Intra-annual wood formation in tropical forests: Case...

Faculty of Bioscience Engineering

Academic year 2014 – 2015

Intra-annual wood formation in tropical forests: Case study in the Mayombe forest, DRC

Selwin Maginet Promoter: Prof. dr. ir. Joris Van Acker Co-promoter: Prof. dr. ir. Kathy Steppe Tutors: dr. ir. Jan Van den Bulcke, ir. Tom De Mil

Thesis submitted in fulfillment of the requirements for the degree of: Master of Science in Bioscience Engineering: Forest and Nature

Management

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Ghent, June 2015

The promoter, The co-promoter, The author,

Prof. dr. ir. Joris Van Acker Prof. dr. ir. Kathy Steppe Selwin Maginet

Acknowledgements

Special thanks goes to Victor for the past three years that we have been studying at the Coupure.

Without him, I would never dare to go on this adventure. We had a great time in the Democratic

Republic of Congo (DRC) together. I think I have found a friend for life during the last 3 years, correct

me if I’m wrong. And if we had not yet been friends before our Congo adventure, the friendship was

definitely born while we were searching charcoal in Manzonzi (#YOLO). Seriously, you’re really a

great guy.

At the same time, there’s only one man that cannot enough be acknowledged. I want to thank him

for the amazing guidance in the DRC, because without him, Victor and I would just have been two

monkeys running in the jungle. Also, I want to thank him because I could always ask him questions,

even though last year has been very busy for him. I admire his diligence and perseverance. Those

characteristics make him a very good tutor and PhD student. I’m talking about the man who soon will

deliver the best PhD dissertation ever made, special thanks to Tom De Mil.

Further, I want to thank the man who currently has written the best PhD dissertation ever, my other

tutor Jan Van den Bulcke. He really owns everything that has to do with XCT scanning and is probably

wiser than Gandalf the sorcerer.

I would like to express my sincere gratitude to my two promoters Prof. dr. ir. Joris Van Acker and Prof.

dr. ir. Kathy Steppe for their knowledge and wisdom. Without their help I would never be able to

study this subject. Thanks for having me at your lab!

Thanks to Maaike De Ridder for helping me with the writing of my VLIR-UOS scholarship, and for her

concern about the malaria. Also thanks to all other people involved in the good running of the

fieldtrip in the DRC: Celine De Caluwé to be the big sister we did not have, Hans Beeckman for his

experience, Bhely, Fils, Jean Maron, Olivier and papa Mbambi for their nice company and efforts in

the field, and the local organization INERA, to have us in Luki.

I also want to thank Stijn Willem for all the help with the stem discs and for the nice conversations

we had, Piet Dekeyser for his patience while making the cross-sections, and all other very nice people

of Woodlab UGent I thank Matthijs Vynck for helping me out with the Gompertz model. I thank VLIR-

UOS, the Leopold III-fund, BOF-UGent scholarship for the financial support in the DRC. Also thanks to

the KMMA for the stem discs.

Of course, I want to thank all my family and friends, especially the most beautiful girl in the world,

Hannie, and my mother and father for the amazing support during my studies.

Contents

List of abbreviations ................................................................................................................................. i

Abstract ....................................................................................................................................................ii

Samenvatting ........................................................................................................................................... iii

1. Introduction ..................................................................................................................................... 1

2. Literature ......................................................................................................................................... 3

2.1. Dendrochronology ................................................................................................................... 3

2.1.1. Definitions and history .................................................................................................... 3

2.1.2. Basic principles and concepts .......................................................................................... 4

2.1.3. Growth ring structures .................................................................................................... 8

2.1.4. Tropical dendrochronology ........................................................................................... 10

2.2. Intra-annual wood formation ................................................................................................ 13

2.2.1. Scope ............................................................................................................................. 13

2.2.2. Cambial pinning ............................................................................................................. 13

2.2.3. Dendrometer measurements ........................................................................................ 14

2.2.4. Other methods used in intra-annual growth studies .................................................... 15

2.2.5. Studies on tropical tree growth ..................................................................................... 15

3. Materials and methods ................................................................................................................. 18

3.1. Study site ............................................................................................................................... 18

3.2. Studied species ...................................................................................................................... 19

3.3. Cambial marking and sample preparation ............................................................................ 20

3.4. Characterization of tree rings ................................................................................................ 22

3.5. X-ray tomography as a method to study radial growth ........................................................ 22

3.6. Anatomical study around the pinning zone .......................................................................... 24

3.7. Analysis of climate-growth relationships .............................................................................. 24

3.8. Analysis of phenology-growth relationships ......................................................................... 25

4. Results and discussion ................................................................................................................... 26

4.1. Diameter at breast height ..................................................................................................... 26

4.2. Growth rings and ring anomalies .......................................................................................... 27

4.3. Relative intra-annual growth ................................................................................................. 34

4.3.1. Results per species ........................................................................................................ 34

4.3.2. General discussion ......................................................................................................... 46

4.4. Anatomical features around the pinning zone ...................................................................... 50

4.5. Climate-growth relationships ................................................................................................ 50

4.6. Phenology-growth relationships ........................................................................................... 55

5. Conclusion ..................................................................................................................................... 59

6. Prospective research ..................................................................................................................... 60

6.1. Dendrometer data ................................................................................................................. 60

6.2. Non-destructive visualization of the stem contour ............................................................... 61

6.3. Alternative crown projection method ................................................................................... 62

6.4. 3D-reconstruction of the stem .............................................................................................. 65

6.5. Plot design and measurements around the studied tree ..................................................... 67

References ............................................................................................................................................. 68

Addenda ................................................................................................................................................ 76

Addendum 1: Code to fit the Gompertz function in R (R Core Team, 2015). .................................. 76

Addendum 2: Measurements for the quality of the fitted Gompertz function. ............................... 78

Addendum 3: Meteorological data of the Luki station ..................................................................... 80

Addendum 4: Phenological observations of the pinned trees from January till September 2014. .. 81

Addendum 5: Historical phenological observations in the Luki Biosphere reserve (1948-1957) ..... 84

i

List of abbreviations

DBH

DRC

XCT

RSE

Diameter at breast height

Democratic Republic of Congo

X-ray computed tomography

Residual standard error

ii

Abstract

Intra-annual wood formation has hardly been studied in tropical forests. The purpose of this work is,

therefore, to study tree growth in tropical trees in detail, and the impact of climate and phenology

on wood formation, through a combination of tree ring analysis, wood anatomy and X-ray computed

tomography (XCT) on cambial pinnings of stem discs.

In the Luki Biosphere Reserve (Mayombe forest), 18 selected trees of 6 different species (Polyalthia

suaveolens, Xylopia wilwerthii, Hylodendron gabunense, Newtonia sp., Corynanthe paniculata, and

Aidia ochroleuca) were monthly pinned during one growing season (from September 2013 till July

2014) and harvested in September 2014. The pinned stem discs are digitized with a flatbed scanner

and camera to reconstruct their shape and relocate the marks when destructively subsampled. Tree

rings are characterized on these optical images. For 8 discs, small cylinders are then drilled containing

the pinnings. These cylinders are scanned with XCT, resulting in a three dimensional view of the

wood structure around the pinned zone. Based on XCT analysis, time series of relative intra-annual

growth are measured for these individuals. Furthermore, these XCT volumes provide a detailed

indication where thin cross-sections can be made for microscopy-based wound studies.

This XCT approach allows to show clear variations in growth from one species to another, and among

trees of the same species. Combining growth ring and high resolution pinning data leads to

fundamental knowledge about intra-annual tree growth of tropical trees. Furthermore, analyses of

cambial pinnings using XCT considerably reduces the time spent in the laboratory.

The results are valuable for management of tropical forests and predicting changes in tree growth as

a result of climate change. This stepwise approach can also be used for the study of pinned

Terminalia superba trees from the Nkulapark in Luki that are equipped with dendrometers, and thus

is suitable for other studies where cambial marks are available.

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Samenvatting

Intra-annuele houtvorming is nauwelijks bestudeerd in tropische bossen. Daarom is het doel van dit

werk om gedetailleerd tropische boomgroei, en de impact van klimaat en fenologie op houtvorming

te bestuderen via een combinatie van jaarringanalyse, houtanatomie en ‘X-ray computed

tomography’ (XCT) op cambiale pinningen in stamschijven.

Achttien geselecteerde bomen die behoren tot zes verschillende soorten (Polyalthia suaveolens,

Xylopia wilwerthii, Hylodendron gabunense, Newtonia sp., Corynanthe paniculata en Aidia

ochroleuca) werden maandelijks gepind (lees: verwond) gedurende één groeiseizoen (van september

2013 tot juli 2014) en nadien geoogst in september 2014. De gepinde stamschijven worden

gedigitaliseerd met een flatbedscanner en een camera om zo hun vorm en de locatie van de

pinningen te reconstrueren alvorens op een destructieve manier submonsters genomen worden. Op

deze optische beelden worden de jaarringen gedigitaliseerd. Kleine cilinders die de pinningen

bevatten, worden nadien uit 8 stamschijven geboord. De cilinders worden gescand met XCT, op deze

manier wordt een drie dimensionaal beeld verworven van de houtstructuren rond de gepinde zone.

