The evolution of foliar terpene diversity in Myrtaceae

Amanda Padovan • Andras Keszei •

Carsten Kulheim • William J. Foley

Received: 4 February 2013 / Accepted: 7 October 2013 / Published online: 13 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Plant terpenes play many roles in natural

systems, from altering plant–animal interactions, to

altering the local abiotic environment. Additionally,

many industries depend on terpenes. For example,

commercially used essential oils, including tea tree oil

and lavender oil, are a mixture of terpenes. Many

species of the family Myrtaceae form a key resource

for these industries due to the high concentration of

terpenes found predominately in their leaves. The

frequency of chemotypic differences within many

species and populations can lead to costly errors in

industry. Terpene diversity in Myrtaceae is driven by

variation in the terpene synthase enzymes, which

catalyse the conversion a few common substrates into

thousands of terpene structures. We review terpene

diversity within and between species of Myrtaceae and

relate this to variation in the terpene synthase enzymes

to reconstruct the evolution of foliar terpene diversity

in Myrtaceae. We found that (1) high inter- and intra-

species variation exists in terpene profile and that a-

pinene the most likely ancestral foliar terpene, and (2)

that high concentration of 1,8-cineole (a compound

which is regarded as the signature compound of

Myrtaceae) is limited to just four Myrtaceae sub-

families. We suggest that the terpene synthase

enzymes do not limit terpene diversity in this family

and variation in these enzymes suggests a mode of

enzymatic evolution that could lead to high 1,8-

cineole production. Our analysis highlights the need to

standardise methods for collecting and reporting foliar

terpene data, and we discuss some methods and issues

here. Although there are many gaps in the published

data, our large scale analysis using the results of many

studies, shows the value of a family wide analysis for

understanding both the evolution and industrial

potential of terpene-producing plants.

Keywords Monoterpene � Sesquiterpene �Terpene synthase � Eucalyptus � Melaleuca �Cineole � Evolution

Terpenes are arguably one of the most important

groups of compounds in the plant kingdom because

they form such a large classes of plant secondary

metabolites and play many roles in the lives of plants.

Terpenes facilitate interactions between a plant and its

biotic environment, for example terpenes are involved

in direct and indirect defence against herbivory in the

terrestrial and marine worlds (Andrew et al. 2007;

Maida et al. 1993; Matsuki et al. 2011; Van Poecke

et al. 2001), defence against pathogens (Huang et al.

Electronic supplementary material The online version ofthis article (doi:10.1007/s11101-013-9331-3) contains supple-mentary material, which is available to authorized users.

A. Padovan (&) � A. Keszei � C. Kulheim � W. J. Foley

Research School of Biology, Australian National

University, Building 116 Daley Road, Canberra,

ACT 0200, Australia

e-mail: amanda.padovan@anu.edu.au

123

Phytochem Rev (2014) 13:695–716

DOI 10.1007/s11101-013-9331-3

2012; Levin 1976) and in pollinator attraction (Pi-

chersky and Gershenzon 2002). They slow down

decomposition of leaf litter on the forest floor,

resulting in slower nutrient cycling within the ecosys-

tem (Horner et al. 1988). Terpenes can also alter the

local abiotic environment, through air quality and thus

local climate; for example Australian eucalypt forests

are known to release terpenes, particularly during the

day, changing the ionic composition of the air

resulting in higher local temperatures and lower local

humidity and rainfall (Suni et al. 2008; Vickers et al.

2009).

Terpenes in Myrtaceae

Terpenes are a defining feature of Myrtaceae, which is

renowned for having some of the highest concentra-

tions of foliar terpenes in the plant kingdom (Keszei

et al. 2008). The recent completion of the Eucalyptus

grandis genome, the first genome from Myrtaceae to

be fully sequenced (Grattapaglia et al. 2012), has

revealed the presence of many more terpene synthase

enzymes than in other terpene-rich plant species.

These enzymes catalyse the synthesis of terpene

structures from a relatively small pool of substrates.

Approximately 112 terpene synthases were identified

in the E. grandis genome, with most (*80) being

expressed in young and mature leaves (Kulheim et al.

2013). The next largest terpene synthase family is

from grape (Vitis vinifera), which contains 53 terpene

synthase sequences, however the fruits and leaves of

grape contain neither a diverse profile nor high

concentrations of terpenes (Martin et al. 2010). The

discovery of such a large family of terpene synthases

in the E. grandis genome prompted us to examine the

patterns of terpene diversity in Myrtaceae.

There are 17 tribes of Myrtaceae comprising

approximately 140 genera and 5,500 species (Biffin

et al. 2010; Thornhill and Crisp 2012). Myrtaceae are

predominately found in the southern hemisphere with

species diversity hotspots in Australia and south east

Asia (Thornhill and Crisp 2012). Originally, two sub-

families of Myrtaceae were recognised; the fleshy-

fruited Myrtoideae and the dry capsular-fruited Le-

ptospermoideae (reviewed by (Thornhill and Crisp

2012). However, more recent molecular phylogenies

have shown there are only two distantly related tribes

containing fleshy-fruited species, but these tribes also

contain dry-fruited species: Myrteae and Syzygieae

(Biffin et al. 2010; Thornhill et al. 2012; Wilson et al.

2005). Species from the Xanthostemoneae, Myrteae

and Syzygieae are commonly found in the tropical

rainforests of Australia and south-east Asia (Brophy

et al. 2006), whereas species of Eucalypteae, Mela-

leuceae and Leptospermeae are common in forest

woodlands from the dry tropics through to the

temperate zones (Brophy and Southwell 2002). With

leaf morphology closely related to habitat (Wilson

2011), woodland plants are predicted to defend their

leaves much more strongly than do rainforest plants

(Grubb et al. 1998). Therefore, we might expect the

leaves of rainforest species have fewer terpenes

compared with those of open forest and woodland

species notwithstanding the many other roles terpenes

can play in the plant.

Not surprisingly, people have exploited plants from

this family for many industrial purposes, for example

tea tree oil is extracted from Melaleuca alternifolia

(Butcher et al. 1994; Carson et al. 2006), eucalyptus

oil is extracted from the 1,8-cineole rich leaves of

several Eucalyptus species (Boland et al. 1991) and

guava (the fruit of Psidium guajava) is a common food

crop and important in traditional south east Asian

medicines (Chen et al. 2007).

Terpenes play a number of roles in the interaction

between a plant and its environment, however to date

the terpenes in Myrtaceae have only been implicated

in defensive roles against herbivores and pathogens. If

terpenes are predominately employed in defensive

roles in Myrtaceae, there may have been a co-

evolutionary arms race with herbivores, which would

drive diversification of terpene profiles within and

between species (Kant and Baldwin 2007) and would

explain the significant qualitative and quantitative

variation in terpene profiles within species, popula-

tions and even individuals (Andrew et al. 2010;

Padovan et al. 2012; Wallis et al. 2011). While intra-

specific diversity of terpene profiles present a viable

evolutionary response for survival in a large range of

environmental conditions (for example: Crankshaw

and Langenheim 1981; Langenheim et al. 1980;

Linhart and Thompson 1995), it also presents a

significant problem for those industries that rely on a

specific terpene profile and both quality and quantity

of terpenes affect of the profitability of the essential oil

industries (Homer et al. 2000).

696 Phytochem Rev (2014) 13:695–716

123

How is qualitative and quantitative variation

in foliar terpenes affected by the terpene

biosynthesis pathway?

