The Systematic Distribution of Tannins in the Leaves of Angiosperms: A Tool for Ecological Studies
SIMON MOLE School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588, U.S.A.
Key Word Index--Tannins; systematics; proanthocyanidins; protein precipitating phenolics; plant-defenses.
Abstract--The systematic distribution of tannins in foliar tissues has not been comprehensively reviewed for the Angiosperms in over 20 years. Here their systematic distribution is assessed using data based on protein precipitation or chemically specific tests. Fewer families are characterized by the typical presence of tannins than has previously been reported, and a greater variation in the occurrence of tannins in species sampled from within single plant families has been detected. This study presents the proportion of tannin-containing species in angiosperm families arranged according to the system of Cronquist for the first time. The potential utility of these data in testing ecological ideas about the distribution of tannins, such as those based on plant habit or life-history, is discussed.
Introduction Tannins have featured prominently in ecological studies of plant-herbivore interactions (Rhoades, 1979; Mole and Waterman, 1987a; Bernays etal . , 1989), as well as in related work concerning the ways in which plants allocate resources to defensive and other physiological functions (Bryant et al., 1983; Coley et al., 1985). Systematic information about the distribution of tannins is critical to the investigator who wishes to locate or avoid tannin containing taxa. It is also basic information for evaluating predictions about the distribution of tannins which arise from theoretical work in ecology, such as those developed from the concepts of plant apparency (Feeny, 1976) or carbon/ nutrient balance (Coley et al., 1985). The present study synthesizes the available information on the distribution of tannin in leaves, with these potential uses in mind.
Over 20 years have elapsed since Bate-Smith analyzed the distribution of tannin in the Dicotyledonae (Bate-Smith and Metcalfe, 1958), and Monocotyledonae (Bate- Smith, 1968). Since then, a considerable amount of new data has become available and our ideas about what a tannin is, and what constitutes an acceptable test for a tannin, have also changed. For instance, much of the information upon which Bate- Smith based his analyses was derived from viewing microscope slides in which phenolics were stained with iron salts (Metcalfe and Chalk, 1950). Iron based reagents have long been used by microscopists to detect tannins (Chalker-Scott and Krahmer, 1989) but in these studies the word " tann in" has been used as a generic name to cover a multitude of substances that react with this stain. In ecological studies we are now much more concerned to define tannins as that subset of plant phenolics which precipitate proteins from aqueous solution (Mole and Waterman, 1987b,c). Tannins are thus detected by protein precipitation assays or by tests specific for particular groups of chemicals such as condensed tannins (proanthocyanidins) or hydrolysable tannins, which typically act as protein precipitants.
Bate-Smith (Bate-Smith and Metcalfe, 1958; Bate-Smith, 1968) was aware of this distinction between histochemical and chemical tests for tannins and he made chemical tests for the degradation products of tannins (anthocyanins, ellagic acid) in hydrolyzed samples of plant material. Working within an Englerian taxonomy, he reported such chemical results for 544 dicotyledonous species drawn from 162 (Received 14 October 1992)
833
834 SIMON MOLE
families and 391 monocotyledonous species from 43 families. The present study adds to this chemical data of Bate-Smith using observations from more recent studies using similar chemical but not microscopic techniques. Protein precipitation techniques have also been used extensively to determine the presence or absence or tannins in plant samples in recent years (Mole and Waterman, 1987c) and such data have also been utilized in this study. By combining chemical and protein precipitation based data, the number of species studied in the Dicotyledonae has been increased four-fold while that for the Monocotyledonae has been almost doubled.
On the basis of these data, the systematic distribution of tannins in the angiosperms has been reanalyzed with the taxa arranged according to the system of Cronquist as adopted by Maberly (1987). This is intended to allow the use of this information, in conjunction with Maberly's (1987) dictionary, as a basis on which to estimate the likelihood that tannins will be found in plant taxa.
