C T E R S A N D S P E C I E S D I V E R S I F I C A T I O N 313

Therefore, a trait that promotes the colonization of et al. (1999) compared the diversity of sister clades new habitats, limits dispersal between popula- using the methods of Slowinski and Guyer (1993) tions, or increases the propensity or ability to mate and found a strong overall pattern that animal with phenotypically similar individuals is a good pollination was associated with more rapid candidate for a trait that might affect diversifica- diversification than abiotic pollination. This find- tion by increasing speciation. Certain traits may ing is bolstered by an extensive body of empirical affect speciation, but in almost all cases these research on the role of plant–pollinator interactions effects are thought to be coincidental to adaptation in speciation (reviewed in Stebbins 1974; Grant to local conditions or genetic divergence in isola- 1981; Coyne and Orr 2004; Chapter 16). tion. Thus, although natural selection may cause Similarly, floral nectar spurs may further pro- evolution in these traits, they are not selected for mote specialization on different pollinators; reproductive isolation or higher diversification affecting reproductive isolation, and thus diversi- per se (Chapter 16). Conversely, some traits may be fication (Hodges and Arnold 1995; Hodges 1997a, selected in certain environments and during short b). The presentation of nectar at the base of a periods, but predispose their possessors to a relatively long, thin tube requires a match between higher chance of extinction. A trait that leads a the pollinator and the floral morphology, limiting population to experience greater demographic the number of pollinating species that can stochasticity or lower adaptive genetic diversity manipulate the flower successfully. Hodges and (see Chapter 2) would be a good candidate for a Arnold (1995) and Hodges (1997a, b) found an trait affecting diversification through extinction. association between the evolution of floral nectar Unfortunately, without a detailed phylogeny and spurs and higher diversification in both a com- fossil record, speciation and extinction are parative study among angiosperms and a detailed exceedingly difficult to tease apart. Thus analysis study of columbines (Aquilegia). of diversification requires a hypothesis for how a In contrast, bilateral floral symmetry, or zygo- trait affects either speciation, extinction, or both, morphy, may affect diversification somewhat dif- which can be tested more directly than by simply ferently. Compared with radially symmetric, or assessing its association with the overall diversifi- actinomorphic, flowers, zygomorphy constrains cation rate. the orientation of pollinators while they visit Animal pollination, bilateral floral symmetry, flowers, thereby enhancing the precision of pollen and nectar spurs have all been suggested to exchange between pollinators’ bodies and the enhance speciation rates for seemingly similar sexual organs of flowers (Neal et al. 1998; Sargent reasons, namely, their likely effects on the specifi- 2004). This increased precision could affect repro- city of mating among plants. Animal pollination is ductive isolation if it promotes specialization by one of the most striking features of angiosperms, different pollinators on different types of zygo- with plants using an incredible array of insects, morphic flowers or if flowers diverge in the loca- birds, and mammals to disperse pollen. Because tion of pollen placement on a pollinator (Chapter successful pollen transfer is so important to fitness, 16). Zygomorphy may also limit the number or pollinators exert selection on floral traits (reviewed type of pollinating species that manipulate a spe- in Fenster et al. 2004 and most chapters in this cies’ flowers effectively, which may increase the volume). Spatial and temporal variation in polli- variance in pollinator assemblages and hence nator assemblages can promote evolutionary selection on floral traits across the landscape. divergence in floral traits among populations Although examples of pollen placement affecting (Chapters 8, 15, and 16), and plant populations reproductive isolation between species visited by adapted to different suites of pollinators may be the same pollinator are known (Brantjes 1982; less likely to mate with each other (Thompson Grant 1994; Kay 2006; Chapter 16), the importance 1994). This avenue for reproductive isolation is not of such shifts in speciation remains to be clarified. available to abiotically pollinated lineages, which Nevertheless, in a sister-group study among depend on wind or water to transfer pollen. Dodd angiosperms, Sargent (2004) found accelerated 314 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S diversification in lineages with bilaterally sym- possessors to diversify rapidly and create new metric flowers. niches. Such