The terpene biosynthetic pathway is well understood

(Dudareva et al. 2005; Kulheim et al. 2011; Lichtent-

haler 1999). Briefly, the mevalontate (MVA) and

methylerythritol phosphate (MEP) pathways result in

the production of isopentenyl pyrophosphate (IPP),

the universal precursor to all terpenes (Dudareva et al.

2005, Lichtenthaler 1999). IPP is isomerised to

dimethyl allyl pyrophsphate (DMAPP). Subsequently,

DMAPP and IPP connect to form a small number of

prenyl pyrophosphates. The MVA pathway is local-

ised in the cytosol and produces IPP that is used in the

synthesis of farnesyl pyrophosphate (which is used by

sesqui- and triterpene synthases) and is localised in the

cytosol (Dudareva et al. 2005). The MEP pathway,

localised in the chloroplast, produces IPP that is used

in the synthesis of geranyl and geranylgeranyl pyro-

phosphate (which are used by mono- and di- and

tetraterpene synthases, respectively) and is localised in

the chloroplast (Lichtenthaler 1999). The prenyl

pyrophosphates formed by the connection of DMAPP

and IPP are the substrates for terpene synthase

enzymes. The terpene synthases are a large family,

and catalyse the conversion of a small set of substrates

into thousands of terpene products (Wise et al. 1998).

There are two points in the biosynthetic pathway that

influence the qualitative profile of terpenes. Firstly, the

ratio of monoterpenes to sesquiterpenes is influenced

by the synthesis of geranyl pyrophosphate (GPP) and

farnesyl pyrophosphate (FPP), the precursors for

monoterpene and sesquiterpenes, respectively. Both

are synthesised from the same precursors but by

unique enzymes in different cellular compartments

(Dudareva et al. 2005; Kulheim et al. 2011; Lichtent-

haler 1999). Variation in the efficiency of these

enzymes can influence the monoterpene:sesquiterpene

ratio (Degenhardt et al. 2009; McCaskill and Croteau

1995).

Secondly, the qualitative terpene profile can be

influenced via the action of terpene synthases. Melal-

euca quinquenervia provides a good example of this.

The foliar terpene chemotypes of this species correlate

with differential expression of at least two terpene

synthases (Padovan et al. 2010). Terpene synthases are

often multi-product enzymes, with some accepting

several prenyl pyrophosphate substrates (e.g. GPP and

FPP, for mono and sesquiterpenes, respectively), to

produce terpene skeletons (Keszei et al. 2008; Wise

et al. 1998). The overall amino acid sequence of

terpene synthases is highly conserved across species

(Bohlmann et al. 1998), however these enzymes have

variable catalytic pockets to facilitate the production

of a range of terpene skeletons, and some enzymes

have two areas in the catalytic pocket that are involved

in distinct steps in the reaction cascade (Kollner et al.

2006).

Although many terpenes are direct products of

terpene synthases, some terpenes are formed by the

actions of one or more cytochrome P450 (CYP)

enzymes acting on the product of a terpene synthase-

catalysed reaction. For example, limonene can be

converted into trans-isopiperitenol, trans-carveol,

carvone, and menthol (Bohlmann and Keeling 2008;

Bouwmeester et al. 1999; Croteau et al. 2005) and a-

pinene can be converted to myrtenol (Aharoni et al.

2004) through a variety of CYP enzymes. Whereas

several economically important terpenes require a

CYP step in their biosynthesis, there are few data

available on cytochrome P450 enzymes known to play

a role in terpene biosynthesis in Myrtaceae and the

terpenes that are known to require a CYP for their

biosynthesis are not common in the leaves of Myrt-

aceae species. Therefore we will focus on terpene

synthase enzymes and how these relate to foliar

terpene variation in Myrtaceae.

Understanding terpene variation in plants addresses

many ecological and evolutionary questions, includ-

ing differential herbivory in thyme (Thymus vulgaris)

(Linhart and Thompson 1995) and origins of intra-

specific chemical variation (Butcher et al. 1994).

Because terpene variation is a direct result of enzy-

matic variation, we can explore the evolution of

chemical variation. Comparison of terpene data from

large groups of species will contribute to our under-

standing of their evolution and ecological interactions,

and may elucidate the origin of terpene production in

related species. Foliar terpene data and terpene

synthase sequence data have been published for

thousands of species worldwide and contain a wealth

of ecological and evolutionary information. Yet, it

remains remarkably under explored. One recent

exception for the Myrtaceae was a study of latitudinal

variation of Eucalyptus foliar terpenes in Australia

(Steinbauer 2010), although that only sampled 66 out

of the more than 900 Eucalyptus taxa. Here we collate

Phytochem Rev (2014) 13:695–716 697

123

foliar terpene data from 1,362 species of Myrtaceae

and describe the patterns of foliar terpene production

and the developments of chemotypes across the

family, in order to:

1. identify evolutionary trends in foliar terpene

diversity within and between species of

Myrtaceae

2. identify constraints on terpene production with

and between species of Myrtaceae

3. identify species that may be of commercial

importance and highlight species where caution

should be taken

Search methods and data processing

Terpene diversity

We compiled a database of the foliar terpene diversity of

all species of Myrtaceae for which published data were

available. We included the year of the study, the species,

the number of chemotypes and the foliar terpene

profile(s). We recorded the five most abundant terpenes

but only if these contributed more than 7 % to the total

oil. We documented the order of most abundant

compounds, but did not record the concentration or

proportion of each compound as these were measured

and recorded differently by various authors. We used the

molecular phylogeny described by Thornhill and Crisp

(2012) to determine which sub-family each species

belonged to and used EUCLID (an electronic identifi-

cation tool for Eucalyptus: Slee et al. 2006) to determine

which section each Eucalyptus species belongs to. For

this study, chemotypes are defined by their single most

abundant compound, called dominant terpenes. Due to

differences in methods between publications, it was

difficult to determine if differences in minor compounds

(those that were not the most abundant) are the result of

chemotypic differences or due to differences in methods

of extraction and analysis.

We have collated data from a number from

publications dated between 1920 and 2012. On top

of the technological changes that have influenced the

data quality in that time, the aim of many publications

is different and thus the number of samples collected is

highly variable. To account for this variation in

experimental design and still look at patterns of foliar

terpene variation across Myrtaceae, we used an

artificial weighting system based on the order of

abundance of compounds in each species. We gave the

most abundant compound in a profile a value of 100,

the second most abundant compound a value of 50, the

third most abundant compound a value of 25 and so on

to the sixth most abundant compound which has a

value of 3. This allowed us to calculate the mean of

these values for all species within a tribe (Thornhill

and Crisp 2012) and we presented this in a heat map

(Table 1—all species are included in Supp. Table 1).

The heat map allows us to visualise patterns in large

data sets, in this case we can easily see the distribution

of specific terpenes across the family Myrtaceae. We

have also used this data to investigate the distribution

of chemotypes across the family. However the original

experiments were not designed for this purpose, which

means there is no consistency in the age or develop-

mental stage of the tissues collected. Whilst this is a

limitation, we were able to identify some interesting

patterns that warrant further investigation.

Terpene synthase enzymes

We compiled a separate database containing data from

site-directed mutagenesis studies on angiosperm ter-

pene synthase enzymes. We chose site-directed muta-

genesis studies only as these provide a good system for

investigating enzyme structure and function. We

recorded the year of the study, the species, the name

of the enzyme, all sequence variations of that enzyme

investigated in the study, the product profile and relative

activity of each enzyme and variant and the x-ray

crystallographic structure used to generate a 3D model

of this enzyme. We generated our own 3D models of the

10 proteins, using the crystallographic structure of

bornyl diphosphate synthase from Salvia officinalis

(1n20A—sourced from the ExPASy database through

SPBDV (Guex et al. 1995–2011). The monoterpene

synthase sequences were truncated before the RRX8W

motif to correspond to the truncation of the model

(1n20A). This motif signifies the end of the plastid-

targeting sequence in monoterpene synthases (Wise

et al. 1998). We used the SPBDV program (Guex et al.

