Marine Chemical Ecology - Chapter 3 doc

The various natural kinds of secondary metabolites are classes ofmolecules, and, being classes, they do not evolve any more than does the calcium carbonate thatforms the shells of mollus

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Marine Natural Products Chemistry as an Evolutionary Narrative

Guido Cimino* and Michael T Ghiselin

CONTENTS

I Introduction 115

II The Evolution of Biosynthetic Capacity 117

III Evolutionary Patterns in Different Classes of Organisms 119

IV Taxonomic Survey 120

A Bacteria 120

1 Cyanobacteria 120

B Algae 120

1 Phaeophyta 122

2 Rhodophyta 122

3 Chlorophyta 125

C Metazoa 125

1 Porifera 125

2 Cnidaria 129

3 Sessile Filter Feeders with Symbionts: Ectoprocta and Urochordata 131

a Tentaculata: Phoronida and Brachiopoda 131

b Tentaculata: Ectoprocta = Bryozoa s.s 131

c Urochordata 132

4 Sessile Filter or Deposit Feeders Evidently without Symbionts 133

a Annelida: Polychaeta 133

b Hemichordata: Enteropneusta and Pterobranchia 135

5 Slow-Moving Grazers and Predators 136

a Echinodermata 136

b Platyhelminthes and Nemertea 137

c Mollusca: Gastropoda 138

V General Discussion 142

Acknowledgments 143

References 144

I INTRODUCTION

Chemistry is generally looked upon as one of the “nomothetic” sciences, i.e., one that seeks to establish the laws of nature and does not concern itself with particular objects or events Natural

* Corresponding author.

3

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116 Marine Chemical Ecology

products chemistry is an exception, being very much concerned with what are called “individuals”

in a broad metaphysical sense.1 Like geology, paleontology, and systematic zoology, it is very much

a natural history discipline The various natural kinds of secondary metabolites are classes ofmolecules, and, being classes, they do not evolve any more than does the calcium carbonate thatforms the shells of molluscs What do evolve are individual populations and lineages, which changewith respect to the properties of the organisms and their parts, including the enzymes that producesecondary metabolites Metabolism has a history and we ought to be able to reconstruct that historyjust like the chemical and physical aspects of defense There seems to have been an arms racebetween shelled molluscs and the crabs that have preyed upon them.2 A chemical arms race involvingmolluscs in which the shell has become reduced is a straightforward extrapolation

It is no longer fashionable to dismiss secondary metabolites as mere waste products or assubstances that no longer play an important role in the lives of organisms They are distributed inthe body very much like other features that have obvious value in the struggle for existence.3 Eventoday, however, much of the discussion of the supposed function of natural products continues totreat natural selection as little more than background material Indeed, historical situations are ofteninvoked to explain away anomalies where efforts to find adaptation fail Adaptation can be treated

as if it were nothing more than a condition or state At least implicitly, however, the product isdefined in terms of the process In other words, when we claim that something is an adaptation,

we presuppose a historical narrative, even if the narrative is concerned only with the very recentpast If we really want to understand the adaptive significance of secondary metabolites, we need

to ask some truly historical questions

These authors’ contributions in this area have mainly dealt with the evolution of chemicaldefense in opisthobranch gastropods.4,5 Faulkner and Ghiselin6 addressed the question of whetherthe reduction of the shell in these animals preceded the evolution of chemical defense (a post-adaptive scenario) or whether the loss of the shell was made possible by the presence of chemicaldefense (a pre-adaptive scenario) The latter hypothesis was preferred on the grounds that in groups

in which the shell is relatively well developed, chemical defense is already present The reasoning

is basically a matter of plotting features on the branches of a phylogenetic tree and inferring thesequence of events But the biological plausibility of the sequence in question may provide anadditional line of evidence This is a traditional mode of reasoning that goes back to Darwin andhis follower Anton Dohrn, who founded the Zoological Station at Naples.7,8 Evolution proceeds bysteps; in each step the functioning of the organism as a whole is conserved, but particular functionsoften succeed one another over time

Various patterns in the evolution of chemical defense have been documented, including ification, modification and sequestration of metabolites, and their positioning in places where theywill more effectively repel predators Of particular interest is the evolution of de novo synthesis.The work of these authors has suggested how this might happen It also suggests that askingquestions about the evolution of biosynthetic capacity might provide a unifying theme for the study

detox-of natural products chemistry

This chapter first discusses how chemical defense might be acquired The authors then suggestreasons why it should evolve differently in various kinds of organisms such as autotrophs andheterotrophs The chapter then gives some examples, presented in an order that is not, strictlyspeaking phylogenetic, although the taxonomic groups discussed are generally thought to be naturalones in the sense that they represent genealogical wholes (clades) The opisthobranchs are discussed

in more than one place because they derive metabolites from various sources, including de novo

synthesis The acquisition and use of some metabolites rather than others provides evidence thatthey are, as suspected, defensive chemicals However, they may be defensive in a broad sense thatincludes dealing with fouling organisms, spatial competitors, and various other things

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Marine Natural Products Chemistry as an Evolutionary Narrative 117

II THE EVOLUTION OF BIOSYNTHETIC CAPACITY

It is well understood that the synthesis and modification of metabolites is under enzymatic control.The enzymes may function as catalysts, and the reactions themselves are not restricted only toliving systems So the evolution of biosynthetic capacity is largely the result of changes in enzymes

by mutation, gene duplication, and other familiar processes The organisms synthesize and modifysecondary metabolites in a stepwise fashion, much as organic chemists do, and in neither case arethe laws of nature violated