effects should be relatively consistent Conversely, a trait may increase the chances of across lineages in which they evolve. Ideally, extinction, even if it is favoured by selection in the identification of key innovations requires knowl- short term (Chapter 2). The dioecious sexual sys- edge of the evolutionary relationships among taxa tem, with separate male and female individuals, and the timing of all critical events, such as spe- may be an example of this. Dioecy has evolved ciation, extinction, and the origins of the trait of multiple times and occurs in approximately 6% of interest. Unfortunately, barring an exceptionally angiosperm species (Renner and Ricklefs 1995). In detailed fossil record, these factors are usually a comparative study across angiosperms, Heilbuth incompletely known, dictating the use of less (2000) found a striking association between dioecy powerful statistical tests for an association between and lower species richness of sister clades. Dioecy a trait and diversification rate. may retard diversification for several reasons. The simplest technique for testing whether a Because they lack the reproductive assurance of trait alters diversification compares the numbers of being able to self-pollinate, dioecious plants may species in two sister taxa differing in the trait of have a higher risk of dying without reproducing interest. By definition, sister groups are the same and may have a lower colonization ability (Baker age, so any difference in species numbers must be 1954; Bawa 1980; Chapter 12). However, self- a result of differences in rates of speciation and/or incompatible species should be subject to the same extinction. These differences are compared to a constraint and have lower species diversity than null model of equal diversification to determine their self-compatible sister taxa, but no such asso- whether they are sufficiently large to indicate a ciation has been found (Heilbuth 2000). Vamosi change in the diversification rate with the origin of and Otto (2002) proposed that differential selection the putative key innovation or whether they on male and female flowers can lead one sex to occurred stochastically during speciation and become more showy than the other (typically extinction (Sanderson and Donoghue 1994, 1996). male), resulting in poor pollination during years of Such an analysis can be implemented with only a low pollinator abundance. Dioecious plants can rudimentary phylogeny showing sister-group also suffer increased variance in both pollination relationships, and may therefore be feasible in and seed dispersal, because, unlike hermaphro- many diverse and poorly characterized lineages; dites, not every individual is a potential mate and however, it has low statistical power and can only females disperse seeds (Heilbuth et al. 2001; detect only extremely large differences in diversi- Wilson and Harder 2003). Because reproductive fication rates (Sanderson and Donoghue 1996). success varies nonlinearly with pollination and More powerful inferences can be drawn for seed dispersal, this increased variance can reduce traits that evolve repeatedly, which can provide the average reproductive performance of dioecious replicated evidence for changes in diversification. species relative to that of otherwise similar her- Most simply, numbers of species between pairs of maphroditic species (Wilson and Harder 2003). sister clades can be compared with a sign test. However, the sign test ignores the magnitude of 17.3 Common tests for key innovations differences in species numbers, and thus provides limited statistical power. Consequently, the sign Discovery of the mechanisms by which particular test should be used only when the relative sizes traits influence speciation and extinction is fun- and not the species numbers of sister groups are damental, but phylogenetically based comparative known. Perhaps the most commonly used method studies are necessary to identify the importance of has been that of Slowinski and Guyer (1993), a trait to angiosperm diversification in general. which compares the difference in species richness Many traits can influence diversification in some between individual sister groups with a null circumstances, but certain traits have been sug- model based on random speciation and extinction, gested to act as key innovations, allowing their and then combines probabilities from multiple F L O R A L C H A R A C T E R S A N D S P E C I E S D I V E R S I F I C A T I O N 315 comparisons. However, several researchers have For example, the proportional contrast between identified severe shortcomings with this method clade A with 10 species and clade B with 5 spe- (e.g., de Queiroz 1998; Goudet 1999; McConway cies shown in the hypothetical phylogeny of Fig. and Sims 2004). Vamosi and Vamosi (2005) 17.1 equals 0.167. This test is prone to errors if recently reviewed