1995–2011) to generate a 3D homology model of the

enzymes and submitted this to the ExPASy sever for

optimisation using default parameters (Arnold et al.

2006). We then generated 2D ‘maps’ of the genes from

the start to stop codons. On these we plotted the amino

acids that contribute to the internal surface of the

698 Phytochem Rev (2014) 13:695–716

123

catalytic pocket, those that were altered in the site-

directed mutagenesis study and the highly conserved

DDXXD motif (Fig. 1).

A brief description of the data

We collected data from 1,393 species of Myrtaceae

from 67 genera of 13 tribes (Tables 1, 2). For some

species, there was more than one chemical profile

reported and we have designated these as putative

chemotypes. This means we have terpene data for

1,694 samples across the family. These samples are not

evenly distributed across all tribes (Table 1). Mela-

leuceae, Eucalypteae and Syncarpieae are well sam-

pled for foliar terpenes, with data available for more

than 75 % of species in each tribe. Despite being some

of the most specious tribes, very few species of

Chamelaucieae (3 %), Myrteae (6 %) and Syzygieae

(1 %) have been sampled for foliar terpene analysis

(Table 1). Some species of Myrtaceae are much better

investigated than others and this unavoidably intro-

duces bias into our analysis. Irrespective of the number

of species in a tribe, those species whose leaves are

industrially important are more extensively sampled

for foliar terpenes than those that contain species

whose leaves are less industrially important.

What patterns are there in foliar terpene

chemotypic development across the family

Myrtaceae (Tables 2, 3)?

There are 175 species of Myrtaceae with more than

one chemical form or chemotype in this study (14 %

Table 1 A heat map of the most abundant foliar terpenes in all species of Myrtaceae

4 4 4 4 4 4 4 4 5 5 5 6 6 6

- - - - - - - - + - + - - -

- - - - - - - - - - - - - -

1.7 0.3 1.9 - 4.1 - 0.4 0.2 0.3 - 0.0 - - 0.6

20.0 - - - - - - - - - - - - -

1.4 0.1 8.4 0.0 0.7 - - - 2.0 0.6 0.2 0.4 1.9 4.7

1.6 - 1.6 - 3.6 - - - - - 0.6 - - -

7.1 - 14.3 - - - - - - - - - - -

- - - - - - - - - - - - - -

0.4 0.0 0.6 0.7 1.3 0.8 0.9 0.7 - - 0.4 - - 0.0

- - - - - - - - - - - - - -

- - - - - - - - - - - - - -

- - - - - - - - - - - - - -

- - - - - 13.3 - - - - - - - 3.3

- - - - - - - - - - - - - -

Carbocation 0 0 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4

P450 + + - + - - + - + - - - - - + - + - - + - - - + -

Xanthostemoneae 2 7 - - - - - - - - - - 12.5 - 25.0 - - - - - - - - - - - -

Melaleuceae 3 404 - 0.5 - 0.9 0.0 0.2 0.3 0.5 - 4.5 0.2 0.2 37.3 3.5 0.0 4.9 0.6 0.2 9.2 - 58.9 6.7 0.2 - 2.0

Syncarpieae 1 4 - - - - - - - - - - - - 120.0 - - - - - - - - - - - -

Eucalypteae 3 797 - 0.1 0.2 0.3 0.4 0.1 0.3 - 0.4 0.2 0.3 0.1 37.5 2.9 0.3 3.7 0.1 - 3.2 0.3 50.2 3.3 0.4 0.6 0.1

Leptospermeaea 4 77 - 1.3 1.3 0.6 1.5 - 0.2 0.3 - 1.3 0.6 0.6 40.3 0.3 - 10.9 1.3 - 3.2 - 15.6 0.6 - - -

Chamelaucieae 5 13 - - - - - - 14.3 - - 7.1 - - 92.9 3.6 - - - 21.4 21.4 - 35.7 - - - -

Lindsayomyrteae 1 1 - - - - - - - - - - 25.0 - - - - - - - - - - - - - -

Myrteae 35 355 1.8 - 0.7 0.6 - - - 0.6 - 2.6 1.4 2.0 24.8 0.6 - 5.2 0.1 - 9.0 0.1 9.8 1.7 0.6 - 0.4

Kanieae 5 12 - - - - - - - - - - - 15.4 76.9 - - 15.4 - - 23.1 - - - - - 7.7

Tristanieae 2 2 - - - - - - - - - - - - 75.0 - - - - - - - - - - - -

Backhousieae 1 3 - - - 33.3 - - 33.3 16.7 - - - - - - - - - - - - - - - - -

Syzygieae 4 15 - - 6.7 - - 0.8 - 3.3 - 1.7 - - 15.0 - - 10.0 - - - - - 7.5 - - -

Metrosidereae 1 1 - - - - - - - - - - - - - - - - - - - - - - - - -

CarbocationP450 - - - - - - - - + - - - - - - - - - - - - - - - +

Xanthostemoneae 2 7 - - - 6.3 - 12.5 - 37.5 - 6.3 - 37.5 - - - 25.0 - - 25.0 25.0 - - - - 18.8

Melaleuceae 3 404 - 0.0 0.3 - 0.5 - - 3.9 0.9 0.3 - 0.2 - - 0.6 0.1 - - 1.2 0.6 4.6 0.2 2.3 - 5.2

Syncarpieae 1 4 - - - - - - - - - - - - - - - - - - - - - - - - -

Eucalypteae 3 797 0.7 - 0.6 - 0.6 - - 0.9 0.2 0.2 - 0.3 - - 0.1 0.1 - - - - 8.0 0.3 6.6 - 4.3

Leptospermeaea 4 77 1.9 - 1.3 1.3 2.9 - - 7.5 - 3.7 - 0.3 - - - 0.3 - 0.3 0.2 0.6 12.7 - 3.9 - 3.2

Chamelaucieae 5 13 - - - - - - - - - - - - - - - - - - - - - - - - -

Lindsayomyrteae 1 1 - - - - - - - 100.0 - 50.0 - - - - - - - - - - - - - - -

Myrteae 35 355 0.3 0.8 0.7 0.7 2.4 1.7 0.6 17.8 5.3 1.8 0.6 2.1 0.7 0.8 2.1 1.2 0.7 2.7 1.8 1.2 1.0 - 7.4 0.7 7.5

Kanieae 5 12 - - - - 19.2 - 15.4 46.2 - - 7.7 7.7 - - - - - - - - - 7.7 7.7 - 15.4

Tristanieae 2 2 - - - - - - - - - - - - - - - - - - - - 50.0 - - - -

Backhousieae 1 3 - - - - - - - - - - - - - - - - - - - - - - - - -

Syzygieae 4 15 - - - 3.3 - - - 5.0 13.3 0.8 - - - - - 3.3 - 10.0 - - - - 25.0 - 10.0

Metrosidereae 1 1 - - - - - - - - - - - - - - - - - - - - - - - - -

0 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 4 4 4 5 5 6 6 6 6 6 6 6 6 6 6 6- - - - - - - -