Materials and Methods Original studies reporting both the presence and the absence of tannins in plant species have been used as the sources of data in this work. Studies only reporting the presence of tannins in plants have been ignored to avoid a bias against data on plants which do not contain tannins. In keeping with Bate-Smith and Metcalfe (1958) and Bate-Smith (1968), the data only relates to observations made on leaves with the exception of a few non-woody herbs where whole plant examples were used. Specifically excluded were data on stems, twigs, bark, roots, flowers, fruits, seeds, etc.
The sources of data are specified below (see Results). Typically they are systematic surveys or studies of the food preferences of highly polyphagous mammalian herbivores. Most of the data used was originally reported as presence/absence data, which is almost exclusively the case for data from protein precipitation tests and that from Bate-Smith (Bate-Smith and Metcalfe, 1958; Bate-Smith 1968). However 8% of the whole data set was derived from the results of spectrophotometric assay procedures such as the proanthocyanidin or vanilin assays. These data were of a continuous nature and they were scored as p~esence/absence values with zero or trace absorbances being treated as absences. The original authors are assumed to have correctly identified each species to at least the generic level. Maberfy (1987) has then been consulted to assign genera to families where the original authors did not follow Cronquist's system or where no family level classification was given.
Results Protein precipitating phenolics in the Dicotyledonae Bate-Smith's original survey used chemical analyses aimed at detecting condensed tannins and ellagitannins and the assumption is made here that both of these types of tannin act as protein precipitating phenolics. These chemical data (Bate-Smith and Metcalfe, 1958) have been combined with observations from reports in which protein precipitation assays have been used to detect tannins. ]he reports used were those by Atal et aL (1978), Bate-Smith (1976, 1977), Hungund and Pathak (1971), Jung et aL, (1979), Marks et aL (1988), Wall et al. (1959), Saxena (1975) and Valenzuela and Gracie (1982). Of the 2277 species reported, 35.2% were positive for the presence of tannins.
The present data set is over four times as large as that available from the chemical data of Bate-Smith and Metcalfe (1958) and so the number of repeat observations is much improved for most families. Monotypic families or those with very few species are rare in the data which is a sample and not even close to a census for most families (see Appendix 1 for sample number per family). As an illustration of the increased sampling rate in the present study, chemical measurements were made on only single species samples for 56 families in Bate-Smith's study while this number is reduced to 26 in the present survey which encompasses 181 families including 19 not previously covered. In the present survey, analyses for tannins have been made on five or more species for 57% of all families in contrast to 27% in Bate-Smith's study. A more complete comparison is presented in Fig. 1 which shows the frequency distributions for the number of observations made per family in both the present study and that of Bate-Smith and Metcalfe (1958).
Figure 2A presents a histogram of the proportions of species testing positive for the
SYSTEMATIC DISTRIBUTION OF TANNINS
08==
P~ 0.6
2 04
£ o - 0.2
A
~ m . - ~ 60 120 180 24O
Number of species exemined per forniLy
835
0 4
B
0 3
"~ 0 2
°°'o n
0 12 24 36
Number of species exomined per fomi ly
FIG. 1. FREQUENCY D~STRIBUTIONS OF THE NUMBER OF SPECIES EXAMINED PER FAMILY IN (A) THE COMPLETE DATA SET USED IN THIS STUDY AND (B) THE DATA FROM BATE-SMITH AND METCALFE (1958).
presence of tannins in each family. From this it can be seen that families tend to have high or low but not average numbers of tannins containing species. Given the presence/absence nature of the data, simple binomial probabilities can be used to test the significance of hypotheses concerning the proportion of tannin containing taxa in a particular family. For instance, all four species from the Myricaceae tested positive for tannins and the probability of obtaining four positive results where the overall proportion of plants containing tannin is 0.35 is low (P<0.015; Beyer, 1966). Thus one can infer that species in the Myricaceae are more likely to contain tannins than the overall 35% frequency seen for the dicots as a group. For families where more species have been examined, confidence can be placed in results indicating large deviations from the 0.35 overall proportion for the dicots. Some of these more robust results are exemplified by the Grossulariaceae where all 51 species tested positive for tannins or for the Umbelliferae where none of the 26 species tested positive.