and compared statistical tests the proportion of species with the trait of interest for sister-group comparisons and showed clearly for each contrast has a U-shaped distribution. In that the Slowinski–Guyer method is prone to type this case, tests for effects of either character state I errors, because a few large differences in species can result in significant test statistics. In contrast, numbers can result in a significant test statistic, this approach applied to a data set with an L- regardless of the direction or magnitude of the shaped frequency distribution, in which most of remaining contrasts. This problem is especially the large contrasts in species counts fall in the severe for datasets in which some sister-group same direction, will yield a significant result for comparisons have large differences in species only one of the character states. Finally, ‘‘log’’ counts that favour the hypothesis, but other sister contrasts compare sister-group diversity based on group comparisons have large differences in the the ratio of the log number of species in the opposite direction. Such a dataset results in a larger group to that for the smaller group. In this U-shaped frequency distribution of the proportion case, the contrast between A and B in Fig. 17.1 of species in each sister-group pair possessing the equals 1.43. Log contrasts may favour small or trait of interest. In these cases, the Slowinski– young sister groups, and therefore should be Guyer method can give the nonsensical result that used cautiously if replicate sister groups differ the trait both promotes and retards diversification systematically in phylogenetic age according to significantly. For these reasons, Vamosi and the direction of their contrast. Tests based on log Vamosi (2005) recommended against the use of the contrasts yield the same result if a specific Slowinski–Guyer method, and suggested more character state promotes or retards diversific- suitable, less biased techniques. They also recom- ation; however, the direction of the effect can be mended that plots of the data accompany any identified from a plot of the contrast distribution statistical tests, making it possible to check (Vamosi and Vamosi 2005). Isaac et al. (2003) visually for data with a U-shaped frequency distribution. Instead of the Slowinski–Guyer test, contrasts 10 A between sister clades can be analysed using a trait absent trait present 5 B Wilcoxon signed-rank test. Vamosi and Vamosi equivocal (2005) reviewed various methods for calculating the contrasts to be tested (also see Isaac et al. 20 C 2003). ‘‘Simple’’ contrasts based on the absolute 40 D difference in species numbers between sister clades may seem straightforward, but do not 50 E account for the overall species richness of the pair and can be misleading. For example, Vamosi and 10 F Vamosi showed that simple contrasts of 1020 versus 1010 species and 20 versus 10 species 5 G result in the same test statistic, but represent two intuitively contrasting cases. Two alternative meth- 15 H ods of calculating the contrasts avoid this problem. Figure 17.1 Hypothetical phylogenetic illustrating the ‘‘Proportional’’ contrasts are calculated as the pro- mapping of both the character state for a trait (present, absent or portion of all species in the sister group represented equivocal) and the species numbers for each clade (A–H) at the by the clade possessing the trait of interest minus tips of each branch. The construction of specific contrasts is explained 0.5, so that it ranges from 0.5 to 0.5. in the text. À þ 316 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S used a simulation study to examine the perfor- group. In such cases, we used the species counts mance of these three contrast methods and from the clades that would be most conservative recommended using either log contrasts, when with regard to the hypothesis of contrasting sister groups are of similar age, or proportional diversification rates (i.e., the results are biased contrasts. against finding an effect). Because families are Note that all techniques based on the Wilcoxon arbitrary constructs and do not represent a well- signed-rank test necessarily rely on phylogenies, defined evolutionary unit, we used total species which in themselves constitute hypotheses. Thus, a numbers in our higher-level contrasts, instead of sister-group analysis should ideally incorporate averaging across the species richness of the con- the confidence in each phylogenetic hypothesis. Of stituent families, as is typically done in nested particular concern are large-scale phylogenies with contrasts of continuous variables. For example, in limited sampling and ability for analysis of statis- the contrast of E and F versus G and H in Fig. 17.1, tical support. Incomplete sampling can impact the we would contrast species counts of 60 and 20, nature of the comparisons profoundly. For