Op basis van XCT-scan analyse worden tijdsreeksen van relatieve intra-annuele groei opgemeten.

Bovendien geven de XCT-beelden een gedetailleerde indicatie van de positie waarop anatomische

coupes kunnen worden gemaakt.

Dankzij deze methode zijn duidelijke groeivariaties tussen de verschillende boomsoorten en ook

tussen de individuen van dezelfde soort bepaald. De combinatie van de karakterisatie van de

groeiringen en de data van de pinningen met een hoge resolutie leidt tot fundamentele kennis over

intra-annuele houtvorming van tropische bomen. Bovendien zorgt de analyse van de cambiale

pinningen via XCT voor een aanzienlijke reductie van de gespendeerde tijd in het laboratorium.

De resultaten zijn waardevol voor het beheer van tropische bossen en voor het voorspellen van

veranderingen in boomgroei als reactie op de klimaatsverandering. De stapsgewijze procedure kan

ook gebruikt worden tijdens het onderzoek over de gepinde Terminalia superba in het Nkula park in

Luki, die ook zijn uitgerust met dendrometers. De voorgestelde methode kan dus zeker en vast

gebruikt worden in andere studies waar cambiale pinningen beschikbaar zijn.

1

1. Introduction

Dendrochronological studies analyze growth rings in trees, and examines events through time that

are recorded in these growth rings or can be dated by these rings (Speer, 2010). Therefore, the use of

dendrochronology can determine annual growth dynamics in trees, and relate them to climate

variables and phenological observations (Fritts, 1976). The existence of tree rings is a prerequisite for

dendrochronological studies. Annual formation of growth rings is widely assumed for temperate and

semi-arid zones (Norton and Ogden, 1992; Worbes, 2004). On the contrary, the complexity of

tropical tree growth structures and the widely assumed lack of seasonality in tropical areas resulted

in a general pessimism among dendrochronologists about the existence of tropical tree rings during

the 20th century (Jacoby, 1989; Speer, 2010; Wils et al., 2011). Due to the development of new

techniques, dendrochronology also has found his way to the tropics, and for many tropical species

the appearance of growth rings has been approved (Worbes, 2002). However, tropical tree ring

analysis still deals with some misunderstandings and misinterpretations, caused by ring anomalies

(Speer, 2010). Thus, together with the improvement of the available methods, new techniques are

necessary in order to determine growth dynamics in tropical tree species.

A more detailed intra-annual growth study splits up radial increment of one growing season into

shorter time units, which can result in better insights on tree growth and a more direct association

between tree growth and climate (Seo et al., 2007). Cambial pinning is one of the techniques that are

used to determine intra-annual growth dynamics (Wolter, 1968). Although this technique provides

valuable information on more detailed tree growth, the analyses is a time consuming process:

carefully selected high-quality thin cross-sections have to be made for a microscopic evaluation of

the wood features. Therefore, a stepwise and faster method to analyze the pinnings is necessary in

order to optimize intra-annual studies.

Intra-annual wood formation has hardly been studied in tropical forests. The purpose of this work is,

therefore, to study tree growth in tropical trees, and the impact of climate and phenology on wood

formation in detail, through a combination of tree ring analysis, wood anatomy and X-ray computed

tomography (XCT) on cambial pinnings of stem discs. Three objectives should be achieved:

1. The determination of intra-annual growth dynamics of tropical species based on a unique

combination of pinnings, XCT, thin cross-sections and optical stem disc images, in order to

obtain fundamental knowledge on tropical tree growth.

2. Evaluation of XCT as a tool to study intra-annual growth via cambial pinnings.

3. An assessment on climate- and phenology-growth relationships of the studied species.

2

When proven valuable, the combination of tree ring analysis, wood anatomy and XCT on cambial

pinnings of stem discs, as described in this work, could be performed for Terminalia superba in the

Nkula park of Luki. Moreover, dendrometers, that are installed on these trees, could add high

temporal data to study intra-annual wood formation more in detail.

3

2. Literature

2.1. Dendrochronology

2.1.1. Definitions and history

Dendrochronology is “both an old and modern science” mainly developed over the last century with

the help of expansion of techniques like isotope analysis, X-ray densitometry and specific statistical

analysis (Worbes, 2004). Some interesting references on the subject have been published (Fritts,

1976; Schweingruber, 1988; Cook and Kairiukstis, 1992; Worbes, 2004; Speer, 2010). A broad

definition of dendrochronology was mentioned by Speer (2010):

“Dendrochronology is one of the most important environmental recording techniques for a

variety of natural environmental processes and a monitor for human-caused changes to the

environment… Dendrochronology examines events through time that are recorded in the tree-

ring structure or can be dated by tree rings” (Speer, 2010).

As defined by Speer, the dating of past events requires the presence of tree rings in wood samples

for further dendrochronological analyses. When tree age can be determined, tree ring analysis could

be regarded as retrospective biomonitoring1. For the lifetime of a tree, records can be obtained of

ancient or recent climatic variables, sudden events like fires, hurricanes etc. and human interventions

in nature. Crossdating tree records can even provide examination of events that go way back in time

(Section 2.1.2). Trees monitor past events based on the principle that any environmental factor limits

a process that influences tree growth from one year to the next (Worbes, 2004; Speer, 2010).

Dendrochronology has been used as a tool to date artwork and archaeological material for a long

time. Tree ring research has nowadays lots of applications, for example to settle conflicts (Sellards et

al., 1923). Dendrochronology is even considered as an independent discipline in science with its own

principles, theories and techniques (Worbes, 2004; Speer, 2010). Dendrochronology has developed

several subdisciplines. One of the first was the reconstruction of past and recent climate, called

dendroclimatology (Fritts, 1976; Fritts et al., 1980; Worbes, 2004). Hughes (2002) pointed out the

strengths and weaknesses of using tree rings to study climate variability. Other subdisciplines are

dendroecology, dendrochemistry, dendroarcheology and dendrogeomorphology (Grissino-Mayer,

1996; Speer, 2010).

In 1937 Andrew Ellicott Douglass was the founder of the first Laboratory of Tree Ring Research at the

University of Arizona. This formed the start of dendrochronology as a modern scientific discipline

1 As mentioned by Worbes (2004), “Biomonitoring is the reflection of growth factors by biological organisms and their change in time.”

4

(Speer, 2010). However in 322 BC Theophrastus already suggested that trees produce annual rings

(Studhalter, 1956). More than 1500 years later, Leonardo da Vinci realized the relationship between

the growth of tree rings and climate (Sarton, 1954). In the 1800s in Germany, Theodor Hartig

broadened the knowledge of the ecological and climatic aspects of dendrochronology

(Schweingruber, 1996; Worbes, 2004). Douglass developed the discipline during the first half of the

20th century. He was the first to largely use and refine the crossdating technique and continuously

developing chronologies2 (Douglas, 1941; Web, 1983; Nash, 1999). He is considered to be the “father

of dendrochronology” (Schweingruber, 1988).

Several milestones of dendrochronology were listed by Worbes (2004). Speer (2010) represented a

chronological review of the history of dendrochronology. Cook and Kairiukstis (1992) described in

detail the development of the discipline separately for different continents.

2.1.2. Basic principles and concepts

Dendrochronologists maintain some basic principles and concepts in the field of tree ring analysis

while collecting samples, analyzing tree rings and drawing conclusions. Fritts (1976) and Grissino-

Mayer (1996) suggest 7 principles, while Speer (2010) defines 6 principles, amplified with 3 concepts.

The latter is used here.

Principle of uniformitarianism

This principle is used to reconstruct past climate, based on the words of James Hutton on 1785: “The

present is the key to the past.” It means that processes that link current environment parameters

with current tree growth must be the same in the past. Based on the knowledge of present-day

relationships between tree growth and climate, the reconstruction of past climate conditions that

were not recorded, is possible (Fritts, 1976; Grissino-Mayer, 1996; Speer, 2010). In addition,

dendrochronological studies of past climate provide a prediction about climatic variability in the

future. In other words: “the past is the key to the future” (Grissino-Mayer, 1996; Vaganov et al.,

1999; Briffa, 2000; Cook et al., 2004).

Principle of crossdating

Crossdating is the main tool of dendrochronology. Patterns in rings widths or density of several tree

ring records are matched so the exact year of growth of each annual ring can be determined and

longtime chronologies can be constructed (Figure 1). Crossdating implies more than just counting

rings: the detection of false and missing rings (Section 2.1.3) provides accurate dates for every single

2 Speer (2004) defined a chronology as “a site-level representation of tree growth”. A site is “a spatially proximal group of trees with similar environmental conditions such as slope, aspect and climate.”