- - - - - - - -

5.0 0.8 0.2 0.1 2.5 - - -

20.0 20.0 - - - - - -

5.0 5.9 0.3 0.4 0.6 0.3 - 0.7

5.5 1.3 0.6 - 3.4 - - -

10.7 - - - - - - -

- - - - - - - -

4.4 3.0 1.4 0.8 0.6 - 1.0 0.9

23.1 - - - - - - -

- - - - - - - -

- - - - - - - -

4.1 - - - - - - -

- - - 100.0 - - - -

On the x-axis are the foliar terpenes and on the y-axis are the tribes of species and a phylogeny defined by Thornhill et al. (2012). The

first table contains monoterpenes and the second table contains sesquiterpenes. The most abundant terpenes are shown in a darker grey

and the least abundant terpenes are shown in a lighter grey. Genera refers to the number of genera represented in each tribe and

samples refers to the number of samples represented in each tribe. Carbocation refers to which carbocation is acted on by a terpene

synthase to result in a particular terpene starting from the FPP or GPP in Figure 3 from Keszei et al. (2008). P450 refers to whether a

cytochrome P450 enzyme is involved in the production of each compound: ? = P450 involved, - = P450 not involved

Phytochem Rev (2014) 13:695–716 699

123

Fig. 1 Gene maps of the terpene synthases from angiosperms

investigated through site directed mutagenesis. The black solid

boxes represent the gene from start codon to stop codon. The

conserved DDXXD domain is represented by a raised black bar

in about the middle of the genes. The raised grey bars represent

sites that have been altered in that enzyme during site-directed

mutagenesis studies. The white bars represent amino acids that

contribute to the internal surface of the catalytic pocket

Table 2 The proportion of species captured in the 13 tribes of Myrtaceae in this study

The phylogeny is a simplified version of that reported by Thornhill and Crisp (2012). The column ‘‘Species’’ refers to the number of

species identified in each tribe (Govaerts 2008). The column ‘‘Sampled’’ shows the number of species for which we collected foliar

terpene data and this as a proportion of the total number of species. The columns ‘‘1,8-cineole’’ and ‘‘a-pinene’’ indicate if these

compounds are common and abundant compounds in the tribe: ? symbolises this and - symbolises these are not common or

abundant

700 Phytochem Rev (2014) 13:695–716

123

of species), some of which have not previously been

reported as chemotypes but rather two independent

studies have identified different chemical forms.

Species that are better characterised, either through

multiple studies or heavy sampling within a single

study, have more chemotypic variation. The chemo-

type is defined by the most abundant compound and

most often there are just two chemotypes of each

species, with an average of 2.4 chemotypes per

species. 73 species have chemotypic patterns involv-

ing both monoterpenes and sesquiterpenes, 90 species

have chemotypic patterns involving only monoter-

penes and 11 species have chemotypic patterns

involving only sesquiterpenes. Only 35 (*20 %)

species from this list have neither an a-pinene nor a

1,8-cineole chemotype, despite these commonly being

the most abundant foliar terpene.

In about half of the species, there is a major

remodelling of the terpene biosynthetic pathway

among the two chemotypes, switching between a

monoterpene-dominated profile and a sesquiterpene-

dominated profile. This would involve repartitioning

substrates between the cytosol and the plastid, possible

through unidirectional transport of IPP across the

chloroplast membrane (Webb et al. 2013), and a

change between high expression of (1) FPPS ? ses-

quiterpene synthases and (2) GPPS ? monoterpene

synthases. The majority of the genes involved in

terpene biosynthesis should be regulated differently

between the two chemotypes, possibly with the

exception of some MEP pathway genes, since there

is evidence of substrate sharing across the plastidal

membrane model plants (Dudareva et al. 2005) and as

well in Melaleuca alternifolia (Webb et al. 2013).

Of the 13 tribes, Meleuceae and Eucalypteae have

the largest number of species with foliar terpene

chemotypes and most often there are just two chem-

otypes in a species. In the vast majority of species

(80 %) there is an a-pinene or 1,8-cineole foliar

terpene chemotype. It is less common to see both

chemotypes in a single species, with this scenario

happening in only 20 % of species with chemotypes

(Table 2). High concentrations of foliar a-pinene is

likely to have appeared first in the common ancestor to

all Myrtaceae, whilst high concentrations of foliar 1,8-

cineole is likely to have appeared in the common

ancestor to Melaleuceae and Eucalypteae.

Although this data set is not ideal for exploring

chemotypic diversity across Myrtaceae (the data were

collected using different methods and none of the

experiments were designed for chemotype detection,

with most sampling from one or a few individuals within

a population), we have identified several species with

multiple chemical forms. In many cases, the chemical

differences indicate changes in how terpene biosynthe-

sis proceeds amongst chemotypes. This poses the

question: what benefits does a species get from vastly

different chemical forms? How is terpene biosynthesis

regulated given such a large remodelling has occurred

within a species? Although these questions have been

pursued in other systems, there are not enough data on

Myrtaceae to allow answering the question with any

confidence (see a recent review of this topic by Moore

et al. (2013)). The first step would be to determine the

chemical profile of entire species of Myrtaceae, with

many hundreds of samples from natural populations in

different climates, as has only been done in a few species

for example: Eucalyptus globulus (Wallis et al. 2011),

Eucalyptus tricarpa (Andrew et al. 2013), M. alterni-

folia (Butcher et al. 1994; Homer et al. 2000) and M.

quinquenervia (Ireland et al. 2002).

What evolutionary trends exist in foliar terpene

diversity within the family Myrtaceae (Table 2)?

a-Pinene and 1,8-cineole are the most common

terpenes amongst the Myrtaceae, with more than

50 % of the samples and species having a-pinene or

1,8-cineole as a dominant compound in their foliar

terpene profile. These compounds are also the most

abundant, with approximately 25 and 35 % having a-

pinene and 1,8-cineole, respectively, as the most

abundant compound in the foliar terpene profile of

species and samples. 1,8-cineole is not found as a

dominant compound in any sample in eight tribes

(Xanthostemoneae, Syncarpieae, Lindsayomyrteae,

Kanieae, Tristanieae, Backhousieae, Syzygieae and

Metrosidereae), whereas a-pinene is not a dominant

compound in samples of just three tribes (Lindsayo-

myrteae, Backhousieae and Metrosidereae). The foliar

terpene profiles of species from the latter three tribes is

often dominated by sesquiterpenes, particularly spath-

ulenol, globulol and bicyclogermacrene. The tribes

Melaleuceae, Eucalypteae and Myrteae show the

greatest monoterpene and sesquiterpene diversity in

terms of the number of unique dominant compounds in

the foliar terpene profile.

Phytochem Rev (2014) 13:695–716 701

123

Ta

ble

3T

he

do

min

ant

com

po

un

do

fch

emo

typ

esfr

om

17

5sp

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fM

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acea

e

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ecie

s(*

den

ote

sfi

rst

rep

ort

of

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tio

n)

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oty

pes

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qu

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pen

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es

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3

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mp

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ea*

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ne

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ma

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inia

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pp

leO

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sd

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ate

nsi

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yn

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caly

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iga

nte

a)

sub

tasm

an

ien

sis*

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hel

lan

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ne

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des

mo

l

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sd

um

osa

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ath

ule

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l

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caly

ptu

sd

wye

ri*

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eole

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ath

ule

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l

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ptu

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stig

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des

mo

l

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caly

ptu

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lia

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ycl

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acre

ne

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sg

un

nii

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eole

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ath

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l

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caly

ptu

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pin

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des

mo

l

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caly

ptu

sle

uco

ph

loia

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ineo

leG

lob

ulo

l

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caly

ptu

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acu

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-Pin

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eole

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des

mo

lG

uai

ol

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caly

ptu

sm

ela

no

ph

loia

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ene

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ycl

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acre

ne

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caly

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sn

eso

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ila

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idifl

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ulo

l

702 Phytochem Rev (2014) 13:695–716

123

Ta

ble

3co

nti

nu

ed

Sp

ecie

s(*

den

ote

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rst

rep

ort

of

po

ssib

lech

emo

typ

ic

var

iati

on

)