rather than the average counts of 30 and 10. instance, in Fig. 17.1, A and B are sister clades and A, possessing the trait of interest, is also more 17.4.1 Trait datasets species rich. However if B was not sampled for the phylogenetic analysis, one would erroneously We first compared species richness between sister conclude that A and C are sister clades and that clades with biotic and abiotic pollination at the the clade lacking the trait is more diverse. Studies family level and higher. We used the data of Dodd based on comparisons of multiple sister groups et al. (1999), with species counts taken from Davies implicitly assume that such errors are not biased in et al. (2004). Additional pollination information one direction or another. was obtained from Watson and Dallwitz (2005), or from literature searches on the ISI Web of Science using the word ‘‘pollination’’ and the family name 17.4 Methods in the topic field. Pollination mode for each family was coded as either primarily biotic, primarily Our general approach in reanalysing the effects of abiotic, both modes present, or unknown, and we the four key traits on diversification was to review excluded families in the latter two categories from the plant groups identified in previous studies, analyses. identify new groups that could be added, update To assess the role of zygomorphy in diversifica- the taxonomic circumscriptions to reflect APGII tion, we expanded the dataset constructed by Sar- (2003), and update the phylogenetic information gent (2004). Character state determinations were according to Davies et al. (2004) and other available taken from Sargent (2004), Watson and Dallwitz phylogenies for lower taxonomic ranks. Indepen- (2005), Takhtajan (1997), and Mabberley (1997). dent contrasts between groups possessing and Families were considered zygomorphic if they were lacking the trait of interest were identified starting described as primarily having zygomorphic, bilat- at the tips of the phylogeny and proceeding erally symmetrical, irregular or bilabiate corollas, towards the root. Nested contrasts were removed whereas actinomorphic families were described as from higher-level contrasts, so that no group was having radially symmetrical, polysymmetric, or used in more than one contrast. For example, in regular corollas. Only animal-pollinated families Fig. 17.1 we would calculate a contrast between A are considered in this analysis, because the and B, and then remove that contrast from the tree hypothesis for how floral symmetry affects diver- and calculate another contrast between C and D. sification depends on plant–pollinator interactions. We used the consensus tree presented by Davies To be conservative in finding an effect, Sargent et al. (2004) for family-level and higher contrasts. (2004) subtracted actinomorphic genera from This tree includes several unresolved nodes, which zygomorphic families, but did not subtract zygo- causes uncertainty about the appropriate sister morphic genera from actinomorphic families. As F L O R A L C H A R A C T E R S A N D S P E C I E S D I V E R S I F I C A T I O N 317 this method could bias the results, we did our from each sister group possessing the trait of analysis both with and without these subtractions. interest to examine qualitatively whether the data For nectar spurs, many contrasts between spur- exhibited a U-shaped distribution. We then per- red and non-spurred groups occur within families. formed one-tailed Wilcoxon signed-rank tests on Therefore we searched the literature to find as simple, proportional and log contrasts (see Section many spurred taxa as possible, regardless of rank, 17.3). For datasets including monotypic groups, we and to determine their putative sister clade. added one to all species numbers before log- Spurred lineages surveyed previously by Hodges transformation. For each contrast, we assigned a (1997a, b) and Hodges and Arnold (1995) were positive to contrasts matching our hypothesis and reviewed for more recent evidence regarding sis- a negative to those opposing it. We excluded cases ter-group relationships. In addition, we searched for which the focal and sister clades have equal the literature for phylogenetic data identifying species numbers, because they are uninformative. sister groups for additional spurred lineages that were unavailable in previous analyses. We exclu- 17.5 Results ded groups possessing nectarless spurs, which may not function in the hypothesized manner, and 17.5.1 Pollination mode groups with flowers described as only saccate. We Of 379 families included in this analysis, we included the Marcgraviaceae, which does not have identified 39 with abiotic pollination, 202 with spurs within flowers, but rather highly modified animal pollination, 17 with both modes present, floral bracts that form elaborate extrafloral nec- and 121 for which there is insufficient