5

ring in a tree ring sequence, which is a requisite when comparing annual data with tree ring patterns

or dating past events (Fritts, 1976; Grissino-Mayer, 1996; Speer, 2010). Successful crossdating

indicates that some common external climatic or environmental information is recorded in different

trees (Fritts, 1976; Pilcher and Gray, 1982; Worbes, 1995; Cherubini et al., 1998).

Figure 1: The construction of a tree chronology. Rings widths are measured and presented in a diagram. The diagrams are transformed into curves, so ring width series can be matched together based on overlapping sequences (Worbes, 2004).

Skeleton plotting is a classic method of dating tree rings developed by Douglas. Each growth year is

assigned to a vertical line and the length of the line represents the relative importance of the ring to

the series. Individual tree skeleton plots result in a master chronology of a stand (Speer, 2010).

Important growth rings are referred to as ‘pointer years’, annual years that differ notably from

adjacent rings. Clear differences between rings can be found in ring widths, proportion of latewood,

density, traumatic tissue and any other tree ring feature (Schweingruber et al. 1990; Schweingruber,

1992).

Principle of limiting factors

This principle states that a biological process, such as growth, is constrained by the most limiting

environmental factor that effects the process. Environmental factors limiting growth are

precipitation, temperature, competition for nutrients and light, etc. In arid areas for example, tree

growth is often bounded by the amount of precipitation (Fritts, 1976; Grissino-Mayer, 1996; Speer,

2010).

The limiting factor determines the tree ring width to some extent in a relative long period of the

lifetime of the tree. However, the abundance of factors can change over time, so others factors could

become limiting. The physiological response of the tree get complex if multiple limiting factors

appear (Speer, 2010). As noted by Fritts (1976): “… Rings widths can be cross-dated only if one or

6

more environmental factors becomes critically limiting, persists sufficiently long, and acts over a wide

enough geographical area to cause ring widths or other features to vary the same way in many

trees.”

Principle of the aggregate tree growth model

Tree growth depends on multiple limiting factors so each tree ring holds information about a

complex array of variables. The aggregate tree growth model provides a conceptual model that

analysis the individual effect of those different variables (Cook, 1992):

𝑅𝑡 = 𝑓(𝐴𝑡 , 𝐶𝑡, 𝐷1𝑡, 𝐷2𝑡, 𝐸𝑡) (1)

Where Rt is the ring width at year t, At is the age- or size-related growth trend due to normal

physiological aging processes, Ct is the climatically related environmental signal such as precipitation

and temperature, D1t is the endogenous disturbance within the stand such as the loss of light-

competitive trees, D2t is the exogenous disturbance from outside the stand such as a disease

outbreak, and Et is the signal that is not covered by the above factors (Cook, 1992; Grissino-Mayer,

1996; Speer, 2010). Cook (1992) and Speer (2010) explained the variables more in detail.

Concept of autocorrelation

Autocorrelation states the correlation of a variable with itself over successive time intervals. This

process occurs in biological organisms. The current year’s tree growth is not only affected by

variables such as climate (cf. Principle of the aggregate tree growth model), but also by the previous

year’s growth (Figure 2). Environmental factors of previous years such as heat, rainfall, carbon

dioxide and nutrient competition, influence the development of new buds, sugars and hormones.

Even a few years later the amount leaves, roots and fruits that could be formed, effect tree growth

(Fritts, 1976; Speer, 2010).

Concept of ecological amplitude

Sampling trees at the margins of their natural range is the best way to perform climate-related

research. A species is assumed to be more stressed at the edge of its range, so more information on

the variable of interest can be collected. This range, in which a certain species can grow and

reproduce, is called the ecological amplitude (Fritts, 1976; Grissino-Mayer, 1996; Speer, 2010).

Principle of site selection

Trees record information about a complex series of variables (cf. Principle of the aggregate tree

growth model). On the one hand, dendrochronologists must select sites that will produce tree ring

7

Figure 2: Diagram that shows tree growth often includes autocorrelation, where t is the current year (Drawing by M. Huggins, in: Fritts, 1976).

series sensitive to environmental variable of interest in order to maximize the signal. E.g. if past

drought conditions must be reconstructed, a sampling in an arid environment should be performed.

On the other hand trees with the same ring size are considered to have complacent growth. In this

case growth is limited by biological factors instead of the environmental variability and trees are

located at the center of their ecological amplitude. The selection of this type of sites is for example

interesting for masting research (Stokes and Smiley, 1968; Grissino-Mayer, 1996; Speer, 2010).

Principle of replication

The principle of replication suggests that a dendrochronologist must collect multiple samples of one

tree and sample multiple trees per site. The replication facilitates the detection of missing and false

rings, so valid crossdating is possible. Moreover, the environmental signal of interest can be

maximized by increasing the number of samples, because the amount non-desirable signals or ‘noise’

is reduced (Fritts, 1976; Grissino-Mayer, 1996; Speer, 2010).

Concept of standardization

Long-term variability in tree ring series such as age-related growth trends can be detected using

standardization techniques. This variability can be considered as noise when defining shorter-term

processes. On the contrary, for long-term climate change studies, the noise is regarded useful

information. The concept of standardization fits curves to tree ring series. The negative exponential

curve is deterministic, based on a same amount of wood that is added on the surface of an increasing

cylinder year after year. Cubic smoothing splines are examples of empirical curves (Fritts, 1976; Cook,

1985; Speer, 2010).

8

2.1.3. Growth ring structures

The aspect of tree rings

A tree ring can be defined as:

“A layer of wood cells produced by a tree or shrub in one year, usually consisting of thin-

walled cells formed early in the growing season (called earlywood) and thicker-walled cells

produced later in the growing season (called latewood). The beginning of earlywood

formation and the end of the latewood formation form one annual ring, which usually

extends around the entire circumference of the tree” (Grissino-mayer, 1996).

The existence of tree rings is a prerequisite for dendrochronological studies. Annual formation of

growth rings is widely assumed for temperate and semi-arid zones (Norton and Ogden, 1992;

Worbes, 2004). Until the end of the 20th century the existence of growth rings in the tropics was

questioned because the seasonality of climatic variables was believed not to be strong enough.

Studies mainly focused on temperate zones (Whitmore, 1990; Worbes, 2002). However the last

decades tropical trees with annual growth rings are being studied at larger scale, so the assumption

of the existence of tropical growth rings increases (Worbes, 1995, 2002; Fichtler et al., 2003, 2004;

Trouet et al., 2006; Speer, 2010).

According to Worbes (2004), tree growth is generally influenced by three different types of climatic

seasonality:

- Annual temperature variation with the occurrence of cambial dormancy as a consequence of

low temperatures in winter.

- Great rivers in the tropics that flood annually cause anoxic conditions in the soil. These

conditions hinder the root respiration and water uptake so the cambium becomes dormant.

- Tree growth in the major part of the tropics is influenced by variation in precipitation due to

a rainy and dry season.

Tree rings can be classified based on four basic growth zone features (Worbes, 2004):

Type 1: Density variations occurring in gymnosperms as a unique feature and in broadleaved trees

together with one or more of the following features: cell wall thickness becomes greater and the cell

lumen becomes smaller from earlywood to latewood (all coniferous species, Annonaceae,

Verbenaceae, Lauraceae, and many other families) (Figure 3a; Worbes, 2004).

Type 2: Marginal parenchyma bands run around the entire cross-section and consist usually of one or

few cell rows (Annonaceae, Bignoniaceae, Leguminosae, Meliaceae) (Figure 3b; Worbes, 2004).

9

Type 3: Rings are characterized by periodical patterns of parenchyma and fiber tissue. The bands

usually become narrower towards the end of a growth zone (Euphorbiaceae, Moraceae, Sapotaceae,

Bignoniaceae, in Ulmus spp., Fraxinus spp., and many others) (Figure 3c; Worbes, 2004).

Type 4: Ring porousness is widely distributed in the temperate zones (e.g., Quercus) but occurs in

only a few examples in the tropics (e.g., Tectona grandis, and some Meliaceae) (Figure 3d; Worbes,

2004).

Figure 3: Illustrations of the four basic growth zone features (Worbes, 2004).

Anomalies

The process of crossdating detects anomalous tree rings, so age dating of each tree ring is possible.

Nevertheless the presence of anomalies can impede ring counts, even in species with annual growth.

A well-polished full cross section enables the identification of ring boundaries and anomalous rings

(Norton and Ogden, 1992; Speer et al., 2004).