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no

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ene

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oty

pes

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qu

iter

pen

ech

emo

typ

es

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1C

h2

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1C

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3

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caly

ptu

sn

itid

a(s

yn

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myg

da

lin

asu

b

nit

ida

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uca

lyp

tus

rad

iata

Var

D)*

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hel

lan

dre

ne

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des

mo

l

Eu

caly

ptu

so

bli

qu

a*

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men

eb

- Ph

ella

nd

ren

e

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ycl

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acre

ne

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btu

sifl

ora

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ger

mac

ren

e

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caly

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sp

au

cifl

ora

(sy

nE

uca

lyp

tus

cori

ace

a,

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caly

ptu

sp

hle

bo

ph

ylla

)*

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men

eE

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ol

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caly

ptu

sp

laty

pu

ssu

bh

eter

op

hyl

la*

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inen

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ne

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oly

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ene

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ren

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gen

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hy

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gen

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hy

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gen

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yll

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gen

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on

ene

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pa

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alo

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ph

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men

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end

ren

e

Phytochem Rev (2014) 13:695–716 703

123

Ta

ble

3co

nti

nu

ed

Sp

ecie

s(*

den

ote

sfi

rst

rep

ort

of

po

ssib

lech

emo

typ

icv

aria

tio

n)

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no

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ene

chem

oty

pes

Ses

qu

iter

pen

ech

emo

typ

es

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1C

h2

Ch

3C

h4

Ch

1C

h2

Ch

3

Lo

ph

om

yrtu

sla

cifl

uu

sa

-Pin

ene

Aro

mad

end

ren

e

Lo

ph

om

yrtu

ssu

ave

ole

ns

a-P

inen

eb-

Car

yo

ph

yll

ene

Mel

ale

uca

cord

ata

Cin

eole

Eu

des

mo

l

Mel

ale

uca

inca

na

sub

inca

na

Cin

eole

Glo

bu

lol

Mel

aleu

cale

iop

yx

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inal

oo

lC

ineo

leE

ud

esm

ol

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ale

uca

ner

vosa

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ineo

leS

pat

hu

len

ol

M.

qu

inq

uen

ervi

a*

Cin

eole

Ner

oli

do

la

-Sel

inen

eV

irid

iflo

rol

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ale

uca

thyo

ides

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inen

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pat

hu

len

ol

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ale

uca

tub

ercu

lata

var

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ub

ercu

lata

Lin

alo

ol

Eu

des

mo

l

Mel

ale

uca

vim

inea

sub

vim

inea

a-P

inen

eE

ud

esm

ol

Myr

ceu

gen

iacu

cull

ata

a-P

inen

eN

ero

lid

ol

a-S

elin

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ceu

gen

iam

yrci

oid

es*

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inen

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pat

hu

len

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cia

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rea*

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rcen

eb-

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yo

ph

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cia

ga

le*

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rcen

eE

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ol

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cia

nth

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ag

an

sa

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ene

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on

ene

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adin

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pu

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ens

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eole

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hy

llen

e

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cia

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ella

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hy

llen

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tifo

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ph

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enta

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hy

llen

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mg

ua

java

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on

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2en

alB

isab

ole

ne

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ary

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hy

llen

e

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terh

ou

sia

mu

lgra

vea

na

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inen

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icy

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mac

ren

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ecie

s(*

den

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rst

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of

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ssib

lech

emo

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tio

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5C

h6

(B)

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ckh

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itra

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ania

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ph

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inen

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llis

tem

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vim

ina

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tra

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an

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mp

om

an

esia

ad

am

an

tiu

ma

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anio

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e

Ch

am

ela

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uu

nu

nci

na

tum

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ron

ella

la-

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ene

Lim

on

ene

704 Phytochem Rev (2014) 13:695–716

123

Ta

ble

3co

nti

nu

ed

Sp

ecie

s(*

den

ote

sfi

rst

rep

ort

of

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ssib

lech

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aria

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n)

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no

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ene

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oty

pes

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qu

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pen

ech

emo

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es

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3C

h4

Ch

1C

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Ch

3

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hy

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asp

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hu

mil

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sa

cced

ens*

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inen

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ptu

sa

mp

lifo

lia

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ineo

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ptu

sa

myg

da

lin

a(s

yn

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caly

ptu

ssa

lici

foli

a)*

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eole

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inen

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ne

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sa

nd

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sii

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an

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lan

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sa

ng

op

ho

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enu

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men

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inen

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caly

ptu

scu

rtis

ii*

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men

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caly

ptu

sd

ives

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eole

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lan

dre

ne

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erit

on

e

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caly

ptu

sel

ata

(sy

nE

uca

lyp

tus

an

dre

an

a,

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caly

ptu

sn

um

ero

sa)*

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lan

dre

ne

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on

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caly

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rys*

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glo

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lus

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laye

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nE

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eole

a-P

inen

e

Phytochem Rev (2014) 13:695–716 705

123

Ta

ble

3co

nti

nu

ed

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ecie

s(*

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ote

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rst

rep

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of

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lech

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aria

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n)

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no

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ene

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oty

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qu

iter

pen

ech

emo

typ

es

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1C

h2

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3C

h4

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1C

h2

Ch

3

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caly

ptu

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pto

ph

leb

a*

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men

ea-

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ene

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caly

ptu

sm

aca

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uri

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eran

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ineo

le

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ns*

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ta(s

yn

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au

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a,

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caly

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dia

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caly

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letr

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carv

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caly

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dw

ayi

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lmo

no

ph

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ym

ene

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erit

on

e

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caly

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der

oxy

lon

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eole

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cia

lis*

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men

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aig

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eran

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any

lace

tate

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eole

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on

ene

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men

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eole

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men

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do

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gen

iasp

ecio

saL

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nen

ea-

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ale

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cio

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sub

als

op

hil

aa-

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men

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op

hil

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alt

ern

ifo

lia

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pin

ole

ne

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pin

en-4

-ol

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eole

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ale

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an

aa-

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ene

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pin

en-4

-ol

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arg

ente

aL

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nen

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inen

-4-o

lN

ero

lid

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ale

uca

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iia-

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eole

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ole

ns

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ania

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ron

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hy

lcin

nim

ate

706 Phytochem Rev (2014) 13:695–716

123

Ta

ble

3co

nti

nu

ed

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ecie

s(*

den

ote

sfi

rst

rep

ort

of

po

ssib

lech

emo

typ

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tio

n)

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no

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chem

oty

pes

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qu

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pen

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1C

h2

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pin

en-4

-ol

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dis

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l

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ale

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alo

ol

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ale

uca

exu

via

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eole

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pin

en-4

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ale

uca

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lop

hil

aC

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leT

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inen

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l

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ale

uca

ha

ma

taC

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l

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ale

uca

leu

cad

end

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yl

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eug

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pin

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pin

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ar.