information. taries (Ward and Price 2002), which pollinators For these data we found 16 independent contrasts probe to access to nectar in a similar manner to between pollination modes. Because the animal- probing nectar spurs. We also considered whether pollinated clade contained more species than the each group is zygomorphic or actinomorphic (e.g., abiotically pollinated clade for 11 of the 16 Plate 7d versus 7e) to test whether spurs correlate contrasts (Electronic Appendix 17.1, http://www. with diversity for the subset of instances in which eeb.utoronto.ca/EEF/), the frequency distribution nectar spurs evolved independently of floral of the proportion of biotically pollinated species in symmetry. sister groups was strongly L-shaped (Fig. 17.2a). For our analysis of dioecy, we reviewed the Indeed, regardless of the contrast measure used, dioecious taxa identified by Heilbuth (2000). animal pollination seems to promote significantly Lineages were considered dioecious if most or all higher diversification (Table 17.1). A notable of the species exhibit separate sexes on different exception to this pattern is the contrast between individual plants, whereas lineages were con- the animal-pollinated Bromeliaceae and an abioti- sidered non-dioecious if most or all of the species cally pollinated clade including the Poaceae, Jun- exhibit both sexes on the same individual plants. caceae, and Typhaceae. To avoid inflating the number of species in the non-dioecious sister group, we either subtracted any dioecious genera from the non-dioecious 17.5.2 Floral symmetry families, or subtracted the estimated number of dioecious species, if this information was available. We found 22 independent contrasts in floral sym- Information on dioecious genera was taken from metry among animal-pollinated angiosperms Mabberley (1997), Takhtajan (1997), and the data- (Electronic Appendix 17.2). Only 16 of these con- base of Renner and Ricklefs (1995). trasts involve the predicted higher species richness in clades with asymmetric flowers and the fre- quency distribution of the proportion of species in 17.4.2 Analyses sister groups with asymmetric flowers is distinctly For each trait of interest, we first constructed a U-shaped, with most contrasts being either frequency distribution of the proportion of species strongly positive or strongly negative (Fig. 17.2b). 318 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S

10 12 (a) (b) 8 10 8 6 6 Count 4 Count 4

2 2

0 0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Proportion Biotic Pollination Proportion Zygomorphic

10 12 (c) (d) 8 10 8 6 6 Count 4 Count 4 2 2

0 0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Proportion Spurred Proportion Dioecious

Figure 17.2 Frequency distributions of the proportion of species in a sister group represented by the clade exhibiting (a) biotic pollination, (b) zygomorphic floral symmetry, (c) presence of floral nectar spurs, and (d) dioecious sexual system.

results suspect. Furthermore, the sister group for Table 17.1 Results of Wilcoxon signed-ranks tests for the effects of four reproductive characters on diversification. one contrast, with 10 zygomorphic families including Marantaceae (total of 1549 spp., Elec- Character Method of Ranking tronic Appendix 17.2), was unresolved and could involve either the relatively species-rich Arecaceae Difference in Proportion Log (high)/ (2500 spp.), or the relatively species-poor Rapata- species numbers log (low) ceae (80 spp.), or both combined. In our analysis Biotic pollination P 0.033 P 0.005 P 0.005 (Electronic Appendix 17.2) we used the Rapataceae ¼ ¼ ¼ Zygomorphy P 0.035 P 0.009 P 0.010 ¼ ¼ ¼ as the sister group, as this relationship supported Spurs P 0.137, ns P 0.019 P 0.007 ¼ ¼ ¼ the hypothesis of greater diversity in the zygo- (P 0.010) (P 0.007) (P 0.007) ¼ ¼ ¼ morphic clade; however, use of either other pos- Dioecy P 0.019 P 0.025 P 0.020 ¼ ¼ ¼ sible sister group reduces statistical support for For each character, we considered three methods for calculating the zygomorphic flowers promoting diversification difference in diversity between clades, as described in the text. For and amplifies the U-shaped distribution. floral nectar spurs, the results in parentheses represent tests that considered only comparisons for which both sister groups have the 17.5.3 Floral nectar spurs same floral symmetry. Probabilities represent the results of one-tailed tests for each comparison, because test comparisons addressed a We found 16 independent origins of floral nectar specific directional hypothesis. spurs for which the sister group can be identified (Electronic Appendix 17.3). For 12 cases, the The statistical tests for proportional and log con- spurred group includes more species than its sister trasts are both significant (Table 17.1); however, clade. A one-tailed Wilcoxon signed-rank test of the U-shaped distribution