False rings or intra-annual growth bands are a consequence of extreme environmental conditions

(Fritts, 1976; Schweingruber, 1980). Limiting factors impede tree growth during a period of the

growing season. A narrow band of thick-walled cells occurs in the middle of the growth ring as

pseudo-latewood. When the limiting resource returns the tree resume the growing process and cell

walls gradually thin back out (Norton and Ogden, 1992; Speer, 2010).

When optimal growing conditions persist between consecutive growth seasons, some trees form

diffuse ring boundaries. Cambial activity will not cease so the tree continues to produce thick-walled

latewood cells during the second year. The feature occurs especially in the tropics where seasonality

is weak (Speer, 2010).

The amount of rings and growth of studied trees should ideally be the same around the full stem

circumference. This facilitates the comparison of cores taken at the same stem height. However

partial, locally absent or missing rings are a common feature of many tree ring series. These types of

10

rings occur in trees growing near the edge of their ecological amplitude. Growth rings are then often

incompletely formed during environmentally limiting years. Another feature is called ring wedging.

This phenomena occurs when over several years in certain segments of the circumference radial

increment occurs rapid, while growth is slow or absent in others. Changes in the appearance of major

branches can cause these effects (Fritts et al., 1965; Fritts, 1976; Norton et al., 1987; Speer, 2010).

Wedging occurs more often in small understory trees than in canopy trees or emergents. In general

changing light conditions as a consequence of variation in competitive pressure explain wedging

(Worbes, 2002).

Micro rings are bands of only two cells wide. They are difficult to distinct on a cross section that is

not good polished. Other ring anomalies are frost rings and fire scars, discussed in Speer (2010).

2.1.4. Tropical dendrochronology

During the 20th century the complexity of tropical tree growth structures and the widely assumed

lack of seasonality in tropical areas resulted in a general pessimism among dendrochronologists

about the existence of tropical tree rings (Jacoby, 1989; Speer, 2010; Wils et al., 2011). However with

the help of innovative techniques to determine tree growth periodicity (Section 2.2), successful

dendrochronological studies in tropical areas have been published over the last decades. A review on

tropical dendrochronology is represented by Jacoby (1989), Worbes (2002), Speer (2010) and

Gebrekirstos et al. (2014).

History

Seasonality of tropical tree growth was observed in the 19th century. Consequently, the existence of

annual tree rings in tropical trees was assumed (Ursprung, 1900). However, in the early 20th century

the existence of tree rings in the tropics was disputed controversially, based on a great number of

single observations on non-seasonal and asynchronous phenological features (e.g. Klebs, 1912;

Koriba, 1958). At the same time Coster (1927, 1928) and Berlage (1931) successfully identified

tropical tree rings on the isle of Java. Two different climate types occur on Java: seasonal monsoon in

the east and almost everwet conditions on the west (Geiger, 1915). The growth structures of species

living in both habitats could be compared. The research resulted in a chronology for Tectona grandis

as the first tropical one and a clear connection between ring widths and precipitation (Berlage,

1931).

As a milestone, Mariaux (1967, 1969) introduced the Mariaux-window to determine annual growth

of important tropical timber species in western Africa (Section 2.2.2). Later, the workshop on ‘Age

and Growth Rate of Tropical Trees’ at the Harvard Forest in 1980 discussed past and future

knowledge and techniques of tropical tree ring analysis (Borman and Berlyn, 1981; Jacobi, 1989;

11

Worbes, 2002). From then until now, tropical dendrochronology increased and developed in several

applications (see Applications, in 2.1.4).

The number of dendrochronological studies in Africa was rather small until the start of the 21st

century (Speer, 2010). However the last 15 years the occurrence of tree species that form annual

growth rings has been explored, in order to fill up the data gap on the continent (Fichtler et al.,

2004; Verheyden et al., 2004; Trouet et al., 2006; Couralet et al., 2010b; De Ridder et al., 2013).

Détienne et al. (1998) studied the opportunity to estimate the radial increment of tropical trees

through growth ring analysis. For this purpose 22 African tree species were marked periodically. The

presence of growth rings appeared to be more dependent on species type than on the climate. The

possibility of growth ring analysis and the presence of wedging and false rings for each of the 22

species was presented.

It has been proven so far that a high number of African species show datable tree rings, but capacity

building of universities and research organizations is needed to maintain and amplify

dendrochronological research (Gebrekirstos et al., 2014).

Applications

Until today dendrochronological research in a new geographical location starts with a period of

describing growth periodicity and proving the occurrence of tree rings (Worbes, 2002; Speer, 2010).

The existence of annual tree rings in the tropics widely has been proven, so a search into literature is

recommended in future research. Worbes (2002) listed some meaningful references for tropical

Africa.

Once chronologies are established, dendrochronologists could investigate climate-growth relations

of tropical trees. For many species in many parts of the tropics, a positive correlation between the

amount of precipitation and ring widths is determined (Worbes, 1995). However, climatological

orientated tree ring research meets scarce observational climate date in Africa. Tree ring analysis can

build high resolution climate proxies going far back in time, which is needed for understanding future

trends and uncertainties in climate change (Nicholson, 2000; Gebrekirstos, 2009; Department for

International Development, 2004). It is predicted that global climate change will affect patterns of

precipitation and vegetation composition. For the vulnerable dry regions of Africa, forestry and

agroforestry need a sustainable management in light of future changes, therefore a trees’ response

on longer time scale is required. Relations between tree growth and past climate events can provide

an insight into the effect of future events such as extreme droughts (Fichtler et al., 2004;

Intergovernmental Panel in Climate Change, 2007; Gebrekirstos, 2014).

12

Future tropical dendrochronological studies can provide information on growth dynamics of natural

forests and asses trends in biomass production, so tree ring research is also linked to carbon

accounting. A more detailed review of possible applications of tropical dendrochronology was

pointed out by Worbes (2002) and Gebrekirstos (2014).

Challenges and opportunities

However the existence of annual growth rings in tropical regions has been proven, tropical tree ring

analysis still deals with some misunderstandings and misinterpretations. The major problem is the

presence of indistinct or diffuse ring boundaries and high abundance of ring anomalies in the tropics

(cf. false, missing and wedging rings). Stem discs have to be used to detect this features so

crossdating is possible (Worbes, 2002; Brienen and Zuidema, 2005; Trouet et al., 2006; Speer, 2010).

Secondly, uncertainty about the triggering factor for tropical growth periodicity has not been settled

so far (Worbes, 2002). Finally, dendrochronologists should be aware that the trees’ response to the

environment does vary with age. During the juvenile growth stage, trees produce wider-than-average

growth rings, because young trees are more sensitive to environmental changes, e.g. site conditions

(Speer, 2010). The inner part of the stem (juvenile wood) should not be used for climatic studies,

since young trees can experience high competition of neighbours and differ in physiological

responses from mature trees (Worbes, 2002).

Despite the ‘principle of site selection’ (Section 2.1.2) tropical trees should not always be chosen at

the edge of their ecological amplitude. Because the sensitivity to limiting factors increases the

amount of ring anomalies in tropical species, it is sometimes better to select trees in a more mesic

environment (Wils et al., 2011).

The advancement of technology and new techniques (see 2.2) offers support to research on growth

dynamics of tropical trees. Worbes (2002) noted that intra-annual stable isotope ratio measurements

would become increasingly important. This assumption was recently been proven correct by Speer

(2010) and Gebrekirstos et al. (2014). A more unique method determines the content of radiocarbon

in individual growth zones of a tree, called radiocarbon dating (Worbes, 1995). An artificial increase

of 14C in the atmosphere between 1960 and 1965 as a result of atomic bomb explosions can be

detected in trees growing at that time (Worbes and Junk, 1989). This so called ‘bomb peak’ (Worbes,

1995) or ‘bomb spike’ (Speer, 2010) can date growth zones of the mid-sixties.

The integration between dendrochronology and other methods such as plant physiological

measurements, cambial wounding, phenological descriptions, isotope measurements and remote

sensing techniques, is valuable to draw comprehensive scientific conclusions across temporal and

spatial scales.

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2.2. Intra-annual wood formation

2.2.1. Scope

Intra-annual growth research endeavors to split up the radial increment of one vegetation period

into shorter time units. This can result in a more direct association between climate and tree growth

(Seo et al., 2007).

In temperate regions trees have generally distinct growth ring boundaries because of clear

seasonality (Worbes, 2004). The use of growth ring widths to get an insight into wood formation

dynamics leads to a temporal resolution of maximum one growth season (Seo et al., 2007). Despite

the use of tree ring research, the existence of periodical growth patterns in tropical regions is still

difficult to determine (Worbes, 2002). The detection of growth rings provides a one year resolution

of cambial growth dynamics. To obtain a higher temporal growth resolution, other tools and

techniques are necessary (Seo et al., 2007).