The term “secondary metabolite” is generally understood to mean that the chemicals in questionare not directly involved in the basic maintenance of the organism Secondary metabolites areproduced from a remarkably limited range of starting materials known as primary metabolites.There are three major classes of secondary metabolites of interest here (Figure 3.1) Acetogeninsare produced by head-to-tail condensation of acetic acid into linear units starting with acetyl-CoA.Terpenes are made from acetate units that are turned into isoprene units and then put together intolarger units by head-to-tail condensation via isopentenyl diphosphate, often followed by cyclization.Finally, alkaloids are usually formed by the Mannich reaction in which amino acids are transformedinto amines and aldehydes There are, of course, less common metabolites, sometimes very inter-esting ones These include the polypropionates, discussed later in this chapter As shown inFigure 3.1, they form similar to acetogenins, but the starting compound is propionyl CoA Aceto-genins and polypropionates are often referred to in the literature as polyketides One should bear

in mind that much of the diversity of metabolites can be explained as a result of stepwise synthesis

of larger and larger units, with some divergent variants in skeletal structure and a lot of ments and other modifications of the basic structures Such patterns of synthesis can be explainedhistorically, and stepwise modification of biosynthetic pathways through time is a basic phylogenetictheme The fact that the same compound may be synthesized by different pathways is not animpediment to such historical analysis, but rather an opportunity Different pathways often reflectseparate historical origins

rearrange-Before discussing how organisms might evolve such pathways, it is convenient to consider howthey might acquire pathways from other organisms One such possibility is through symbiosis,especially mutualism Such mutualism is well documented in the phylum Porifera, or sponges,which often contain bacteria within their tissues that produce some of the metabolites that defendthe sponge and presumably the bacteria as well The sponges did not have to evolve the chemicalsthat defend them Another possibility is lateral gene transfer The well-known spread of antibioticresistance between lineages of bacteria makes such a transfer seem highly plausible Lateral genetransfer may be quite common among marine microorganisms.9 It can enable them to acquire thecapacity for biosynthesis without having to evolve it through the modification of pre-existingmetabolic apparatus Such capacity means that the organisms are not constrained by the necessity

of obtaining the metabolites from symbionts or food But whether such a transfer is not just possible,but has in fact occurred, has to be established on the basis of empirical evidence For multicellularanimals it is mere conjecture

We have suggested elsewhere that there are two modes by which the capacity for biosynthesis

of secondary metabolites might evolve.5 The first of these is the straightforward and well-documentedanasynthetic mode in which more and more steps are added and, perhaps, a molecule of increasingsize is produced Such evolution has been well documented in terrestrial plants, and some marineexamples are discussed below In some cases it has been shown that the end product of the mostderived evolutionary stage is accumulated in the tissues, but that the intermediates are present inlesser concentrations These intermediates, however, may be the metabolites that are concentrated

in the tissues of related forms that represent ancestral conditions in a historical sequence This casepresents a chemical analogue of the traditional notion that ontogeny recapitulates phylogeny.10 Suchrecapitulation occurs only under restricted conditions, namely, where there has been terminal addition9064_ch03/fm Page 117 Tuesday, April 24, 2001 5:16 AM

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of new stages There is nothing to prevent the secondary loss of developmental or biosynthetic stages,and it is possible that earlier steps in a pathway might be affected

Another possibility is called the retrosynthetic mode We recognized this possibility becauseopisthobranchs have evolved the capacity for de novo synthesis of metabolites similar to those thatthey originally derived from food How could they evolve an entire pathway that supposedly hadnot been part of their evolutionary heritage, a process that usually takes a long time and a lot ofunusual events? One possibility was lateral gene transfer However, the metabolites in questionsometimes did not have the same chirality as those in the food organisms, suggesting that different

FIGURE 3.1 Main classes of secondary metabolites Fatty acids and acetogenins are derived from acetyl Coenzyme A, which forms the isopentyl diphosphate that forms terpenes The (unusual) polypropionates derive from propionyl CoA Alkaloids are typically modified amino acids.

O

H3C C

C

C OH

O

O C

CH C OH

O

O C

n

fatty acid

N H

N

acetogenin (a) acetyl CoA

H3C C O

C O

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enzymes of different historical origins are involved The chirality is just one example of the factthat when nudibranchs have evolved de novo synthesis, the metabolites that they produce, althoughsimilar, are almost never identical to the originals So, elaborating on some earlier ideas,11,12 weproposed that the predators first evolved the ability to modify the last stages of a biosyntheticpathway while still relying upon intermediates that are available in food Given that the intermediatesare present in the food, any mutation that increases their concentration would be selectivelyadvantageous Steps in the synthesis could be added backward until replacing a point in which aprecursor was available that the predator could synthesize on its own This would clearly not be asituation in which ontogeny recapitulates phylogeny

III EVOLUTIONARY PATTERNS IN DIFFERENT CLASSES

OF ORGANISMS

Classes of organisms refers here to abstract kinds, as opposed to taxonomic groups, which areconcrete historical units Classes here means groups of organisms that share such properties as themanner in which food is obtained, whether they are sessile or motile, large or small, etc Large,sessile autotrophs have evolved separately and independently upon numerous occasions So havefilter-feeders, grazers, and predators These groups of unrelated organisms have many of the sameecological requirements and functional constraints They often display some evolutionary conver-gences that help us infer what the important selection pressures have been

Microorganisms include prokaryotes (bacteria such as Cyanobacteria) and unicellular otes (Protozoa and algae of various taxa) Because of their small size and considerable biosyntheticversatility, they are predisposed to assume the position of mutualistic symbionts within the bodies

eukary-of other organisms Lateral gene transfer is, eukary-of course, particularly well documented in bacteria.Unicellular organisms that remain together as a group and form clones can defend the group as awhole in the same way that an entire multicellular organism does Part of the unit can be sacrificed,leaving the rest still able to survive and reproduce

Sessile autotrophs in marine environments are almost all algae Many algae remain unicellular,but there are several lineages which are multicellular organisms crudely convergent with terrestrialplants Because the source of nutriment is photosynthesis, multicellular plants have no dietarysource of secondary metabolites, and, furthermore, the opportunities for evolving symbiotic rela-tionships that might provide defense are quite limited So, we would expect the anasynthetic mode

to be virtually the only way of acquiring chemical defense in such organisms On the other hand,the sessile life style, where organisms cannot move away from either grazers or spatial competitors,makes chemical defense a particularly important mechanism There are, to be sure, examples ofterrestrial plants with defensive symbiotic relationships, for example, with ants, and there may besome marine examples as well