makes the statistical simple contrasts rejected an association higher F L O R A L C H A R A C T E R S A N D S P E C I E S D I V E R S I F I C A T I O N 319 diversification with nectar spurs, whereas tests of results to previous analyses occurred despite proportional and log contrasts are highly sig- our use of a different phylogenetic tree, which nificant (Table 17.1). Furthermore, the proportion represents angiosperm families much more com- of species with nectar spurs in sister groups has a pletely, and new statistical methods. The general distinctly L-shaped frequency distribution robustness of these results to new analyses pro- (Fig. 17.2c) that is consistent with spurs promoting vides strong support to the conclusion that a diversification. Restriction of the dataset to con- variety of floral traits thought to affect the like- sider only comparisons for which the sister groups lihood of speciation or extinction contribute to have the same floral symmetry resulted in 10 species diversification. However, note that every independent comparisons, of which 9 have more trait that we considered had contrasts that span the species in the spurred group compared with its full range of outcomes (Fig. 17.2). Thus, the effect sister clade. Each of the signed-rank tests for this of any of these traits on diversification is likely to restricted analysis detected significantly greater be context dependent, with other factors influen- diversity in spurred clades (Table 17.1). cing specific cases. Also, the general pattern found for any trait need not explain the true causal factor for diversity in any specific contrast, even those 17.5.4 Dioecy strongly supporting the general trend. As we We identified 29 independent contrasts in sexual emphasize below, even when a multiple sister- system among angiosperms (Electronic Appendix group analysis supports a hypothesis for diversi- 17.4). For 18 contrasts, the dioecious clade has fication, these correlations should represent start- lower species richness, whereas the opposite is ing points of more thorough phylogenetic and true for 9 contrasts. Two comparisons, Barbeya- population analyses. ceae versus Dirachmaceae, and Myricaceae versus In contrast to the expectation that changes in Juglandaceae, involve equivalent species numbers phylogenetic reconstruction should not favour one between sexual systems, and we excluded them hypothesis or another, the difference between our from the analysis. The frequency distribution for results and those of Sargent (2004) suggest that the proportion of species in sister groups with a even a strong association between the evolution of dioecious sexual system is L-shaped (Fig. 17.2d), trait and subsequent diversity should be treated with dioecious clades being less diverse cautiously. Such caution is especially necessary in than hermaphroditic clades. Regardless of the the absence of additional data supporting the contrast measure used, dioecy is associated functional hypothesis (see below). In this specific with significantly lower diversification rates case, we found that although most contrasts sup- (Table 17.1). port the hypothesis that the evolution of zygo- morphic flowers enhances diversification, a 17.6 Discussion substantial number of the contrasts support the exact opposite conclusion. With one exception, our reanalysis supports pre- The results for flower symmetry should be vious findings that the evolution of floral traits can treated prudently, as zygomorphy may influence alter subsequent species richness within clades. diversification in some instances, but not others. Like previous analyses, we found that the evolu- As noted in Section 17.2, zygomorphy may influ- tion of animal pollination (Dodd et al. 1999) and ence reproductive isolation by constraining the floral nectar spurs (Hodges and Arnold 1995; orientation of pollinators during flower visitation, Hodges 1997a, b) enhanced species diversification, encouraging precise placement of pollen on their whereas the evolution of dioecy retarded diversi- bodies. However, some species with actino- fication (Heilbuth 2000). In contrast, our results morphic flowers may have other traits that restrict cast some doubt on Sargent’s (2004) conclusion the position of pollinators while they visit flowers, that the evolution of bilaterally symmetric flowers such as inflorescence architecture and flower affects diversification. The overall similarity of our orientation. For example, bees visiting flowers 320 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S arranged in a raceme inflorescence (e.g., Chamerion context in which it evolves. If the evolution of angustifolium) do so primarily from the bottom nectar spurs promotes diversity by facilitating towards the top and so approach each flower from pollinator transitions, this role can be played only a similar angle (Routley and Husband 2003), in the