2.2.2. Cambial pinning

Seo et al. (2007) listed a brief review of intra-annual growth tools. Mork (1928) separated early and

latewood radial increment of conifers and Eckstein et al. (1974) practiced the same method on ring-

porous trees. Secondly, the development of X-ray densitometry resulted in wood density profiles

(Polge, 1963). Almost simultaneously the anatomical structure of tracheids was used to obtain more

insight in the intra-annual cambium activity (Vaganov and Terskov, 1977). On the other hand Sass

and Eckstein (1995) integrated the vessel area of deciduous trees into the growth ring research.

The previous methods deliver high resolution data, but the date of onset and cessation of the

cambial activity during a certain period in the vegetation season can still not be linked with the

formed wood. The need to implement a timer in this process remained. The following tools can fulfill

the timer function (Seo et al., 2007): the periodic extraction of small cambium samples was created

by Eckstein (1983). Small wood cores are extracted by using a cutting tube or injection needle

(Mäkinen et al., 2008). This destructive method is still being used and is nowadays called micro-

sampling (Rossi et al., 2006). A less destructive method is the so called ‘Mariaux’-window. By cutting

window-like wounds in the cambium, the periodical cambium activity in tropical trees can be

investigated (Mariaux, 1969; see 2.2.5). Finally, Wolter (1968) wounded the cambium with a thin

needle, similar to the Mariaux-window principle.

The cambial pinning technique of Wolter (1968) is based on the experience that the cambium is very

sensitive to an external impact. Due to the wounding typical structures will be formed (Larson, 1994).

The needle impact stops the cambium forming new cells around the pinning canal. The

differentiation of young cells stops and these cells can indicate the place to where the xylem had

14

been formed at the timing of the pinning. The surrounding living cells will react and modify. Wound

tissue could be formed around the pinning canal (Seo et al., 2007). This tissue is variable in size and

structure according to species, individuals and time of wounding (Couralet et al., 2010a).

2.2.3. Dendrometer measurements

Dendrometers are instruments which are used to provide time series of stem radius fluctuations.

These fluctuations are composed of diurnal water storage depletion and seasonal tree growth, so the

assessment of daily hydrological swelling and shrinking, and radial tree growth at different time

scales can be obtained (Deslauriers et al., 2007; Mäkinen et al., 2008; Drew and Downes, 2009;

Krepkowski et al., 2011). Dendrometers have proven their value, several studies have been published

representing stem growth phenology and the relationships between tree growth and climate

variables (Dünisch and Bauch, 1994; Downes et al., 1999; Nicault et al., 2001; Tardif et al., 2001;

Deslauriers et al., 2003; Mäkinen et al., 2003; Yoda et al., 2003; Bouriaud et al., 2005; Steppe et al.,

2006).

To understand the tree’s reaction to short-term changes in the environment and climate,

dendrometers need to measure continuously (Deslauriers et al., 2007). Resolution of dendrometers

can be separated in time and space resolution. High temporal dendrometers provide measurement

taken at short intervals, whereas high spatial instruments measure small changes in stem diameter.

Different resolution combinations are possible (Drew and Downes, 2009). Dendrometers are

classified into three classes: radial instruments that measure the stem size in a single radius,

diametric instruments that monitor across the diameter of the stem and the band dendrometers that

measure that measure the circumference at a particular height (Breitsprecher and Hughes, 1975).

The latter class has been widely used (Détienne and Mariaux, 1975, 1976, 1977).

Drew and Downes (2009) listed four general applications of high resolutions dendrometers:

- Resolving sub-diurnal stem size variation into mathematically defined phases.

- Linking tree diameter fluctuations with sap flow, leaf water potential and atmospheric

demand.

- Understanding growth responses to climate and silviculture.

- Temporal analysis of wood properties.

Only using dendrometer measurements to draw conclusions about true wood formation is difficult.

Dendrometers monitor the stem radius variations which also enclose the daily water storage

fluctuations (Mäkinen et al., 2003). Different studies compared dendrometer data with micro-cores

and cambial pinning (Mäkinen et al., 2003; Yamashita et al., 2006). For example, Ohashi et al. (2001)

stated that the radial growth measured by dendrometers was larger than the results of the pinning

15

method. The combination of dendrometer measurements and wood anatomical research is a

prerequisite to differentiate between swelling and shrinking by water on the one hand and the cell

formation on the other hand (Deslauriers et al., 2003; Krepkowski et al., 2011; Steppe et al., 2015).

2.2.4. Other methods used in intra-annual growth studies

During the first studies on intra-annual wood formation of tropical trees, phenological3 observations

were made (Détienne and Mariaux, 1975). Because of its non-destructive character and simplicity to

implement, phenological measurements give a first indication of the growth rhythm of a tree

(Worbes, 1995).

Wood density is an important parameter in anatomical wood analyses. Polge (1963) invented the

technique of X-ray densitometry. Recently, Van den Bulcke et al. (2014) represented the potential of

X-ray computed tomography (XCT) for 3D tree-ring analysis. These techniques can be used for fast

and non-destructive study of intra-annual growth dynamics.

Finally, stable isotope analysis is considered as one of the new frontiers in tree ring research (Speer,

2010). McCarroll and Loader (2004) presented a first overview of stable isotope research in trees,

explaining the theory and methodology.

2.2.5. Studies on tropical tree growth

A sustainable tropical forest management and utilization under changing climate conditions is only

possible when wood formation of indigenous tree species is understood. Knowledge of periodical

tree growth dynamics is needed (Krepkowski et al., 2011). In the early 1960s Mariaux (1967)

introduced the technique of wounding the cambium to increase this knowledge. During the 1960s

and 1970s lots of research based on this techniques was done (Détienne and Mariaux, 1975, 1976,

1977).

In Cameroon and Ivory Coast, eight Tarriela utilis (Niangon) were marked yearly between 1965 and

1972. A Mariaux-window with a width of 0.5 cm and a height of 3 to 4 cm was implemented to

wound the cambium. The variation of the stem diameter was also measured using a dendrometer

band and phenological observations were made every 15 days. The main difficulties experienced in

determining the periodical growth and age of the tree were the absence of distinguishable growth

rings (especially during the first growth years and periods of rapid increment) and the appearance of

ring anomalies (Détienne and Mariaux, 1975).

3 Phenology is the study of recurring life-cycle events including leaf fall, flowering, fruiting and seed dissemination (Fenner, 1998).

16

During the same period Détienne and Mariaux (1977) marked 57 trees of 7 species of African

Meliaceae to detect growth rings and measure tree age. To improve analysis dendrometer bands

were implemented and phenological studies were performed. Accurate results were found for

almost all the species. The radical increment during the study period is shown in Figure 4. The main

problems to detect growth arise in the heartwood and in slowly growth periods. In contrast to the

study of T. utilis, false rings and wedging showed less difficulties (Détienne and Mariaux, 1977).

Worbes (1999) used the technique of cambial marking to determine successfully the presence of

annual growth rings in 37 tree species in Venezuela. Tree ring widths were proven correlated with

precipitation patterns. At the same time, dendrometer measurements showed intra-annual growth

rhythms. Deciduous trees stopped growing at the end of the rainy season, whereas growth in

evergreen species stopped for a shorter period at the end of the dry season (Worbes, 1999).

Couralet et al. (2010a) recently studied the intra-annual wood formation in the western Democratic

Republic of Congo (DRC) using the cambial pinning technique for Prioria balsamifera, Terminalia

superba, Xylopia wilwerthii, Corynanthe paniculata and Aidia ochroleuca. Two interesting conclusions

were made. Firstly, the cambium of most of the studied trees was never completely dormant,

because wound-reaction was formed all year round, even when the normal xylem growth was

absent. So both climate and other endogenous factors are a potential trigger to cambial activity.

Secondly, it appears that only wood formation of P. balsamifera was positively associated with

temperature and rainfall, while growth of understory species may be influenced by other factors

than climate. The upperstory acts like a buffer of external climate variations (Couralet et al., 2010a).

In the semi-deciduous Mayombe forest of the DRC, the evergreen species Aidia ochroleuca,

Corynanthe paniculata and Xylopia wilwerthii dominate the understory. Distinct annual growth rings

in these species were observed. Moreover, precipitation during the rainy season has been proven to

be a common external factor for radial tree growth of the observed species. For A. ochroleuca

growth associates with precipitation during the early rainy season, whereas for C. paniculate and X.

wilwerthii, it associates at the end of the season. This study showed the possibility of

dendrochronology for determination of growth-climate relations of tropical understory species. In

addition a different response to a common external factor between species was found. Monthly

diameter at breast height (DBH) measurements (15-months long time series) supported these

results. The cambial reactivation occurred at the onset of the rainy season for A. ochroleuca, but for

C. paniculate and X. wilwerthii the cambial growth peaked in January/Februari (Couralet et al.,

2010b).