Sessile animals (and ones that are virtually so) have much the same problems in defendingthemselves and warding off predators as terrestrial plants In fact, many of these animals supplementtheir food supply by means of symbiotic unicellular organisms, as do a few animals that movefrom place to place, but slowly Most sessile marine animals feed upon material that is eithersuspended in the water or deposited on the substrate This material sometimes contains metabolitesthat might be put to use defensively However, the food is usually heterogeneous and relativelyunpredictable as to content; therefore, it does not supply a reliable source of metabolites It isgenerally not used defensively, and such use is facultative Some bivalves, however, do concentratesaxitoxin, derived from dinoflagellates, in tissues that are exposed to predators.13

A large number of sessile filter feeders do have defensive metabolites that are not derived fromfood Some of these metabolites are synthesized by the animals themselves, and some are synthe-sized by symbionts In the latter case, there must be a reliable way of providing a supply ofsymbionts for the next generation This is especially important because sessile and sedentary marine9064_ch03/fm Page 119 Tuesday, April 24, 2001 5:16 AM

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animals generally disperse as larvae or as in even earlier developmental stages Special adaptationsthat transmit the guest symbionts from host to host across generations are presumptive evidencethat the host species actually receives a benefit, i.e., that the host is either a mutualist or a parasite

Of course, unwelcome guests often do succeed in getting transmitted from parent to offspring, butthis occurs in spite of the noncooperation of the host In that case the parasite is the exploitingorganism, and is not necessarily contained within the other organism, so an ectoparasite couldactually surround its host

Slow-moving grazers are also in a poor position to flee from predators and require some sort

of suitable protection, either mechanical, chemical, or both Spatial competitors and fouling isms are less of a problem Depending on what they eat, it may or may not be easy for them toobtain defensive metabolites from food If they do obtain such metabolites from food, one wouldexpect them to be specialists that feed upon organisms of those taxa that contain a copious andreliable supply of such metabolites Obtaining defensive metabolites from symbionts is a distinctpossibility, but there is little evidence of that in these organisms The difficulty of overcoming thefood organism’s chemical (and mechanical) defenses combined with relying upon that same organ-ism as a source of protective metabolites tends to produce specialization, yet it constrains the animal

organ-to a narrower range of food items Hence one might predict shifts of host and the evolution of acapacity for de novo synthesis

IV TAXONOMIC SURVEY

The bacteria of interest to us here are restricted to one major clade of Eubacteria: the Actinobacteriaand the closely related Cyanobacteria They are particularly significant as sources of metabolitesthat are used by other organisms, either because the other organisms are their symbionts or feedupon them A few fungi are also significant for the same reason The possible origin of their use

of such compounds has been subject to some speculation A reasonable possibility is that themetabolites were used in competing with other prokaryotes, perhaps in or on the surface ofsediments The bacterial metabolites of interest here are macrolides, polyketides, cyclic lactones,cyclic peptides, and alkaloids (Figure 3.2) Macrolides of sponges are produced by symbioticbacteria in their tissues Polyketides of urochordates are also produced by symbiotic bacteria, andthese are concentrated by the opisthobranch gastropods that feed upon them Figure 3.2 showstypical metabolites from bacteria and fungi

This section discusses the three groups of algae within which multicellular, mainly sessile lineageshave evolved All three groups have members rich in secondary metabolites that are pressed intoservice defensively by opisthobranchs that feed upon them Presumably, they are also used9064_ch03/fm Page 120 Tuesday, April 24, 2001 5:16 AM

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FIGURE 3.2 Selected metabolites after marine bacteria and fungi 3.2.1 Saxitoxin 173 3.2.2 Swinholide from

uniden-tified deep-sea bacterium 176 3.2.5 Loloatin B from Bacillus sp isolated from an unidentified tube worm 177 3.2.6 Penochalasin A, from Penicillum sp., symbiotic with the green alga Enteromorpha intestinalis 178 3.2.7 6- Bromindole-3-carbaldehyde from the ascidian Stomozoa murrayi and an associated Acinetobacter sp 179 3.2.8 Gymnastatin A from a strain of Gymnasella dankaliensis isolated from the sponge Halichondria japonica 180

HO HO

O OMe

OH O

MeO

OH OH O

OMe

HO O

O OH

HN NH

O

N H O

N H Ph

NH HOOC

O Ph

N O O

H N O

OH O

N H

O

H N

H N O

N H HN O

H O

NH

O

N H Br

CHO

Cl O Cl

O N H

O

N N H

N H

H N

3.2.2

OH OH

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defensively by plants, though, again, the wider range of effects upon other organisms under thatrubric is included The differences between these three groups in the kinds of metabolites thatthey elaborate suggest separate historical origins The possibility that these metabolites could betraced back to an earlier common ancestry cannot be ruled out, but there seems to be no evidencefor that theory What has been documented is the elaboration and diversification of metaboliteswithin each of these major algal groups

1 Phaeophyta

The first algal lineage considered here is the brown algae, or Phaeophyta, which include the familiar,macroscopic kelps This chapter does not go into the fascinating and highly controversial issue ofwhether polyphenolics in brown algae are used defensively (see Chapter 6 in this volume) Thesecompounds are not expropriated by algivorous animals, and indeed, there would seem to be nodefensive use of these compounds by any animals Terpenoids are another matter altogether.Dolabellanes (Figure 3.4.7), for example, are highly toxic compounds They are named after thegenus Dolabella, which are opisthobranchs of the order Anaspidea (see below)