As sample sizes increase, so does the probability that false positive or negative results are present. However, exceptional positive or negative results cannot be assumed to be mistaken. For instance, in the Compositae where only 4% of the 228 species tested positive for tannins, Bate-Smith (1980) demonstrated the unusual presence of tannins for one of these positive cases. The importance of such results
836 SIMON MOLE
60
50
40
20
60
50
4o
so
10
O I 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9
Proport ion of tGnnin conta in ing species
I 0
0 O I O ? 0 5 0 4 0 5 0 6 0 7 O[q 0 9 I i
Proporbon of tann in conta in ing species
FIG. 2. HISTOGRAMS OF THE PROPORTION OF TANNIN CONTAINING SPECIES PER FAMILY IN (A) THE COMPLETE DATA SET USED IN THIS STUDY AND (B) THE DATA FROM BATE-SMITH AND METCALFE (1958).
lies in demonstrating that even families such as the Compositae contain members able to express a tannin containing phenotype.
Table 1 presents the occurrence of tannins in the leaves of all dicotyledonous plants. Families where the frequency of tannin is 50% or more in the species samples are distinguished from those where this frequency is lower than 50%. With the families placed in the arrangement of Cronquist (Maberly, 1987), it is clear that the distribution is far from random. Most striking is that for all the 26 families tested in the Asteridae, all are typically tannin free. In 18 of these families the proportion of species containing tannin is 10% or less. There are only two families where tannins are at all frequent these being the Rubiaceae (44%) and the Caprifoliaceae (42%), The one other subclass with notably few tannin containing taxa is the Caryophyllidae. Here there is a useful dichotomy in that tannins are found in the single families present in the Polygonales and Plumbaginales while tannins are entirely absent from seven families in the Caryophyllales and are present in less than 20% of the species in the two other families surveyed.
Something of the reverse is the case for the Hamamelidae where tannins are frequent in all but one of fourteen families tested. The one exception is the Moraceae which may be a family where condensed tannins are typically present but not at
SYSTEMATIC DISTRIBUTION OF TANNINS 837
TABLE 1. DISTRIBUTION OF TANNINS AMONG SUBCLASSES, ORDERS AND FAMILIES OF DICOTYLEDONOUS PLANTS*
*Subclass and order names are given in full in the order they appear in Maberly (1987). Numbers in the body of the table refer to the numbers of the families as given in Maberly (1987). "Prop" refers to the proportion of species in which tannins occur. Nrefers to the total number of families in an order.
sufficient levels to preciptate proteins efficiently. Such problems of methodology, which may affect the results, are discussed below.
For the three subclasses considered so far, the relatively ordered distribution of families with members that typically do or do not contain tannins allows for some confidence that Cronquist's system may be of predictive use. In the remaining three subclasses no such order can be seen: There are just a few orders where every family surveyed typically does or does not contain tannins. Examples of the former case are the Theales and the Proteales while the Capparidales and the Papaverales exemplify the latter. Beyond this, predictions about whether a plant is likely to contain tannins depend on information about that family alone which is detailed in Appendix 1. In overview, the finding here is that some but certainly not all of the subclasses and orders of Cronquist's system are homogeneous with respect to the likely occurrence of tannins in their subordinate taxa.
Condensed tannins in the Dicotyledonae By consulting studies where specific chemical tests rather than protein precipitation
assays have been used to detect condensed tannins, information on this specific type of tannin has been obtained. The key distinction here has been to include data based on methodology involving the detection of anthocyanins released from the cleavage of proanthocyanidins, and to exclude data based on other methods. Thus, here we are dealing with a chemically defined subset of the protein precipitating phenolics present in the Dicotyledonae. The studies used were those by Burrer et aL (1981), Coley (1983), Daniel and Sabnis (1977), Gartlan et al. (1980), Janzen and Waterman (1984), Julkunen-Ziitto (1986), Marks et al. (1988), Oates et al. (1977, 1980), Cooper and Owen-Smith (1985), Rogers et aL (1990), Satyvathi et al. (1984) and Waterman et al. (1983, 1988). These data have been used to provide an independent check on Bate- Smith and Metcalfes' (1958) results for condensed tannins (leucoanthocyanins) exclusive of his data on hydrolysable tannins (ellagic acid).