17

Phenological data for the understory species were gathered in the Mayombe forest from 1948 to

1957. Couralet et al. (2010b) also presented personal observations on some species. These data can

be analyzed (Couralet et al., 2010c) and compared to results of the intra-annual wood formation.

Other studies on cambial growth dynamics and periodicity of wood formation in the tropics were

represented by Amobi (1974), Shiokura (1989), Nobuchi et al. (1995), Worbes (1995), Dünisch et al.

(2002) and Krepkowski et al. (2011). Gebrekirstos et al. (2014) referred to more recent studies, also

using intra-annual stable isotope measurements to reveal seasonality in wood formation of species

without distinct growth ring boundaries.

Figure 4: Radial increment during the marking period of the examined species of Meliaceae (Détienne and Mariaux, 1977).

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3. Materials and methods

3.1. Study site

The Luki Biosphere Reserve is located at the extreme west of the DRC (5°28’-42’ N, 13°14’-18’ E), 120

km of the Atlantic Coast and 30km north of the city of Boma (Figure 5). The forest reserve covers

about 32,700 ha and the altitude varies between 150 and 500m above sea level. It’s the most

southern part of the Mayombe forest and may be considered as representative of the flora of the

Mayombe (Lubini, 1997; Couralet et al., 2010b).

Figure 5: Location of the Luki Biosphere Reserve with simplified vegetation cover of the area from Global Land Cover 2000 (Mayaux et al. 2004): darker zones are a mosaic of evergreen, deciduous and mixed forests with a minimum of 15

percent tree cover.

The Mayombe forest stretches along the Atlantic Ocean from the central coast of Gabon till the Luki

reserve in the DRC. It’s part of the Central African rain forest of the Congo Basin (Lubini, 1997). Only

the Amazon forest occupies a bigger area of continuous rain forest (Ruiz Perez et al., 2005). Tropical

forests are poorly understood, because of their structural and biological complexity, the extended

time scale due to the longevity of trees and political stability in tropical Africa (Couralet, 2010). In

Luki, scientific research was already done, for example, by Donis (1948), Lubini (1997) and more

recently by Couralet (2010) and De Ridder et al. (2013).

The Luki reserve can be classified as tropical semi-evergreen forest of the Guineo-Congolean forest

domain and consists of a mixture of evergreen species and deciduous species in the upper-stratum

and mostly evergreen species in the understory (Lubini, 1997). The soils are generally determined as

ferrallitic, acid and with poor chemical content (Senechal et al., 1989). The climate is assigned

tropical savanna climate or tropical wet and dry climate, corresponding to the ‘Aw’-climate of the

‘Köppen climate classification’ (Peel et al., 2007). Climate records (1959-2006) were provided by the

Luki meteorological station (Figure 6). The mean annual temperature is 24.6°C and the mean annual

19

rainfall is 1180 mm/year. From June to September the monthly precipitation is less than 50 mm, this

period defines a distinct dry period. Generally these low precipitation values would not be favourable

for the presence of the dense humid Mayombe forest (Couralet et al., 2010b). However, the cold

Benguela stream in the gulf of Guinea creates a thick, low-level, non-precipitating cloud layer during

the dry season (Pendje and Baya ki, 1992). As a consequence the solar irradiance is blocked,

temperature is lower and relative air humidity remains high all year long (above 80 percent). Plants

may thus not experience extreme water stress during an occurring dry period (Lubini, 1997; Couralet

et al., 2010b).

Figure 6: Climate diagram of the Luki meteorological station, Democratic Republic of Congo: monthly means of rainfall (±SD), temperature, air humidity (1959–2006) and solar irradiance (1959–1994) (Couralet et al., 2010b).

The reserve of Luki is selected based on the dendrochronological principle of site selection (Section

2.1.2). Luki is the most southern part of the Mayombe forest. This principle states that species are

most stressed at the edge of the forest range. In Luki, intra-annual wood formation of typical

Mayombe forest species are thus considered to be sensitive to environmental factors. Consequently,

the site of Luki promises valid results on the climate-growth relationships. Nevertheless, in some

cases it is more appropriate to select tropical tree species in a more mesic environment, because the

amount of ring anomalies increases to the edges of the ecological amplitude (Wils et al., 2011).

3.2. Studied species

Six species Polyalthia suaveolens Engl. & Diels, Xylopia wilwerthii De Wild. & T. Durand (both

Annonaceae), Hylodendron gabunense Taub. (Caesalpiniaceae), Newtonia sp. (Fabaceae), Corynanthe

paniculata Welw., and Aidia ochroleuca (K. Schum.) E. Petit (both Rubiaceae) were selected in the

Luki Biosphere Reserve, to study tropical intra-annual wood formation. These species are evergreen

mesophanerofytes (8-30 m in height), except for the megaphanerofyt (more than 30 m in height) P.

20

suaveolens, and are widespread in the Guineo-Congolean forest domain. Climate-growth

relationships, seasonal characteristics of wood formation and phenological data of the understory

species X. wilwerthii, A. ochroleuca and C. paniculata were observed by Couralet et al. (2010a, 2010b,

2010c). Also the other species occur in the upper canopy and understory of the Mayombe forest

(Fouarge and Gérard, 1964; Lubini, 1997), but previous research on their growth dynamics was not

found.

The selected individuals all grew in the same primary forest stand close to the enclave of Kiobo,

located close to the research station of Luki. Species were selected based on their ability to form

growth rings (Fouarge and Gérard, 1964; Couralet et al., 2010b; Détienne, n.d.). Macro- and

microscopic characteristics of the wood of Mayombe forest species, inter alia distinct or indistinct

growth rings, were pointed out by Fouarge and Gérard (1964). Selection criteria were health,

maturity, and roundness and straightness of the stem to limit the amount of anomalies in the wood

structure.

3.3. Cambial marking and sample preparation

In the reserve of Luki, 18 selected trees of 6 different species (4 P. suaveolens, 3 X. wilwerthii, 4 H.

gabunense, 1 Newtonia sp., 3 C. paniculata and 3 A. ochroleuca) were pinned monthly during one

growing season (from September 2013 till July 2014). Because of the timing of the field trip to the

DRC, the planned pinnings in August are missing. As a consequence the length of the experiment

covered only 11 months. Trees were harvested in September 2014.

Cambial pinning technique

The cambial pinning technique, first introduced by Wolter (1968), was used to wound to cambium

monthly (Section 2.2.2). A thin needle (approximately 1 mm) was then inserted through the bark and

cambium into the xylem of the tree. The wounding initiates the formation of wound tissue and

modified wood reaction cells around the pinning canal (Couralet et al., 2010a). As mentioned by Seo

et al. (2007), the wound reaction can be used as a timer to monitor the amount of wood formation

within a certain time period, in this case almost one growing season. Intra-annual growth dynamics

for the studied trees will be discussed. In addition wound initiated features around the pinning canal

will be described, and difficulties and uncertainties will be taken into account.

On the 21st of each month from September 2013 till July 2014, a pinning was executed at breast

height on each of the 18 trees. Each pinning was repeated three times in a spiral arrangement. Each

following monthly pinning was made around the stem trunk at the same height about 5 cm to the

right. All pinnings were made on sufficient distance of one other to avoid any interference of

wounding reaction around nearby pinnings. Around each pinning a red circle was painted and used

21

as a visible marking to localize the pinning when the needle had disappeared. Figure 7 shows how

pinnings were indicated on the stem. When the first pinning was executed, the DBH was measured as

well.

Figure 7: Example of how the monthly cambial pinnings and their repetitions ware performed on P. suaveolens in the Luki Biosphere Reserve.

Sample preparation

In September 2014, all trees were felled and stem discs were taken at the level of the visible red

markings. The DBH was measured using a tape-measure and the height of the felled tree was

estimated using a tape measure. Subsequently, stem discs were dried at room temperature (about

25°C) during a period of 4 months. When the samples had been dried, the big stem discs were sliced

into 3 smaller discs each containing 11 monthly consecutive pinnings (Figure 8).

Figure 8: Sawn stem disc of 14 pinned trees.

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3.4. Characterization of tree rings

The pinned stem discs were digitized with a flatbed scanner and camera to reconstruct their shape

and relocate the marks when destructively subsampled. Using stem discs that cover the complete

circumference provides a more convenient way to identify matching rings and anomalies than using

small core beams. Successful analysis of the macroscopic tree ring structures demands a careful

preparation of the sample surface (Worbes, 2004). Therefore the stem disc surface was sanded (50-

600 grid size). To ensure orthogonal digital projections of the stem discs, flat transversal stem

sections were prepared. Tree rings could be measured on these optical images using an in-house

developed toolbox (Van den Bulcke et al., 2014).