presence of a diverse pollinator fauna. Per- especially if the flower face is roughly vertical. haps Halenia encountered a sufficient pollinator Similarly, hummingbirds visiting Aquilegia formosa diversity for nectar spurs to affect diversification do so by probing the nectar spurs in a precise only after invading South America (von Hagen manner causing their chin to brush against the and Kadereit 2003). Unfortunately, little is known anthers and stigmas. Thus, not all zygomorphic about the pollination biology of this group, so that taxa may have more precise pollen placement or this latter hypothesis remains untested. pollinator specificity than their actinomorphic sis- A second type of phylogenetic analysis explores ter groups. We encourage studies concerning how a trait may affect diversification. For instance, whether the evolution of zygomorphy enhances the hypothesized effects of both animal pollination diversity in specific cases and especially studies of and floral nectar spurs on diversification involve how zygomorphy may enhance pollinator and an increased likelihood of transitions to novel pollen placement specificity and reproductive iso- pollinators, thereby promoting reproductive isola- lation, which remain uncertain, as it has never tion and thus speciation (Hodges and Arnold 1995; been tested explicitly. Dodd et al. 1999). Therefore, a species-level phy- Speculation about how a trait affects rates of logeny should reveal frequent transitions to novel species diversification points to a general problem pollinators, especially for recent radiations for with tests such as those performed here. Associa- which extinction is less likely to influence clade tions of diversification with specific traits are, of diversity. Unfortunately, few species-level phylo- course, just associations. Any additional factor that genies are currently available for entire groups, co-varies with the trait of interest could be the true particularly for those in which a trait correlated causal factor for increased diversification, even if it with species diversification has evolved recently is not recognized. Thus, although identification of (although see Beardsley et al. 2003; Kay 2005; characters that associate significantly with diver- Whittall 2005). In addition, although transitions sification is an important first step, additional between major pollinator types (e.g., bee and analyses of how a particular trait affects speciation hummingbird) are most convincing, transitions or extinction are essential for testing whether a between different species within a major pollinator true causal relationship exists. type may also provide reproductive isolation. One such analysis requires more detailed phy- Thus, detailed knowledge of pollination in multi- logenetic sampling for specific examples of the ple species will be needed for a full analysis. origin of a trait. For instance, von Hagen and Lack of phylogenetic information also restricted Kadereit (2003) examined a detailed phylogeny for our analyses because the sister-group relationships Halenia, which possesses floral nectar spurs. Sim- could not be determined for many groups. This ple sister-group analysis shows that Halenia has difficulty is especially problematic for our review many more species than its non-spurred sister of floral nectar spurs, because spurs are commonly group; however, von Hagen and Kadereit (2003) generic, rather than family, traits and or showed that diversification did not follow the species-level phylogenetic information is com- evolution of nectar spurs immediately. Rather, paratively rare. Consequently, we had to exclude diversification seems to have increased after the many groups from our analyses, reducing the invasion of South America by a subclade of the power of our tests. In addition to reducing the genus. Although contradicting an immediate number of comparisons available for simple tests, diversification effect of nectar spurs, this pattern is such as those described here, this lack of phylo- consistent with a general hypothesis of a key genetic information precludes more detailed ana- innovation (Simpson 1953), which considers two lyses needed to tease apart the effects of multiple factors, the origin of the trait and the ecological characters. F L O R A L C H A R A C T E R S A N D S P E C I E S D I V E R S I F I C A T I O N 321