Amico14 studied the secondary metabolites in brown algae of the genus Cystoseira (familyCystoseiraceae, order Fucales) from a phylogenetic point of view He was able to arrange metab-olites in series, such that the more evolutionarily derived algae have metabolites that require moresteps in their biosynthesis (Figure 3.4) Linear diterpenoids in his scheme are succeeded by what

he calls a pool of open-chain meroditerpenoids, and these in turn are elaborated into several groups

of derived and divergent ring systems His arrangement agreed well with morphological cations and made sense in terms of the biogeography of the group Because the genus originated

classifi-in the late Cretaceous period (ca 80 MYBP), it seems clear that the elaboration and diversification

of the metabolites occurred during the Cenozoic Period

Opisthobranchs of the order Anaspidea (sea hares) feed upon a diversity of algae That theysometimes obtain metabolites from blue-green algae (Cyanobacteria) and brown algae (Phaeophyta)has already been mentioned They also feed upon green algae (Chlorophyta, see below), but these

FIGURE 3.3 Selected metabolites from cyanobacteria 3.3.1 Laingolide from Lyngbya bouillonii 181 3.3.2 Malyngamide from Lyngbya majuscula 182

C7H15

Me N

Cl O

OH O

O

3.3.1

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are not known to provide them with an important source of metabolites A remarkably wide range

of chlorinated terpenoids has been recovered from red algae, especially from the genus Laurencia

and from sea hares of the genus Aplysia that feed upon them The ability to feed upon algae thatare rich in halogenated terpenoids seems to have been a major innovation within the Anaspidea,but the group in general is adept at feeding upon plants with a broad range of algal metabolites

To what extent the anaspideans are expropriating the metabolites and using them defensively,and to what extent they are merely obtaining them from food and disposing of them, has been atopic of controversy It has been pointed out that the metabolites are mostly concentrated in the

FIGURE 3.4 Metabolites of brown algae 3.4.1–3.4.6 Selected metabolites from the Cystoseira showing simple and derived conditions 183 3.4.7 Dictyotatriol A from Dictyota dichotoma 184

OH H HO

H HO

3.4.7

OH

OMe O O H

HO

3.4.6

O OMe

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digestive gland, which is not where they would most effectively deal with predators.15 However,evolution from a relatively ineffective mechanism of defense to a more effective one is what isexpected and has precedents in other groups of opisthobranchs Some oversimplifications aboutselective mechanisms may be implicit in such discussions It is generally assumed that a defensiveadaptation cannot evolve if the organism that possesses it is killed and therefore fails to reproducemore than conspecifics without it This stricture would apply to the anaspideans in question were

it a scenario with metabolites becoming present in the digestive gland because of their defensiverole It does not apply, however, to a scenario in which the metabolites were initially present inthe food and, therefore, in the digestive gland An animal containing such metabolites in its digestivegland would not be protected from predators that devoured it But if the predators were sickened

or killed, they would tend to leave the survivors alone Selection could then favor putting themetabolites in a more effective position Kin selection might also operate in spite of some theoreticalconsiderations first put forth by Faulkner and Ghiselin,6 and subsequently by other authors.16,17 Theproblem with kin selection in opisthobranchs is that most of them have larval dispersal, and,therefore, the juveniles and adults do not live with close relatives Under such circumstances, kinselection does not seem to be a good explanation for the aposematic coloration that is so common

in the group One important point, however, has been overlooked Opisthobranchs are internallyfertilizing hermaphrodites that store sperm, and they often occur in aggregations of conspecifics

FIGURE 3.5 Selected metabolites from red algae 3.5.1 Pannosallene from Laurencia pannosa 185 3.5.2 Pantafuranoid A from Pantoneura plocamioidea 186 3.5.3 Rigidol from Laurencia rigida 187 3.5.4 10-Epide- hydroxythyrsiferol from Laurencia viridis 188 3.5.5 Almazole D from Haraldiophyllum sp 189

N H

O

N O

H

H H Br

HO Br

3.5.2

O O

Br Br

3.5.1

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Killing such an organism eliminates its ability to lay eggs, but its sperm may survive and fertilize

another organism’s eggs

Furthermore, the present-day situation in anaspideans turns out to be a bit more complicated

than expected According to some recent field studies, Aplysia juliana ate only green algae and had

no defensive metabolites, whereas A parvula, which fed upon red algae, used diet-derived

metab-olites quite effectively in defense.18,19 The latter species is considered the most primitive

represen-tative of the genus,20 and it seems likely that the former has ceased to accumulate metabolites or

was not doing so when the study was conducted It is noteworthy that metabolites typical of

Rhodophyta have been recorded from what has been identified, at least, as A juliana.21 The

prevailing pattern of feeding mainly upon taxa with metabolites, coupled with occasional shifts to

taxa that also contain secondary metabolites, likewise points toward a defensive role

3 Chlorophyta

Green algae, or Chlorophyta, which are genealogically closer to vascular plants than they are to

other algae, have well-documented chemical defense in the order Bryopsidales In this order

(Figure 3.6), the main defensive metabolites are diterpenoids and sesquiterpenoids Minor

metab-olites of interest include cyclic peptides (Figure 3.6.1), and bicyclic lipids (Figure 3.6.2) Of

particular interest are the closely related families Udoteaceae and Caulerpaceae.22,23 The Udoteaceae

include several genera (Udotea, Halimeda, Chlorodesmis) which have a combination of chemical

defense and mechanical defense based on calcification The combination has been shown to be

more effective against the grazing animals that occur in the largely tropical or subtropical habitats

of the algae.24 Caulerpaceae consists only of the genus Caulerpa, and its separation evidently does

not have a genealogical basis, so it seems likely that the absence of calcification is secondary The

metabolites of these algae have much in common Their biological activity has been shown to

correlate with the structure of the molecules, which generally have a protected 1,4-conjugated

aldehyde moiety which would react with primary amines This was established by comparing a

series of natural and synthetic analogs.25

The green algae are fed upon by opisthobranchs of the order Sacoglossa, which pierce the cells

and suck out their contents The evolution of this relationship has recently been treated in

consid-erable detail by these authors.4 Only those aspects that are germane to the theme of this review are

included here Although it is not clear which genus among these algae was the ancestral food, shifts

to other groups of algae are obviously secondary Thus, although most species of Elysia continue

to feed upon and utilize algae of the family Udoteaceae, with the typical sesquiterpenoid metabolites,

kahalalides, as they are called, are suspected of coming from epibiotic cyanobacteria rather than

from the alga.27 Even within the Udoteaceae, Avrainvillea longicaulis is the source of a brominated

diphenylmethane derivative found in Costasiella ocellifera (= C liliane), which is in a different

family of sacoglossans; this compound is an effective deterrent to feeding by fish.28