3.5. X-ray tomography as a method to study radial growth

X-ray computed tomography

Drill cores of approximately 1.5 cm in diameter and 1 cm in length containing the pinnings, were

sampled from the stem discs. These cylinders were scanned with XCT, resulting in a three

dimensional view on the wood structure and around the pinned zone. The digital wood volumes

could be used in growth related studies. This method is less time consuming compared to making

thin sections of each sample.

The method of data acquisition was based on Van den Bulcke et al. (2014). The scanner used at

Woodlab-UGent is a setup developed at UGCT, the Ghent University Centre for X-ray Tomography.

The system offers a large range of operational freedom, all combined in versatile acquisition routines

(standard or fast scanning, tiling, helix, etc.). It has a generic in-house developed XCT scanner control

software platform (Dierick et al., 2010) that allows full control of the scanner hardware. Samples

were scanned with standard cone-beam XCT with a scan time of approximately 21 min.

Reconstruction is performed with Octopus, a tomography reconstruction package for parallel, cone-

beam and helical geometry as well as phase correction and retrieval (Vlassenbroeck et al., 2007)…

Beam hardening correction was applied, both by hard- as well as software filtering. The obtained

approximate voxel pitch is 10 μm (Van den Bulcke et al. (2014).

Relative growth measured on XCT images

Density variations between regular wood structures and wound-induced features enable the

localization of the edge between cells before and after pinning of the cambium. The increment from

the beginning of the growth season up to the pinning date was measured. According to Seo et al.

(2007) the increment up to the harvesting date was additionally measured to correct for eccentric

tree growth. The measurements provide a time series of relative radial growth during almost one

23

growth season. This method is based on the measurements of Seo et al. (2007) on light micrographs

of cross-sections of Pinus sylvestris and Picea abies. Relative growth is defined as (Equation 2):

RG =A+B

C+D∗ 100% (2)

Where A and B are the distances that equal the increment from the onset of the growth season up to

the pinning date, and C and D are the distances that equal the increment from the onset of the

growth season up to the harvesting date (Figure 9).

Figure 9: Relative growth measurements on a XCT-scan image of a pinned zone of Newtonia sp. (pinning No. 8). A and B are the distances measured to obtain the increment from the onset of the growth season up to the pinning date. C and D

are the distances measured to obtain the increment from the onset of the growth season up to the harvesting date.

Fit of the Gompertz function

A Gompertz function (Gompertz, 1832) was fitted in R (R core team, 2015) to the time series of

relative growth data. The code to fit the function in R is presented in Addendum 1. Many applications

of the Gompertz function for the study of radial xylem growth have been performed. Previous

research has been performed in boreal and temperate regions (Deslauriers, 2003; Rossi et al., 2003;

Mäkinen et al., 2008), but applications for tropical regions were not found. Because of its flexibility

and asymmetrical shape the Gompertz equation has been proven successful to describe growth-time

relationships (Zeide, 1993; Mäkinen et al., 2008). According to Cheng and Gordon (2000) the

Gompertz equation is defined as (Equation 3):

RG = a ∗ e−eβ−κ∗t

(3)

Where RG is the relative growth according to Equation 3, a is the upper asymptote, β is the x-axis

placement parameter, κ is the rate of change parameter and t is the time expressed as the number

of days from the first cambial pinning (in this case September 21, 2013).

24

3.6. Anatomical study around the pinning zone

XCT images provide first insights into the wound-induced features of the wood. Based on the XCT

analysis, thin cross-sections were taken for detailed microscopic wound study. When the results of

XCT were insufficient to measure relative growth, cross-sections should provide more reliable

information. This approach allows also to show differences in formation of wound tissue and wood

reaction cells between the studied species, and also between different times during the growing

season within an individual. References to previous anatomical studies of the studied tropical species

is listed in Table 1.

Table 1: References to previous anatomical studies of tropical species.

Species Literature

P. suaveolens Détienne (n.d.)

H. gabunense Fouarge and Gérard (1964) and Détienne (n.d.)

X. wilwerthii Fouarge and Gérard (1964) and Couralet et al. (2010b)

Newtonia sp. Fouarge and Gérard (1964) and Détienne (n.d.),

C. paniculata Fouarge and Gérard (1964) and Couralet et al. (2010b)

A. ochroleuca Fouarge and Gérard (1964), Couralet et al. (2010b) and Détienne (n.d.)

Preparation of transversal sections for anatomical study

Based on the XCT analysis, cylinders were selected for anatomical study. Selected cylinders were

reduced to cubes of about 1 cm³ containing the pinned zone. Cubes were heated in the oven for 24h-

48h at 70°C in a mixture of 60% H2O, 30% glycerol and 10% alcohol. Then transversal sections of 20-

30 µm in thickness were cut on a sliding microtome and colored. The process of coloring and

dehydrating is described in Table 2. Finally, cross-sections were mounted using synthetic and

colorless Euparal resin.

3.7. Analysis of climate-growth relationships

Air temperature, precipitation, relative humidity (RH), photosynthetic active radiation (PAR) and

solar radiation (SR) were measured during the growing season 2013-2014 at the meteorological

station of Luki to determine impact of climate on intra-annual tree growth. Measurements were

made with a temporal resolution of 30 minutes. Because pinnings were performed on a monthly

resolution, the climate data were rescaled to monthly values. All the measured data, except for

precipitation, were available for visual comparison with the relative growth measurements. To

compensate for the absence of precipitation data, mean monthly precipitation measurements from

January 1948 to December 2006 were used instead (Table 3).

25

Table 2: Treatment steps in the process of cross-section coloring in chronological order (left column) with the time of each treatment (right column) (P. Dekeyser, technical staff Woodlab UGent, pers. comm., May 29, 2015).

Treatment Time(min)

Staining with safranin (1% in H2O) 5

Washing with H2O -

Staining with Astra blue (1% in H2O + 3 drops of acetic acid) 5

Washing with H2O -

Dehydration with alcohol (50% in H2O) 5




Dehydration with alcohol (100%) 15

Table 3: Average monthly precipitation data from 1948 to 2006 measured at the climatological station of Luki (Prec., mm) (Couralet et al., 2010b).

Month Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov Dec.

Prec. 130.3 145.0 183.4 187.3 59.6 4.5 2.7 5.4 20.9 70.8 209.7 158.5

3.8. Analysis of phenology-growth relationships

Every 21st of the month from January 2014 till September 2014, phenological events were observed

for the 18 pinned trees. The observed events were the presence of old (which means present since

the previous growth season) and new (which means sprouted during the current growth season)

leaves, flowering, defoliation and dissemination. Data for September 2013 till December 2013 were

not available. The timing of phenological events was visually compared to the onset of wood

formation, period of radial stem growth and cessation of wood formation. This study is relevant since

tree growth of tropical species has been found to be linked with phenological events (e.g. Amobi,

1973). Growth-phenology relationships of the six studied species had not yet been explored.

However acquisition of phenological data of these species had been done by Belgian researchers in

the Luki Biosphere Reserve from January 1948 till December 1957 (Couralet et al., 2010b). The

proportion of trees for which a phenological event was observed was calculated for every 10 days of

the 10 year period. To compare the latter data to the recent phenological data, average proportions

for each month from 1948 to 1957 were calculated and plotted.

26

4. Results and discussion

4.1. Diameter at breast height

Table 4 represents DBH of the 18 studied trees measured at the start and end of the pinning period

as well as the estimated height of each tree after felling. The average radial increment (derived from

the DBH increment) measured using a tape-measure can be compared with radial growth

measurements determined on XCT images (Section 3). According to DBH measurements, 2 P.

suaveolens and one X. wilwerthii did not grow during the studied period. Most trees showed an

increment of about 2 mm (range from 0.5 to 4 mm). The radial increment of one P. suaveolens and

one X. wilwerthii was more than 10 mm. Two individuals even showed negative radial increment. A

lack of accuracy of the diameters measured using a tape-measure causes false results of radial

increment. Therefore these measurements are not reliable to determine intra-annual xylem growth,

or they should be taken with high precision, and on regular stems.

Table 4: The diameter at breast height (DBH) and height measurements of the pinned trees in the reserve of Luki. The first diameter measurement was taken using a tape-measure on September 21, 2013 (DBH 2013, cm). The second diameter measurement was taken on September 30, 2014 (DBH 2014, cm) and the height (m) was estimated at the same time. The increment (ΔDHB, cm) was calculated. The mean radial increment (mm) is calculated as ΔDBH. The mark on the stem (Mark) and a reference number (No.) was given for each tree.