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tifo

lia

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rten

ala-

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ale

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plu

mea

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eole

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inen

e

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ale

uca

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iger

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inal

oo

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le

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ale

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ach

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eole

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ale

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tric

ho

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chya

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eole

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pin

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ale

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ylla

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eole

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alo

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cin

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inen

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pin

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rlie

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ino

len

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cia

nth

esrh

op

alo

ides

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alo

ol

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tus

com

mu

nis

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leL

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nen

ea-

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enta

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ica

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ug

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lM

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ug

eno

l

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enta

race

mo

sav

arg

rise

a*

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hy

lis

oeu

gen

ol

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ho

xy

iso

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msa

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ria

nu

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ene

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on

ene

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tan

iop

sis

coll

ina

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rcen

ea-

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ene

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tan

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sis

lau

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ene

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on

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terh

ou

sia

flo

rib

un

da

a-P

inen

ea-

Ter

pin

eol

Phytochem Rev (2014) 13:695–716 707

123

The most striking pattern in foliar terpenes across

Myrtaceae is that a-pinene and 1,8-cineole are both

the most common and the most abundant compounds

across the majority of species in this family (Supp

Table 1). In most cases, 1,8-cineole or a-pinene are

the most abundant compounds in that foliage sample.

However, this pattern is not consistent in the clade

containing the tribes Myrteae, Kanieae and Syzygieae,

as well as the tribes Xanthostemoneae, Syncarpieae

and Lindsayomyrteae. In these tribes, 1,8-cineole (a

monoterpene), is primarily replaced by b-caryophyl-

lene (a sesquiterpene) as the most abundant terpene

and in Lindsayomyrteae, Metrosidereae and Back-

housieae, allo-aromadendrene (a sesquiterpene) is the

most abundant compound rather than the monoterpene

a-pinene (Tables 1, 2 and Supp Table 1). However,

we only have foliar terpene data for one species in

each of the latter three tribes, so this conclusion may

be an effect of sample size, and an avenue for future

research is to fill this sampling gap.

Species of Myrtaceae have some of the highest

concentrations of 1,8-cineole in the plant kingdom. De

Vincenzi et al. (2002) surveyed a range of aromatic

and medicinal plants with the focus on 1,8-cineole,

and found that essential oil from E. globulus (Myrt-

aceae) consists for 70–80 % this monoterpene. Other

published data show that some species of Eucalyptus

contain this compound at greater than 90 % of the total

essential oil (e.g. E. polybractea—Brophy and South-

well 2002). Few other plants outside the Myrtaceae

contain such large proportions of 1,8-cineole, however

there are some exceptions such as Cardamon (Elet-

taria cardamomum (Zingiberaceae)—De Vincenzi

et al. 2002) and camphor laurel (Cinnamomum cam-

phora (Lauraceae)—Stubbs et al. 2004). This suggests

that although 1,8-cineole is not unique to Myrtaceae,

high concentrations are found predominantly within

the Myrtaceae and so high foliar 1,8-cineole is a

defining feature of the family.

Mapping the patterns in foliar terpenes to the

phylogeny (Table 2), there are two evolutionary

trajectories that could have occurred (Fig. 2). The

first evolutionary trajectory is that the common

ancestor to all Myrtaceae had high foliar levels of a-

pinene and low foliar levels of 1,8-cineole. Subse-

quently, high concentrations of 1,8-cineole evolved in

the leaves in the common ancestor to Melaleuceae,

Syncarpieae, Lindsayomyrteae, Eucalypteae, Lepto-

spermeae and Chamelaucieae but was subsequentlyTa

ble

3co

nti

nu

ed

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ecie

s(*

den

ote

sfi

rst

rep

ort

of

po

ssib

lech

emo

typ

icv

aria

tio

n)

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1C

h2

Ch

3C

h4

(C)

Au

stro

myr

tus

flo

rib

un

da

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nes

ol

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nes

yla

ceta

te

Eu

caly

ptu

sn

ova

-an

gli

caA

rom

aden

dre

ne

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oli

do

lG

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gen

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laG

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op

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-Car

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yll

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gen

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teri

cab

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ta*

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cia

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ax

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cia

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ati

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enen

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ino

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abo

len

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ata

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ren

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ech

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(i.e

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emo

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the

do

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ant

chem

oty

pe

inth

esp

ecie

s,n

or

isit

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ance

stra

lch

emo

typ

e).

(A)

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ecie

sw

ith

chem

oty

pes

of

bo

thm

on

ote

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esan

dse

squ

iter

pen

es(i

.e.in

vo

lvin

gtw

ob

iosy

nth

etic

pat

hw

ays)

,(B

)sp

ecie

sw

ith

on

lym

on

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ech

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typ

es,(C

)sp

ecie

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on

lyse

squ

iter

pen

ech

emo

typ

es

708 Phytochem Rev (2014) 13:695–716

123

lost in the Syncarpieae and Lindsayomyrteae lineage.

The second possible evolutionary trajectory is that the

common ancestor to all Myrtaceae had high foliar

levels of 1,8-cineole and a-pinene, then the machinery

leading to high foliar 1,8-cineole was lost several

times in the Xanthostemoneae and the Syncarpieae

and Lindsayomyrteae lineages as well as the clade

containing Myrteae, Tristanieae and Syzygieae. In

each case, the occurrence of high foliar a-pinene is a

trait common in most tribes of Myrtaceae and is

therefore most likely a trait present in the first species

of Myrtaceae. Since a-pinene is common in the leaves

of many species of Myrtaceae (Supp Table 1) and in

many species from closely related families (e.g.

Toudahl et al. 2012; Ogunbinu et al. 2007; Kaur and

Kaur 2010) it is likely that this characteristic was

present in the common ancestor and was lost in a few

species in several tribes of Myrtaceae. Both evolu-

tionary trajectories suggest it was likely that the

capacity to produce high foliar a-pinene was lost three

times, in Lindsayomyrteae, Metrosidereae and Back-

housieae. The major difference between the two

possible evolutionary trajectories is that one involves

a gain-of-function and a loss-of-function and the other

involves three loss-of-function events.

Fahnrich et al. (2011) compiled a review of the

major and minor products from monoterpene syn-

thases from Nicotiana whose major product was

produced by 1,8-cineole synthase. The analysis

showed that the only monoterpene synthases that

produced 1,8-cineole as a minor product were the a-

terpineol synthases of tobacco (Nicotiana langsdorfii

and N. suaveolens) and grape (V. vinifera). These two

monoterpenes are structurally similar and a-terpineol

is produced as an intermediate in 1,8-cineole synthesis

(Keszei et al. 2010), therefore it is possible that an a-

terpineol synthase could gain the ability to produce

1,8-cineole without significantly reducing the activity

of the enzyme (Kampranis et al. 2007). a-Terpineol is

rarely a dominant compound in Myrtaceae, but it is

frequently found in low abundance. Nonetheless, it is

often in the five most abundant compounds when 1,8-

cineole is the most abundant compound (Supp

Table 1). It would be instructive to survey both 1,8-

cineole synthases and a-terpineol synthases in the

leaves of Myrtaceae species and use these in site-

directed mutagenesis studies to obtain an a-terpineol

synthase that produced large amounts of 1,8-cineole.

We predict a simple sequence change will convert an

a-terpineol synthase to a 1,8-cineole synthase, perhaps

in a similar way to the sesquiterpene synthase from

maize (Zea mays) that has two areas in the catalytic

pocket (Kollner et al. 2006). We further hypothesise

that the distribution of these two terpene synthases will

be similar across the family Myrtaceae.

There is a large diversity of terpenes in the leaves of

Myrtaceae where a-pinene and 1,8-cineole are the

dominant compounds. Since many samples contain

Fig. 2 The two possible evolutionary trajectories of Myrtaceae

with respect to high foliar 1,8-cineole concentration. The dotted

lines in the phylogeny represent lineages with the high foliar

1,8-cineole trait, and the black lines represent lineages without

this trait. The crosses represent the predicted loss of high foliar

1,8-cineole, and the star represents gaining this trait. Tribes

whose names are green are those with the high foliar 1,8-cineole

trait

Phytochem Rev (2014) 13:695–716 709

123

high foliar levels of a-pinene, this compound is the

likely ancestral foliar chemotype of all Myrtaceae and

high foliar 1,8-cineole appears to be a defining feature

of the family. The reaction cascade that leads to these

two compounds include the same carbocation inter-

mediate: the a-terpinyl cation (Kampranis et al. 2007),

which suggests only a small change in the amino acid

sequence of one enzyme could allow it to produce

significant quantities of both compounds.