Also secondary are shifts to other kinds of food, abandonment of chemical defense, and de novo

synthesis In any event, the animals almost (but not quite) always continue to specialize upon food

organisms that contain such metabolites When the animals evolve the capacity for de novo

syn-thesis, they may produce variants of the ancestral compound, but these variants are more

physio-logically active than the originals Finally, like some other opisthobranchs, they sometimes use

polypropionates synthesized de novo (see below)

1 Porifera

Porifera, or sponges, are rich in secondary metabolites; a representative sample is shown in Figure 3.7

Some of these are biosynthesized by the sponges themselves, but others are biosynthesized by bacterial

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symbionts.29–31 The metabolites of interest here may conveniently be listed under two categories,

according to whether they are probably synthesized by the sponges or their symbionts Metabolites

produced by the symbionts are often identified as such by their presence in bacteria that are not

symbionts or at least are symbionts of organisms other than sponges

Sponge systematics is a difficult area of research, and, partly for this reason, secondary

metab-olites have been pressed into service in efforts to create a system that more nearly reflects the

phylogeny Secondary metabolites make good taxonomic markers or, better, diagnostic characters,

for lineages of sponges For example, brominated metabolites derived from tyrosine (Figure 3.7.1)

are characteristic of the order Verongida in the subclass Ceractinomorpha,32 and isonitriles

(Figure 3.7.2) are mainly known from the order Halichondrida including the Axinellida.33

Devel-opmental characters have also been used, but the emphasis has been upon anatomy, especially the

morphology of the spicules that help support the organism and may also provide the organism

some protection from getting eaten It seems somewhat enigmatic that Chanas and Pawlik34 did

not get positive results in efforts to corroborate the defensive role of spicules against fish grazing

Similar experiments had given positive results with other organisms Laboratory tests with artificial

food are hard to interpret, and even field studies may not relate to actual selection pressures

Evidence to the contrary is that the Keratosa, a group in which the spicules have regressed, are

particularly rich in biologically active secondary metabolites.32 That fits the notion of an arms race

with a shift from mechanical to chemical defense

FIGURE 3.6 Selected metabolites from green algae 3.6.1 Kahalalide A from Bryopsis sp and the

sacoglo-ssan Elysia rufescens.190 3.6.2 Dictyospharerin from Dictyospheria sericea.191 3.6.3 Halitunal from Halimeda

the sacoglossan Costasiella ocellifera.194

Br

Br OH

OAc

3.6.3

COOH OH

3.6.2

N H O

H N

O

N H

OH

O

O O

H

Ph O

O N

O

HO O

3.6.1

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FIGURE 3.7 Selected metabolites from sponges 3.7.1 Oxohomoaerothionin from Aplysina cavernicola.195

3.7.2 9-Isocyanopupukeanane from Hymeniacidon sp and the dorid nudibranch Phyllidia varicosa.196 3.7.3

Haliclonacyclamine B from Haliclona sp.197 3.7.4 E-chlorodeoxyspongiaquinone from Euryspongia sp.198

3.7.5 (-)-Microcionin from Microciona toxystila.199 3.7.6 Isospongiadiol from Spongia sp.200 3.7.7 Isonitenin

from Spongia officinalis.201 3.7.8 Scalaradial from Cacospongia mollior.202 3.7.9 Pellynic acid from Pellina

OH COOH

10

3.7.9

CHO

CHO AcO

3.7.8

O

O O

O

3.7.7

O HO

Cl O

O

H OMe

Br

OMe Br

OH O N

3.7.1

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The role of symbionts in producing metabolites that occur in sponges has received increasingdocumentation The relationship may reasonably be considered a mutualistic one, i.e., one thatbenefits both parties, rather than parasitism or commensalism There is a good possibility ofcoevolution between the two partners Symbiotic bacteria are passed from one generation of sponge

to the next via maternal cells that surround the egg, even when the animals are oviparous,35 indicatingthat the sponges have evolved adaptations that ensure the presence of the bacteria The spongesand their associated bacterial symbionts are known to produce quite different metabolites The

sponge Dysidea herbacea produces sesquiterpenoids, whereas its symbiotic cyanobacteria produce

polychlorinated amino acids.36 Likewise, the sponge Suberea creba produces quinolines, whereas

its symbionts produce tyrosine metabolites.37

For the order Haplosclerida in the subclass Ceractinomorpha and their close relatives, therehave been two particularly interesting phylogenetic studies on metabolites produced by symbionts.Andersen, Van Soest, and Kong38 studied the 3-alkylpiperidene alkaloids from this assemblage.They placed the molecules in series beginning with a monomeric unit leading to dimers and derivedand rearranged skeletons The resulting tree agreed quite well with more traditional skeletal char-acters It has two branches, one consisting of the viviparous families Callyspongiidae and Niphatidaewith Chalanidae as the sister group, and one consisting of the oviparous families Phloeodictyidaeand Petrosiidae

More recently, Van Soest, Fusetani and Andersen39 studied the straight-chain acetylenes thatoccur in the same group (including close relatives of Haplosclerida) with broadly similar results.Again, they produced a phylogeny based on derived and divergent biosynthetic pathways, a phy-logeny that agrees well with morphological data The preliminary results also suggest that symbioticbacteria have occasionally moved from one lineage to another, much as one might predict It must

be mentioned that the Haplosclerida contain quite a variety of metabolites other than these twoexamples Some of these make their way into nudibranchs that feed upon the sponges; for example,

both sterols and high molecular weight polyacetylenes have been recovered from Peltodoris