Data on a total of 393 species from 81 families was obtained. For those families examined by both Bate-Smith and the other studies consulted here, there is 80% agreement as to whether the species in a family typically do or do not contain condensed tannins. From the 20% of cases where there is a discrepancy between the two sources of data, the eleven families with the largest number of species sampled per family are presented in Table 2. For families not presented in Table 2,
SYSTEMATIC DISTRIBUTION OF TANNINS
TABLE 2. PROPORTIONS OF TANNIN CONTAINING TAXA AS DETERMINED FROM THREE SOURCES OF DATA*
*ECBS refers to data on condensed tannins from Bate-Smith and Metcalfe (1958), CT refers to data from other determina- tions that are chemically specific for condensed tannins. PPT refers to data derived from protein precipitation assays alone.
discrepancies probably arise because small numbers of species, and different species within each family, have been tested by different investigators.
In contrast, the results in Table 2 need some explanation. The problem of different investigators working with different species may still apply here, however there are also families where results for condensed tannins are in agreement with each other but not with those dependent on protein precipitation assays. For example, in 10 out of 13 cases in Table 2, the least proportion of tannin containing species has been found with the protein precipitation based assays. Two technical reasons may account for the lower proportion of positive results with precipitation assays. One is that threshold effects are frequently encountered, where a certain minimum amount of tannin is required before any precipitation is seen (Mole and Waterman, 1987c). This would lead to false negative results. The second is that spectrophotometric data is continuous rather than discrete (presence/absence) and so any absorbance greater than that assayed for control reactions leads to non-zero numerical data which are routinely reported in the literature. Such apparently positive results may be mis- leading if some artifactual reaction leads to the increased absorbance. When condensed tannins are degraded to yield anthocyanins a characteristic red color is produced which Bate-Smith required before reporting the presence of tannins.
It is impossible to tell from assay data based on mechanically determined absorbance readings whether the color reaction was normal or otherwise. For both of these reasons, it is not surprising that fewer positive results are obtained with protein precipitation based tests.
Hydrolysable tannins in the Dicotyledonae ff hydrolysable tannins were an important component of protein precipitating
phenolics, we might have expected a greater success in detecting tannins by protein precipitation than by specific tests for condensed tannins. Instead, the preceding results do not suggest that hydrolysable tannins are a major component of protein precipitating phenolics, although, differences in the sensitivities of the methods preclude a firm statement on this point. With regard to data with which the frequency with which hydrolysable tannin occur can be calculated, we have not progressed much beyond the work of Bate-Smith and Metcalfe (1958). However, very considerable progress has been made in the structural elucidation of these tannins and Okuda et aL (1983) provide a systematic review of the taxa in which these have
840 SIMON MOLE
been found. Cronquist (1981) also covers the likelihood of encountering ellagic acid (ellagitanins) and gallic acid (gallotannins) in each family.
Protein-precipitating phenolics in the Monocotyledonae Substantially less new information has become available about the monocots
relative to the dicots, and in particular there is less information based on protein precipitation assays. It is also the case that hydrolysable tannins appear to be absent from these plants (Cronquist, 1981) so that it may be taken that the following refers exclusively to the occurrence of condensed tannins. The results presented in Table 3 are derived from Bate-Smith (1968) together with information from other studies using the same chemical and protein precipitating techniques used in the studies of dicots. The studies used were by Atal et al. (1978), Harborne (1979), Hugund and Pathak (1971), Jung et al. (1979), Laracine et aL (1985), Marks et aL (1988), Mereh et aL (1986), Wall et aL (1959), Rogers et aL (1990), Saxena (1975), Waterman et aL (1983), Williams and Harborne (1977, 1975) and Williams et al. (1991).
The results in Table 3 may surprise those who hold the view that monocots are typically tannin free: A significant number of families contain species that typically test positive for tannins. See Appendix 1 for details on specific families.