Could the structure of terpene synthase enzymes

constrain the diversity of terpenes found

in Myrtaceae (Fig. 1)?

We found 72 unique terpenes that occurred at high

abundance in the leaves of Myrtaceae (Table 2),

representing all of the carbocation intermediates in

terpene biosynthesis (Keszei et al. 2008). On top of

this, there are 112 functional and expressed terpene

synthases in the E. grandis genome, 80 of which are

expressed to some level in young or mature leaves.

This is the largest terpene synthase gene family

described in a single species (Kulheim et al. 2013).

However, the germacrone-type sesquiterpenes, which

includes elemanes and cadinanes (Iguchi et al. 1969),

are typically rare or missing from the foliar terpene

profile of Myrtaceae (this study, Brophy 2012, pers.

comm.), despite there being a functional and

expressed isoledene synthase that can also be used to

produce cadinanes in significant amounts present in

the genome of E. grandis (Kulheim et al. 2013). This

suggests that terpene synthases impose very few

constraints on the foliar terpene profile.

The presence of a functional enzyme capable of

producing the rare germacrone-type compounds in the

leaves of E. grandis, prompted us to investigate the

evolution of the terpene synthases in Myrtaceae by

examining relevant site-directed mutagenesis studies.

Surprisingly, there are only 10 angiosperm terpene

synthases that have been used in site-directed muta-

genesis studies: 1,8-cineole synthase from Salvia

officinalis (Kampranis et al. 2007); (?)-germacrene

D synthase and a (-)-germacrene D synthase from

Solidago canadensis (Prosser et al. 2006); 5-epi-

sesquithujene synthase and sesquithujene synthase

from Z. mays (Kollner et al. 2004); (E)-a-bergamo-

tene/(E)-b-farnesene synthase from Z. mays (Kollner

et al. 2009); 5-epiaristolochene synthase from

Nicotiana tabacum (Greenhagen et al. 2006); zing-

iberene synthase, b-sesquiphellandrene synthase and

(E)-b-farnesene synthase from Sorghum bicolor (Zhu-

ang et al. 2012). Amino acids contributing to the

internal surface of the catalytic pocket are in approx-

imately the same position in the sequence, relative to

the highly conserved DDXXD motif (Fig. 1), how-

ever, there is a region in the C-terminal domain that

shows variation across the 10 genes. Single amino acid

changes introduced into the terpene synthase lead to

relatively simple changes in the terpene profile such as

changes in stereochemistry or the ratio of products

(Prosser et al. 2004). However, when several amino

acids (in this case up to seven) were changed, the

entire product profile was altered (Kampranis et al.

2007). There is strong evidence that terpene synthases

have diversified within distinct lineages by duplication

and neofunctionalisation resulting in an array of

enzymes across the plant kingdom (Degenhardt et al.

2009; Keeling and Bohlmann 2006). The site-directed

mutagenesis studies show that changes of just a few

amino acids can radically change its production of

particular terpenes in a variety of angiosperms, which

provides a method by which neofunctionalisation of

duplicate terpene synthases can occur. If a mutation

occurs in one copy of the terpene synthase, then it may

not affect the terpene profile since the other copy is

functioning normally. This single mutation could

result in a functionally distinct terpene synthase which

is then exposed to selection pressures. This hypothet-

ical scenario is unlikely to evolve quickly, but the

family Myrtaceae arose at least 66 mya and has been

diversifying since (Biffin et al. 2010).

Most often the amino acid changes induced through

site-directed mutagenesis result in a different ratio of

particular terpenes produced, and in natural systems,

this might lead to different dominant compound.

A Thus, the ability could evolve to produce large

quantities of any compound found in its terpene product

profile through a short series of mutations. Evidence of

this is seen in thea-terpineol synthases of many species,

which are the only characterised terpene synthases, that

are not 1,8-cineole synthases, but produce significant

amounts of 1,8-cineole (Fahnrich et al. 2011).

The limited number of studies available for review

would suggest that enzymatic constraints do not limit

the diversity of terpenes in Myrtaceae, despite the

general absence of germacrone-type compounds in the

leaves. Terpene synthase diversity probably arose

710 Phytochem Rev (2014) 13:695–716

123

through duplication and small, step-wise sequence

changes in ancestral enzymes. At different times these

genetic changes were exposed to different selection

pressures. It is clear that site-directed mutagenesis

studies could be used more widely to tackle evolu-

tionary questions, especially in families that contain

many species with closely related terpene synthases,

such as Eucalypteae and Melaleucaeae. Analysis of

this type of data could be used to better understand

how the diversity in terpene synthases arises and even

to design transgenic plants that could be used to test

the role of different terpenes in mediating ecological

interactions.

What is the role of different species of Myrtaceae

within ecosystems?

Myrteae and Syzygieae are the only two tribes of

Myrtaceae to contain fleshy-fruited species (Biffin

et al. 2010; Thornhill et al. 2012; Wilson et al.

2005) and of the 256 species, which have low foliar

1,8-cineole, the majority (*90 %—Table 1) belong

to the these two tribes. On top of this, these species,

with low foliar 1,8-cineole concentrations, have a

greater diversity of abundant foliar sesquiterpenes

than species with high foliar 1,8-cineole (Table 1).

A recent description of the foliar terpene profile of

two chemically mosaic Eucalyptus trees indicates

that resistance to herbivory is determined by the

concentration of one or a few compounds, whilst

susceptibility to herbivory is determined by the

absence of these compounds (Padovan et al. 2012).

If terpenes are playing a defensive role in Myrta-

ceae, then there are evolutionary forces acting on

these terpenes associated with resistance, and if the

resource allocation to terpene production does not

change, we could expect that the terpene profile of

undefended leaves should contain a greater variety

of compounds than the terpene profile of defended

leaves. Since 1,8-cineole can act as a defensive

compound in Myrtaceae (Butcher et al. 1994;

Lawler et al. 1998) and the foliar terpene profile

of some tribes is more diverse than others, these

data suggest that defence may not be the primary

function of terpenes in species of Myrteae and

Syzygieae. Alternative roles such as attracting

beneficial animals or meditating root-microbe inter-

actions in the soil are possible (Steffen et al. 2012).

Which species should be explored by the essential

oil industry and which species might prove

problematic?

Industrially valuable essential oils are produced from a

number of species of Myrtaceae. 1,8-cineole is sought

from several species of Eucalyptus (e.g. E. polybrac-

tea, E. globulus and E. loxophleba) but also from

Melaleuca cajaputi. A second species of Melaleuca

(Melaleuca alternifolia) produces industrially desir-

able oils rich in terpinen-4-ol. There are two Austra-

lian standards for euclayptus oil. The first standard is

for eucalyptus oil that is 70–75 % 1,8-cineole (AS

2113.1-1998) and states that the terpene profile must

contain 1,8-cineole (70–75 %), limonene (1–15 %)

and a-terpineol (0.5–9 %). Whilst a-pinene, a-phel-

landrene and iso-valeric aldehyde can only be present

in trace amounts. The second standard is for eucalyp-

tus oil that is 80–85 % 1,8-cineole (AS 2113.2-1998)

and states that the terpene profile must contain 1,8-

cineole (80–85 %), limonene (1–15 %) and a-terpin-

eol (0.5–9 %). Whilst a-pinene, a-phellandrene and

iso-valeric aldehyde are only allowed in trace amounts

(\6 % of the oil). Although there are more than 900

species of Eucalyptus, only 20 species have been

exploited for the commercial production of eucalyptus

oil (Boland et al. 1991).