The isonitriles mentioned above as characteristic of Halichondrida (in some classifications thisgroup includes the Axinellidae) are interesting as they provide the basis for a minor radiation indorid nudibranchs Namely, the Porostomata include a lineage of slugs, the family Phylidiidae, thatfeed exclusively on sponges that are rich in such isonitriles The biosynthetic pathways wherebythese isonitriles are produced have been studied, and it is noteworthy that cyanide is incorporatedinto the molecule.42 The nudibranchs have not evolved the capacity for de novo synthesis of these

compounds.43 Some authors have questioned whether they are used defensively by the nudibranchs

It is true that rigorous experiments to this effect have not been done However, the animalsthemselves provide a sort of natural experiment or assay The species that live in the most exposedpositions, readily visible to predators in the tropics, are the ones with the most obvious smell.44

Furthermore, there are mimetic complexes consisting of these nudibranchs, other nudibranchs,polyclad flatworms and sea cucumbers, all with similar color patterns.45

Sponges themselves apparently do biosynthesize a variety of secondary compounds Someexamples from the Keratosa (mentioned above as having lost the spicules) are of particular interest.32

There are three orders in subclass Ceractinomorpha of the class Demospongiae The order Verongidaconsists of the families Aplysinidae, Aplysinellidae, and Ianthellidae, and is distinct from the othertwo by being oviparous and containing brominated tyrosine derivatives, rather than being viviparousand containing terpenes The order Dendroceratida, with families Darwinellidae and Dictyoden-dillidae, has diterpenes Finally in the order Dictyoceratida, there are three families with sesterter-penes — Spongiidae, Thorectidae, and Ircinidae — and one family, Dysideidae, with sesquiterpenes.Although the phylogeny is perhaps debatable, the taxonomic pattern suggests a diversification inthe metabolites that are synthesized by the sponges

Among these keratose sponges, Aplysina aerophoba (Verongida) is said to be at least capable

of biotransformation of the characteristic brominated tyrosine derivatives.46 They are held, evidently

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as inactive precursors, and modified by enzymatic action at the time of release.47 However, there

is some question as to how well the experiments represent in vivo conditions.

There is a significant correlation between the phylogeny of sponges and that of the doridnudibranchs of the Family Chromodorididae that feed upon them.5 The early branches of this group

feed on a wide range of sponges, and some of them are quite euryphagous In general, Chromodoris and Glossodoris have diterpenes and sesterterpenes of the kind found in most Dictyoceratida, whereas Hypselodoris is characterized by sesquiterpenes characteristic of the family Dysideidae There is at least one exception to the rule, H orsini, with a sesterterpene scalaradial (Figure 3.7.8)derived from a sponge of the family Thorectidae.48 The scalaradial is modified by the nudibranch

by selective reduction into deoxoscalarin (Figure 3.14.1), which is less toxic, then by selectiveoxidation into 6-ketodeoxoscalarin (Figure 3.14.2)

Various dorid nudibranchs are thought to have evolved the capacity for de novo synthesis of

metabolites originally derived from sponges, but the evidence is not always compelling The

fol-lowing cases have been well established experimentally Sclerodoris tanya synthesizes tanyolide

(Figure 3.14.3), a terpenoic acid glyceride.49 Archidoris odhneri synthesizes an unnamed farnesic

acid glyceride (Figure 3.14.4).50 Dendrodoris limbata synthesizes 7-deacetoxyolepupuane

(Figure 3.14.5).51 This is the only example we have of a nudibranch biosynthesizing a defensive

metabolite that is identical to one found in potential prey D grandiflora and some other species of

the same genus synthesize drimane terpenoids.52–54 The aforementioned are all Porostomata or their

supposed sister group In the Chromodorididae (discussed above), Cadlina luteomarginata

synthe-sizes albicanyl acetate (Figure 3.14.6), cadlinaldehyde (Figure 3.14.7), and luteone (Figure 3.14.8).55

2 Cnidaria

Representative metabolites from Cnidaria (= Coelenterata s.s.) are shown in Figure 3.8 The Cnidariahave venomous stinging capsules that are beyond the scope of this study Also excluded from thisdiscussion are some occasional secondary metabolites in various groups that are not sufficientlywell enough known to generalize about This discussion is restricted to a single order, Gorgonacea,

in subclass Octocorallia (Alcyonaria) and class Anthozoa These gorgonians, or sea fans and seawhips, are sessile animals that feed on small planktonic organisms Their stinging capsules aresmall relative to those of some other cnidarians, and are less important for defense They haveproven to be a rich source of secondary metabolites, and there are a few interesting connectionswith nudibranchs and other gastropods

Gorgonians are particularly rich in terpenoids, notably sesquiterpenoids and diterpenes.Gerhardt56 produced a biosynthetic tree for these compounds and found that it agreed well withthe existing, morphological system of classification Although rooting the tree under the assumptionthat the ancestral form had no terpenoids at all would seem to be unrealistic, he got two distinct

lineages In the first branch, with the genera Gorgonia, Pseudopterogorgia, Plexaurella, and

Muri-cea, sesquiterpenes were present and diterpenes were absent In the second branch, with the genera Briareum, Eunicella, Eunicea, and Pseudoplexaura, diterpenes are always present, but in one

subclade there are also sesquiterpenes Given that Briareum and Eunicella occupy a more basal

position in the tree, Gerhardt reasoned that the ability to produce the sesquiterpenes had evolvedtwice within this lineage It is a case of parallel evolution, but not the rampant variety The presence

of terpenes in other Octocoralia may perhaps represent parallel evolution as well The terpene

skeletons of Alcyonium (Octocorallia: Alcyonacea) are somewhat different from those of Porifera.57

Another intriguing class of metabolites that occur in Gorgonacea is the prostanoids These pounds are ubiquitous among metazoans, and have a wide range of physiological functions.58–61

com-However, a defensive function, involving a much increased level of concentration and some ification in structure, is unusual Claims that the compounds are allelopathic rather than defensivehave been rejected.62,63 Negative evidence about feeding deterrence by fish turned out to be theresult of the fish not responding immediately but vomiting after a delay.64 The delay, coupled with