Discussion The major findings of this study are that there is litle evidence that all members of a plant family either do or do not contain tannins, and that overall, most dicotyledonous families contain species which do not typically contain protein precipitating phenolics. This position differs from that of Bate-Smith and Metcalfe (1958) who concluded that
TABLE 3. DISTRIBUTION OF TANNINS AMONG SUBCLASSES, ORDERS AND FAMILIES OF MONOCOTYLEDONOUS PLANTS ~
Family with tannin Without taenm
Subclass/order prop. - >0.50 prop. <:050 N
Subclass L Alismatldae 1. Alismatales
2. Hydrocharitales 3. Najadales 1,6
Subclass I1. Arecldae 1. Arecales 1
2 Cyclanthales
3 Pandanales
4 Arales
Subclass III. Commehnidae 1 Commelinales 2
2 Eriocaulales
3 Restonales 1, 3
4. Juncales
5. Cyperales 7. Typhales 1, 2
Subclass I~Z Zingibendae 1 Bromeliales
2. Zingiberales 3, 6
Subclass V. Lihidae 1. LiHales 1, 2, 3, 15
2. Orchida~es
1 3 3 1 1
2, 4, 10 10
1
1
1,2
3,4
1
1
1,2
]
2 ,5 ,7
4, 5, 6, 7, 8, 9, 10, 12, 14 1!5 4 4
*Subclass and order names are given in full in the order they appear in Maberly (1987). Numbers in tile body of the table refer to the numbers of the families as given in Maber]y (1987). "Prop" refers to the proportion of species in which tannins
occur. N refers to the total number of families in an order.
SYSTEMATIC DISTRIBUTION OF TANNINS 841
every species examined contained tannins in 82 families and that species in 52 families gave entirely (38) or mostly (14) negative ,results (see Fig. 2B). Only a relatively small proportion of families gave what Bate-Smith and Metcalfe (1958) considered to be mixed results for the occurrence of tannins.
The present reassessment is based on a consideration of three factors, these being analytical, statistical and taxonomic. The rationale for discounting the microscopic and histochemical evidence employed by Bate-Smith has been explored above. This change in the choice of analytical techniques and the change to Cronquist's system of taxonomy accounts for some of the changes made in this reassessment. The third, statistical, consideration concerns the sampling distributions (Fig. 1). Where only one species from a family is tested, then the result must be either entirely positive or negative for that family. Where two species are tested per family, then even if one samples only from families in which tannins occur with a frequency of 50%, half the results will be either all positive or all negative because of the binomial nature of the "trials". However, as the number of species sampled per family rises the binomial expectation for entirely positive or negative results declines rapidly.
For this reason, the larger size of the data set in this study has considerably increased the number of families which Bate-Smith and Metcalfe (1958) would have classified as mixed, (note horizontal scales in Fig. 1A vs 1B). Again, the reason for this is distributional and concerns what is now a relatively low number of families from which only one or two species have been sampled. This, together with the generally lower detection rate for tannins using assays based on protein precipitation techniques, has reduced the number of families where all species test positive for tannins. As a group, families that typically test positive for tannins are now in the minority relative to those that typically do not (see Fig. 2A vs 2B).
In summary, there would appear to be much more variability in the presence or absence of tannins within a family than was previously thought. For a population of species in which tannins occur with a hypothesized 10% probability, even a run of 20 negative results (P = 0.12) would not be a statistically significant reason to reject the hypothesis in favor of believing that tannins were never found in the family. Very few families have had as many as 20 representative species tested for tannins and so it is likely that the present study continues to underestimate such variation within families.
Phylogenetic constraints to tannin production? A feature of much current research is the exploration of historical factors and the
extent to which they may affect present day ecological processes (Brooks and McLennan, 1991). If Cronquist's system were to approximate to a phylogeny for angiosperms, then at least for some orders and subclasses, there appear to be patterns in the present day ocurrence of tannins that reflect this. Two questions need to be explored in this context. Is there (i) circularity in the argument because the construction of the taxonomy was dependent on the occurrence of tannins and if not then (ii) does the taxonomic system reveal a phylogenetic pattern in the distribution of tannins?