A similar story is true for the tea tree oil. The

Australian standard for tea tree oil (AS 2782-2009; ISO

4730:2004) states that the terpene profile must contain

terpinen-4-ol (30–48 %), c-terpinene (10–28 %), 1,8-

cineole (\15 %) and a-terpinene (5–13 %). Whilst a-

pinene, sabinene, limonene, q-cymene, terpinolene, a-

terpineol, aromadendrene, ledene, d-cadinene, globu-

lol and viridiflorol must be kept to trace amounts

(\8 % of the oil). There are more than 350 species of

Melaleuca, however only four species have been

exploited by the tea tree oil industry (Australian

Standards for tea tree oil—AS 2782-2009).

The major challenges for the essential oil industries

using species from the family Myrtaceae are that

require careful selection of stock to avoid undesirable

chemotypic effects, understanding the degree to which

planting stock is resistant to emerging pathogens (e.g.

Myrtle Rust) and awareness of regulatory issues

around undesirable traits. For example limonene and

its oxidation products are considered undesirable

because of irritant effects on the skin in high

concentrations (Christensson et al. 2009). However,

Phytochem Rev (2014) 13:695–716 711

123

limonene is formed by the same carbocation interme-

diates as is 1,8-cineole and is often the product of

cineole synthases. Understanding the origins and

enzymatic control of limonene formation may indicate

the degree to which these two compounds could be

uncoupled so that 1,8-cineole is produced without

limonene.

The following are a list of species that could be

considered by these two essential oil industries as a

means of diversifying the stock populations. Eucalyptus

oil: Blepharocalyx salicifolius, Callistemon pinifolius,

Callistemon viminalis, Eucalyptus aromaphloia, Euca-

lyptus johnsoniana, Melaleuca armillaris sub. armil-

laris, Melaleuca fulgens sub. corrugata, Melaleuca

hypericifolia, Melaleuca lateralis, Melaleuca paludi-

cola, Melaleuca phoenicea, Myrcianthes cisplatensis,

Myrcianthes pungens, Pimenta obscura, Callistemon

glaucus, Callistemon acuminatus, Callistemon citrinus,

Callistemon recurvus, Eucalyptus angustissima, Euca-

lyptus cinerea, Eucalyptus luehmanniana, Eucalyptus

parvifolia, Eucalyptus risdonii, Eucalyptus robertsonii,

Eucalyptus willisii, Melaleuca acutifolia, Melaleuca

calothamnoides, Melaleuca linophylla, Melaleuca oro-

phila, Melaleuca recurva, Melaleuca sabrina, Melal-

euca stereophloia and Myrcianthes ‘black fruit’. Tea

tree oil: Eucalyptus ovata, Leptospermum polygalifoli-

um Salisb sub. cismontanum, Psidium caudatum (also

known as Calycolpus moritzianus), Melaleuca concre-

ta, Melaleuca ochroma, Uromyrtus sp McPherson

Range, Melaleuca argentea, Melaleuca foliolosa and

Melaleuca dissitiflora. Caution should be taken to

evaluate each species thoroughly before using it as an

industrial source of essential oils.

Future directions

Our database is almost entirely focussed on terpenes

from mature leaves because this is the most common

material studied when considering whether a plant is

suitable for industrial purposes. Terpenes play a

crucial role in protecting leaves from herbivory and

infection (DeGabriel et al. 2009; Macel and Klinkh-

amer 2010; Matsuki et al. 2011), but Myrtaceae must

employ terpenes in important roles in root and wood

tissues since many terpene synthases are highly

expressed in these tissues as they are in leaves

(Kulheim et al. 2013, Rasmann et al. 2012). There is

a distinct gap in our understanding of the role terpenes

play in roots and wood which limits our understanding

of the ecological importance of terpenes to plants. If

we are truly to understand the role of terpenes in

Myrtaceae, this will need to be addressed.

The second significant gap in data we identified was

in the diversity of site-directed mutagenesis studies of

plant terpene synthases. There are only 10 angiosperm

terpene synthases used in site-directed mutagenesis

studies (Greenhagen et al. 2006; Kampranis et al.

2007; Kollner et al. 2009; Kollner et al. 2004; Prosser

et al. 2004; Zhuang et al. 2012), with only a few more

from the gymnosperms (Hyatt and Croteau 2005;

Katoh et al. 2004). These experiments provide insights

into terpene synthase evolution that is unparalleled by

any other method. In Myrtaceae the most useful target

of these approaches would be a detailed examination

of the function of the catalytic pocket, with the focus

on enzymatic evolution.

The final and arguably the most important future

direction is to standardise the method of collecting and

reporting constitutive terpene data in Myrtaceae.

Almost every author cited in this study used a different

method for extracting terpenes and presenting their

data that is independent of the journal requirements.

Most of this revolves around whether data is reported

either as proportions or as concentrations relative to

dry or wet weight. Birks and Kanowski (1993)

reviewed these issues for analysis of conifer resin

and propose standardised statistical approaches for

analysis and reporting proportional composition data.

For a comparison amongst different Myrtaceae, we

believe that terpene data should be presented as

concentration since proportion can be calculated from

concentration (or presented along with concentration)

but concentration cannot be calculated from propor-

tional data. Approximately half of the studies that we

reviewed present concentration data in terms of fresh

weight and the other half dry weight. One of the

problems in presenting dry weight data of terpene rich

leaves is that many drying methods volatilize the

terpenes as well as the water but approaches have been

developed to account for this (Ammon et al. 1985).

Although analyses of leaves for industrial purposes

might favour reporting concentration on a wet weight

basis, most if not all ecological analyses should report

concentration on a dry weight basis and we urge future

studies to standardise on these units or to report both.

There are numerous terpene extraction methods

currently being used, each with different advantages

712 Phytochem Rev (2014) 13:695–716

123

and limitations. The two main methods of terpene

extraction that underlie the data discussed are distil-

lation (steam, hydro or thermo) and organic solvent

extraction (ethanol, pentane, hexane and dichloro-

methane—data not shown). The major disadvantages

of distillation are the loss of thermolabile and highly

volatile compounds. For example, cryptomeridiol and

4-epicryptomeridiol are two eudesmol hydrates that

are not observed after isolation of oil by steam

distillation as they are thermolabile (Cornwell et al.

2000b); and cubenol and epicubenol break down to d-

cadinene, epicubenol and cubenol as a result of the

processes involved in steam distillation (Cornwell

et al. 2000a, b, 2001). Themajor disadvantages of

solvent extraction are inaccurate measures of concen-

trations due to evaporation and the varied success of

extraction of different monoterpene alcohols (e.g.

extraction of a higher amount of linalool compared

with distillation methods, anda lower amount of 1,8-

cineole (Charles and Simon 1990). We believe that

structural re-arrangements are far more detrimental to

a survey of plant terpene diversity than differences in

the proportion of high abundance compounds and thus

solvent extraction methods should be favoured over

distillation methods.

Acknowledgments This research was supported by a grant

from the Australian Research Council to WJF (LP110100184).

We thank our partners in that work (Australian Tea Tree

Industry Association and GR Davis) for their support and

appreciate the advice and comments of many essential oil

chemists.

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