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mod-130 Marine Chemical Ecology

aversive learning, is effective in deterring predators that take a bite and then return to feed again

The prosobranch gastropod Cyphoma gibbosum feeds upon gorgonians that contain high levels of

Prostaglandin A2 The snail feeds preferentially upon gorgonian colonies that have not previouslybeen attacked, apparently because the defense is inducible and more of a deterrent at higher levels.63

It feeds upon a range of gorgonians, and has been found not to be immune to terpenoids from

upon Octocorallia Another of these, Ovula ovum, was thought to detoxify terpenoids from a soft

coral,66 but further investigation showed that this was not the case.67 The snails are generally believed

to use the metabolites defensively

A few nudibranchs feed upon gorgonians, which may represent the original food of the branch(Cladohepatica) that switched from sponges to cnidarians as food Unfortunately, the lineage ofnudibranchs (Dendronotacea) that are the sister group of the other Cladohepatica and include many

of the animals that feed upon gorgonians are among the less common and less studied nudibranchs,especially from the point of view of natural products When Gosliner and Ghiselin68 described

Tritonia hamnerorum, their editor deleted the remark that the animals smelled like camphor.

Despite that, a furan was recovered both from this nudibranch and a gorgonian upon which itfeeds.69 Within the Dendronotacea there is one lineage, the family Tethyidae, that has ceased to

FIGURE 3.8 Selected metabolites from anthozoans 3.8.1 Clavulone 1 from Clavularia viridis.204 3.8.2

(+)-Ancepsenolide from Pterogorgia anceps.205 3.8.3 7β, 8α-dihydroxydeepoxysarcophine from Sarcophiton

O H H

C5H11

3.8.1

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feed upon cnidarians and eats small arthropods instead Tethys has been found to defend itself

with prostaglandins.70–75 We have suggested that this may be no coincidence, and that perhaps theuse of prostaglandins that were derived from food was followed by the use of prostaglandinssynthesized by the nudibranchs themselves.5 Because prostaglandins are so widespread in metazoan

tissues, it is not necessary to invoke the evolution of de novo synthesis in the retrosynthetic mode

for this particular case

3 Sessile Filter Feeders with Symbionts: Ectoprocta and Urochordata

The next two groups to be discussed contain defensive metabolites that are generally acknowledged

to be produced by symbionts, and not by the animals themselves These are the phylum Ectoprocta(= Bryozoa s.s.) and the chordate subphylum Urochordata Although Ectoprocta are lophophorates,and lophophorates have previously been considered related, albeit remotely, to the Chordata andother Deuterostomes, molecular evidence has made this relationship seem quite dubious; rather,lophophorates are closer to annelids and molluscs.76–80 At any rate, the ancestry of lophophorates

is so remote that cross-lineage transfer of symbionts seems much more likely than their havingbeen present since the Precambrian era We treat Ectoprocta and Urochordata together because oftheir feeding relationships with opisthobranchs as well as their chemical similarities The metab-olites in question are mainly alkaloids

The phylum Ectoprocta (= Bryozoa s.s.) is generally thought to be most closely related to the phylaPhoronida and Brachiopoda The three are often placed together into a single phylum or superphy-

lum Tentaculata The phoronid Phoronopsis viridis is a tube-dwelling worm that is abundant in the

mudflats where it was studied It contains the halogenated phenolics 2,6-dibromophenol and tribromophenol.81 These compounds are similar to those secreted by hemichordates of the classEnteropneusta, and in both groups they are thought to suppress the growth of bacteria Otherwise,little is known about their natural products chemistry Brachiopods are said to be repellent,82,83 butthe only marine natural products reported from them are four long-chained glycerol enol ethers(Figure 3.9.3) from the terebratulid Gryphus vitreus.84

2,4,6-b Tentaculata: Ectoprocta = Bryozoa s.s.

Secondary metabolites of Ectoprocta occur in distantly related lineages within the group, indicatingthat they have been acquired since diverging With trivial exceptions, marine ectoprocts are colonialanimals with good mechanical defense in the form of calcareous shells that surround the zooids.There is an excellent fossil record showing a sustained tendency over 100 million years for thecheilostomes to out-compete ctenostomes by overgrowth, though apparently there has not been anarms race.85 Mechanical defense by means of spines has been shown to be inducible.86,87

Although, for example, tetracyclic terpenoid lactones (Figure 3.9.1) have been found,88 themain bryozoan metabolites of interest are alkaloids and macrocyclic ethers.89 The tambjamines are

a good example of alkaloids These are bipyrolles that occur in ascidians as well as in nudibranchsthat derive them from eating ectoprocts, ascidians, or even other nudibranchs (see section below

on Urochordata) The bryostatins (Figure 3.9.2) are the important polyethers from ectoprocts.90

They are macrocyclic lactones

A microbial origin for both the alkaloids and the polyethers has been maintained.91 Dietaryderivation seems dubious, since ectoproct food is not likely to provide a rich and reliable source

of metabolites Ectoprocts are suspension feeders that take up small particles by means of tentacles.Furthermore, most ectoprocts have a reproductive biology that would facilitate transfer of bacteriaacross generations: they brood the developing embryos Bacteria suspected of producing bryostatinswere found in the larvae in some, but not all, of the species surveyed by Woollacott.92 What was

formerly thought to be a single species (Bugula neritina) of ectoproct that varied in its bryostatin

content is actually a complex of cryptic species each with characteristic bacterial symbionts.93