Many characters probably affect the rate of key innovation. As yet, such analyses tend to be either speciation or extinction. For instance, performed on specific groups for which the Vamosi and Vamosi (2004) performed nested necessary phylogenetic information is available analyses of the effect of dioecy, while also con- (e.g., spur evolution in Halenia described above). trolling for woody versus herbaceous growth More detailed tests, such as these, performed on habit, tropical versus temperate distribution, and multiple groups that have evolved the same trait fleshy versus dry fruits. Based on the detailed would be especially useful for exposing how par- analysis of dioecious genera and their non-dioe- ticular traits affect the timing and tempo of cious sister lineages, Vamosi and Vamosi found diversification. significantly slower diversification in dioecious Last, tests of a key-innovation hypothesis can groups, which is ameliorated somewhat for clades focus on whether a particular trait actually affects with a tropical distribution and/or fleshy fruits. speciation and/or extinction. For example, Fulton They attributed these results to opposing effects on and Hodges (1999) showed that aspects of nectar extinction, with dioecy increasing the risk of spurs in Aquilegia (spur length and orientation) extinction, whereas fleshy fruits, a tropical dis- affect pollinator visitation and pollen removal (and tribution, and possibly woody growth reduce the therefore, presumably, pollen dispersal) and risk of extinction. This untangling of multiple fac- Hodges et al. (2004) showed that nectar spur colour tors was possible only with a large number of affects pollinator visitation. Such studies link var- sister-group comparisons derived from a fairly iation in nectar spurs directly to pollinator beha- comprehensive character database and phyloge- viour and reproductive isolation. Several other netic information below the family level. studies suggest that nectar spurs affect pollinator We attempted a similar analysis for floral nectar visitation or pollen dispersal, including studies of spurs, because 6 of 16 sister-group comparisons orchids (Nilsson 1988; Johnson and Steiner 1997) coincide with a change from actinomorphic to and Epimedium (Suzuki 1984). More such studies zygomorphic flowers. Comparisons within sym- are needed, particularly between sister species, to metry classes found even stronger support for the test fully how proposed key innovations affect association of spurs with higher diversification speciation or extinction. Studies such as these are rates (Table 17.1). Although these specific tests particularly amenable in hybrid zones (Chapter 18) involve fewer comparisons, they are more robust, between species that differ in the trait of interest. because a potentially confounding factor has been Our review also revealed the remarkable lack of excluded. knowledge about the floral biology of most plant More detailed phylogenetic information would species and, therefore, the need for more studies of enhance the study of diversification in several floral biology. This lack of information hindered other ways. With more complete taxon sampling our tests of hypothesized associations of floral and information on the timing of lineage diver- traits with diversification rates. For example, we gence, more powerful methods can be used to found no information on the dominant mode of detect changes in diversification rates. Sanderson pollination for over 30% of plant families (121 out and Donoghue (1994) developed a maximum- of 379). This paucity of information probably likelihood approach that employs the diversities of resulted in fewer independent contrasts in our three branches of a clade and determines which data set, inaccurate estimates of species numbers models of changes in diversification best fit these in some sister clades, and misidentification of diversities. Wollenberg et al. (1996) proposed a some sister-group relationships. Although these method for comparing branching patterns in problems need not have biased our tests in favour empirical trees to those generated by a stochastic of increased diversification, we note again how model of speciation and extinction. Ree (2005) also new information can alter the interpretation of proposed using stochastic models of speciation, associations with diversity, as we found for zygo- but allowed for uncertainty in the tree topology morphy. The Eriocaulaceae illustrate this point. and for multiple gains and losses of the putative Although this family is listed as having either 322 E C O L O G Y A N D E V O L U T I O N O F F L O W E R S biotic or abiotic pollination (Watson and Dallwitz them and Tim Holtsford for careful reading and 2005), the pollination system of the family was first suggestions that improved our manuscript, along reported only recently, for two species of Syngo- with an anonymous reviewer who provided thor- nanthus (Ramos et al. 2005). This study found clear ough comments. We gratefully acknowledge grant evidence of animal pollination in these species, support from the NSF (EF-0412727) and a National despite contrary predictions of other authors, and Parks Ecological Research Fellowship to KMK. suggests that other species that have been con- sidered abiotically pollinated in the family may actually be animal pollinated as well. 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