In Cronquist's (1981) work, chemosystematic information has clearly been employed and each family description in his "Classification of Flowering Plants" includes both histochemical and phytochemical data on the presence or absence of tannins. However, this classification is based on a multitude of factors most of which are morphological among which tannins do not appear to be critical and for this reason circularity is largely avoided. The less useful side of this approach is that this system is an evolutionary taxonomy and not the product of phylogenetic systematics sensu Brooks and Wiley (1986). Until such a cladistic system is devised the pattern in which tannins occur within taxa above the family level is likely to remain indistinct and
842 SIMON MOLE
subject to debate, although few would argue against the idea that there is some phylogenetic pattern in the present day occurrence of tannins.
The one generalization that Bate-Smith and Metcalfe (1958) made was that within the Dicotyledonae, modern representatives of more primitive or typically woody families tend to contain tannins. They used the Advancement Index of Sporne (1954, 1975) as their metric of whether a family was primitive or not. An attraction of this index is its basis in fossil evidence which provides for some independence from taxonomic systems. The Pearson correlation coefficient of the index with the propor- tion of tannin containing species per dicotyledonous family found in the present study is --0.445 (P>0.99 for r<0.00) which indicates a loss of tannins with advancement, This is a result that is still in agreement with Bate-Smith and Metcalfe (1958).
The ecological utility of the data The frequencies with which tannins occur in particular families can be used to
predict the likelihood that tannins occur in each species of a plant community, given an enumeration of the species present. With additional data on the species' life history, such as relative abundance, phenology and habitat preference, alternate predictions as to which species should contain tannins can be developed from the expectation of apparency or other theories. Actual analyses of plant materials can then be used to see whether such ecological ideas or the expectations generated from Tables I and 3 (or more accurately from the data in Appendix 1) provide the best predictions of experimental observations at the community level. In the event that the taxonomy is superior then there will be room for argument as to whether historical (phylogenetic) or other factors underlie this result. Whatever the ultimate decision, the plant attributes used in the taxonomy are explicit (Cronquist, 1981 ) and provide for a defined alternate against which to test other hypotheses such as apparency or resource allocation ideas. A pragmatic standard for judging hypotheses developed from ecological theory might be that they make superior predictions relative to those based on taxonomic data alone.
Apparent plants such as oak trees (Quercus) "should" and often do contain tannins (Feeny, 1976). It is less well appreciated that oak trees belong to the Hamamelidae where, with the exception of the Moraceae, the species in every family of every order typically contain tannins. Given this information, it is hard to conclude that apparency is the most obvious explanation for the occurrence of tannins in species of the genus Quercus. In the Violaceae, species range in habit from herbs to tall tropical trees yet tannins appear wholly absent from this family (see Appendix I) and this may be a case where evolutionary history can provide an explanation for the distribution of tannins which apparency and resource allocation based theories cannot.
Such an analysis is anecdotal at best and the future direction of this work will be to analyze the morphological and life-history characteristics of the species surveyed here. For the present, this work is offered as a predictive tool for use by ecologists who wish to locate or avoid tannin containing plants in their work. It has been with this in mind that histochemical data on tannins have been excluded from the study. As such this study may also be of interest to systematists wishing to re-evaluate the distribution of tannins. Whatever the interest of the reader, this work is a guide only, and not a substitute for actually determining the tannin contents of plants of critical importance in empirical work.
Acknowledgements--t am particularly grateful to Tony Joern and Robert Kaul for their encouragement at various stages of this work and to Kathy Keeler for comments on a previous version of the manuscript. This work has been supported by USDA grant CSRS 89-37153-4467.
SYSTEMATIC DISTRIBUTION OF TANNINS 843
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SYSTEMATIC DISTRIBUTION OF TANNINS 845
APPENDIX 1
A TABULATION OF THE NUMBER OF SPECIES TESTED (N) AND THE PROPORTION OF THESE (Prop.) TESTING POSITIVELY
FOR TANNINS IN EITHER SPECIFIC CHEMICAL ASSAYS OR TESTS OR IN PROTEIN PRECIPITATION BASED ASSAYS OR TESTS.
The tabulation is alphabetic by family (first 8 letters given) with subclass (5"), order (O) and family (F) numbers given following