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The phylum Chordata consists of three subphyla: Urochordata, Cephalochordata, and Vertebrata.Among the Urochordata (tunicates), chemical defense has not yet been recorded in the two plank-tonic classes (Appendicularia and Thaliacea), perhaps because of inadequate sampling However,

it does occur quite commonly among the Ascidiacea, which are benthic organisms Ascidians aremainly sessile, and their asexual reproduction has repeatedly led to the formation of highly organizedcolonies The sessile habitus is conducive to the evolution of both mechanical and chemical modes

of defense Ascidians are well defended by a tough but flexible tunic that contains cellulose Theability of the colony as a whole to resist predators even though some members of the colony aredestroyed may further the utilization of particular kinds of defensive metabolites Some (compound)

ascidians of the genus Didemnum produce eicosanoids, evidently of non-symbiotic origin, providing

an interesting parallel with gorgonians (see above).94,95

Symbiosis with algae and bacteria (including Cyanobacteria) is widespread in ascidians and isresponsible for coloration as well as defensive metabolites, which may themselves be colorful.Symbiotic bacteria are also responsible for the (probably defensive) bioluminescence of their pelagicrelatives, the Thaliacea.96 Brooding of the young “tadpole” larva may facilitate the transfer ofsymbionts from one generation to the next Many of the metabolites in ascidians are quite similar

to those known to be produced by bacteria, and the taxonomic distribution of the metabolites in theanimals tends to confirm this.97 Others, however, are evidently produced by the tunicates themselves.Examples of secondary metabolites from Ascidians are shown in Figure 3.10 Ascidians arenoteworthy for the presence of vanadium (and related metals) together with high concentrations ofsulphuric acid and tunichromes, which are unstable hydroquinoid compounds A defensive role forthese has been suspected,98 and it is noteworthy that gastropod molluscs (both prosobranch andopisthobranch) that feed upon tunicates often secrete large amounts of sulfuric acid, used in both

FIGURE 3.9 Selected metabolites from bryozoans and a brachiopod 3.9.1 Securine A from Securiflustra

HO OH

O

O O

OH

OH OH

MeOOC

3.9.2

N H HN

Cl

HN N

Br O

3.9.1

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feeding and defense.99 A fair range of chemicals may be characterized as occasionally present, butnot of major significance, for example, a few terpenoids, isoprenoid hydroquinones, fatty-acidderivatives, and polyethers The predominant metabolites are amino-acid-derived metabolites,including both linear and cyclic peptides and various alkaloids.100

A few groups of gastropods feed upon ascidians The presence of metabolites in the molluscsprovides a fair indication of which ones are actually used defensively In the subclass Prosobranchia,the Lamellariidae are a good, if perhaps understudied, example An unidentified species of Lamel-laria from Palau was found to contain alkaloids called lamellarins (Figure 3.10.9).101 They occur

in the ascidian genus Didemnum, and about 30 of them have been described.102 Another lamellariid,

Chelynotus semperi, contains a series of pyridoacridines, kuanoniamines (Figure 3.10.10), derived

from an otherwise unidentified colonial tunicate.103 Homarine has been found in the lamellariid

Marseniopsis mollis; however, even though it has a defensive role, this metabolite evidently derives

from epibionts, not from the ascidian upon which the snail feeds.104

In the subclass Opisthobranchia, several lineages have switched to feeding upon ascidians

Within the order Notaspidea, which probably fed originally upon sponges, Pleurobranchus feeds upon ascidians, and at least one of them, P forskalli, contains a cyclic peptide.105 Within the orderNudibranchia, originally sponge-feeders, there is one group of “phanerobranch” dorids, the Suc-toria, which evidently switched to bryozoans and (subsequently, it would seem) to ascidians Thepattern here is particularly instructive Tambjamines (Figure 3.10.5) occur in both ectoprocts and

ascidians Nudibranchs of the genus Tambja obtain tambjamines from the bryozoans upon which they feed Roboastra, in the same family, feeds upon Tambja and uses the metabolites defensively.106

Another nudibranch of the same family, Nembrotha, obtains tambjamines by feeding upon

ascid-ians.107 Here, the metabolites are obviously a resource, over and above the nutritional content ofthe food, that provides the basis for adaptive changes in diet The nudibranchs in question areremarkably conspicuous and are often encountered in the open by divers, unlike many tropicalnudibranchs that are cryptic or keep out of sight

Some tunicates are fed upon by flatworms Not much information is available, but there aresome intriguing parallels between the flatworms and the nudibranchs, which resemble each othersuperficially and for which cases of mimicry are known Five alkaloids (heterocycles), including

lepadin B (Figure 3.10.8), have been described from the marine turbellarian Prostheceraeus villatus; they derive from the tunicate Clavelina lepadiformis upon which it feeds.108 Attention has beendrawn to the convergence with nudibranchs: the flatworm is aposematically colored Another

example is staurosporine derivatives, which have been recorded from the ascidian Eudistoma

indolocarbazole alkaloids (Figure 3.10.9) resemble alkaloids known from fungi rather than theusual ascidian alkaloids that are suspected of having bacterial origin

4 Sessile Filter or Deposit Feeders Evidently without Symbionts

be toxic, and some opisthobranchs of the order Cephalaspidea feed upon them They form a distinct

clade that is not recognized in formal classifications, but consists of such genera as Actaeon and

Hydatina Natural products chemists have not yet investigated this relationship.

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FIGURE 3.10 Selected metabolites from tunicates 3.10.1 Namenamicin from Polysyneraton lithostratum.212

3.10.2 Virenamide B from Diplosoma virescens.213 3.10.3 Meridine from Amphicarpa meridiana.214 3.10.4

Patellin 5 from Lissoclinum sp.215 3.10.5 Tambjamine A from Atapozoa sp.216 3.10.6 Woodinine from Eudistoma

and the flatworm Prostheceraeus villatus.219 3.10.9 Lamellarin Z from Didemnum chartceum.220 3.10.10

Kuanoniamine A from an unidentified tunicate and the prosobranch gastropod Chelynotus semperi.221

N

S N

N O 3.10.10

N

OH H

H 3.10.8

O

O 3.10.7

O

OH

3.10.4

N NH

O O

O

O HN

S N N Ph

NH O

NH

O

O O

3.10.3

N

S H

O N Ph

MeS

OH

HO O O

HN OMe

MeSSS

NHCOOMe HO

3.10.1

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