No. Species Mark DBH 2013 DBH 2014 ΔDHB Radial increment Height

1 Polyalthia suaveolens CM1 33.7 34.5 0.8 4.0 25

2 P. suaveolens CM2 22.9 22.9 0.0 0.0 20

3 P. suaveolens CM3 27.5 27.5 0.0 0.0 23

4 P. suaveolens CM4 13.5 15.6 2.1 10.5 17

5 Hylodendron gabunense CM1 33.6 33.7 0.1 0.5 32

6 H. gabunense CM2 34.4 35.0 0.6 3.0 30

7 H. gabunense CM3 27.8 27.7 -0.1 -0.5 28

8 H. gabunense CM4 36.4 33.1 -3.3 -16.5 30

9 Xylopia wilwerthii CM1 29.3 29.3 0.0 0.0 12

10 X. wilwerthii CM2 22.9 25.0 2.1 11.5 11

11 X. wilwerthii CM3 17.8 18.0 0.2 1.0 11

12 Newtonia sp. CM1 20.7 21.2 0.5 2.5 16

13 Corynanthe paniculata CM1 27.2 27.5 0.3 1.5 22

14 C. paniculata CM2 12.1 12.4 0.3 1.5 15

15 C. paniculata CM3 12.8 12.9 0.1 0.5 12

16 Aidia ochroleuca CM1 25.4 26.3 0.9 4.5 12

17 A. ochroleuca CM2 22.0 22.6 0.6 3.0 13

18 A. ochroleuca CM3 22.1 22.6 0.5 2.5 14

27

4.2. Growth rings and ring anomalies

As can be seen from Figure 10 to Figure 15, optical images of the stem discs were taken on a flatbed

scanner with a resolution of 1200 dpi, and growth rings and cambial pinnings were localized on these

images. Digitizing transversal sections of the stem discs is necessary in order to analyze growth rings

and the positions of the pinning on the cross-section, prior to destructive analysis. The sections

provide useful information of ring widths, ring anomalies and the location of the pinnings, which is

necessary for the determination of relative intra-annual growth.

Characterization of the growth rings enables to create a 2D plot on which anomalous growth rings

are clearly distinct (Figure 16).

Figure 10: Characterization of growth rings on an optical image of the transversal section of the stem of H. gabunense (No. 5). The numbers 1 to 11 indicate the location of monthly pinnings, with pinning 1 executed on September 21, 2013

and pinning 11 on July 21, 2014.

28

Figure 11: Characterization of growth rings on an optical image of the transversal section of the stem of X. wilwerthii (No. 9). The numbers 1 to 11 indicate the location of the pinnings, with pinning 1 executed on September 21, 2013 and pinning 11 on July 21,

2014.

Figure 12: Characterization of growth rings on an optical image of the transversal section of the stem of Newtonia sp. (No. 12). The numbers 1 to 12 indicate the location of the

consecutive pinnings, with pinning 1 executed on September 21, 2013.

29

Figure 13: Characterization of growth rings on an optical image of the transversal section of the stem of C. paniculata (No. 13). The numbers 1 to 11 indicate the location of

monthly pinnings, with pinning 1 executed on September 21, 2013 and pinning 11 on July 21, 2014.

Figure 14: Characterization of growth rings on an optical image of the transversal section of the stem of A. ochroleuca (No. 16). The numbers 1 to 11 indicate the location of


30

Figure 15: Characterization of growth rings on an optical image of the transversal section of the stem of P. suaveolens (No. 4). The numbers 1 to 11 indicate the location of


Figure 16: Characterization of the latest formed growth rings of P. suaveolens (No. 4). The numbers indicate the location of monthly pinnings of each disc, with pinning 1 (P1)

executed on September 21, 2013 and pinning 11 (P11) on July 21, 2014.

P1

P2

P3P4

P5

P6

P7

P8

P9

P10

P11-8

-6

-4

-2

0

2

4

6

8

-10 -8 -6 -4 -2 0 2 4 6

Dis

tan

ce t

o c

en

ter

of

the

tre

e (

cm)

Distance to center of the tree (cm)

Growth rings Disc 1 Disc 2 Disc 3

31

Despite the fact that individuals were selected for stem roundness (Section 3.2), all trees showed

clear eccentric growth. Wedging and partially formed rings occur commonly in all trees and can be

characterized on a well-polished disc. Worbes (2002) explained wedging as a growth reaction of the

tree on changing light conditions. This can occur due to variation in competitive pressure between

trees within a stand. The six studied species occur commonly in the lower canopy of the Mayombe

forest (Lubini, 1997), so selecting another site in the Mayombe forest will possibly not reduce the

amount of wedging rings in these species. Partially formed rings occur more often in trees growing

near the edge of their ecological amplitude (Fritts, 1976; Speer, 2010). Luki is the most southern part

of the Mayombe forest, so trees growing on the Luki site are considered to be standing at the edge of

the natural range. As a consequence the studied trees are considered to be stressed, and partial rings

occur more often than if trees would be selected in the center of their distribution. A more advanced

method that takes circumferential variation into account will be necessary if anomalies hinder the

determination of intra-annual growth.

The studied tree species were also selected by the appearance of growth rings, as determined in

previous research (Fouarge and Gérard, 1964; Couralet, 2010b, Détienne, n.d.). In this way it is

possible to characterize rings on the optical stem disc images. Growth ring structures and boundaries

can be seen in Figure 17. Worbes et al. (2003) proved the existence of annual rings of P. suaveolens

by radiocarbon dating and tree ring analysis. Accordingly, growth ring boundaries of P. suaveolens

(Annonaceae) are found distinct (Détienne, n.d.), and can be clearly characterized on optical images,

due to the difference between the light-colored earlywood and the dark latewood. The same

earlywood-latewood contrast is notable to determine ring boundaries of the other species of the

Annonaceae, X. wilwerthii. Dark bands of latewood were also described by Fouarge and Gérard

(1964). However, this feature is not distinct for all boundaries of this species. Some ring boundaries

are characterized by an increasing frequency of parenchyma bands, according to Couralet et al.

(2010b). In addition to the features described by Fouarge and Gérard (1964) and Couralet et al.

(2010b), a minority of boundaries can also be seen as a vessel-free band. X. wilwerthii has a relative

high number of anomalous rings, which results in a highly irregular stem form, and lowers

consistency of ring characterization.

Growth ring boundaries of species from the Caesalpiniaceae are distinguished by terminal

parenchyma bands that are often visible on cross-sections as a fine light-colored band (Höhn, 1999).

H. gabunense (Caesalpiniaceae) shows distinct growth rings due to the presence of those small

parenchyma bands, also described by Fouarge and Gérard (1964) and Détienne (n.d.). The latest

formed rings are difficult to mark, because they are very small. This results in dense tissue of small

vessels and parenchyma bands. The narrow widths of the latest formed growth rings compared to

32

earlier formed rings of H. gabunense No. 5 indicate slow wood formation in H. gabunense in the most

recent years.

Although distinct growth ring boundaries for Newtonia sp. were observed (Fouarge and Gérard,

1964), Détienne (n.d.) stated that they are indistinct and absent. Observations on the stem discs of

Newtonia sp. evidence the presence of distinct boundaries, due to a clear difference between light-

colored earlywood and dark latewood.

The majority of growth rings of C. paniculata (Rubiaceae) are observed as distinct. The number of

vessels decreases to the outer part of the growth rings, and just before the growth ring boundaries

no vessels are formed. Fouarge and Gérard (1964) noted the ‘more or less’ sharp ring boundaries.

Nevertheless some boundaries are indistinct due to lack of clear differences in vessel frequency

between consecutive growth rings. Furthermore characterization of growth rings cannot rely on

color variation, as it can be used for P. suaveolens, X. wilwerthii and Newtonia sp.

Growth ring boundaries in A. ochroleuca (Rubiaceae) were determined as indistinct and absent

(Détienne, n.d.). Similar to C. paniculata, species of the same family, A. ochroleuca shows little color

variation. According to Fouarge and Gérard (1964) this is the main reason that ring boundaries are

difficult to distinguish. Another similarity to C. paniculata is the decreasing number of vessels to the

end of the growth rings (Couralet et al, 2010b). Detachment of the bark during drying process of the

discs resulted in fracturing of some of the outer growth rings, which impedes characterization of the

outer boundaries.

According to features at ring boundaries, Worbes (2004) classified growth zones into four basic

anatomical types (Section 2.1.3). P. suaveolens and X. wilwerthii (both Annonaceae), and Newtonia

sp. can be classified into type 1, because their fibre cell wall thickness increases and the cell lumen

volume decreases from earlywood to latewood. This feature determines color variation. X. wilwerthii

can also be part of type 3, due to the increasing frequency of parenchyma bands towards the end of

the growth rings. The presence of marginal parenchyma bands in H. gabunense that run around the

entire cross-section, classifies this species into type 2. Type 4 is based on the vessel distribution. C.

paniculata and Aidia ochroleuca can be included in this type because of the decreasing number of

vessels towards the end of the growth rings, but they are not ring porous.

33

Figure 17: Tree ring structures and growth ring boundaries of P. suaveolens, H. gabunense, X. wilwerthii, Newtonia sp., C. paniculata and A. ochroleuca (from left to right, and from top to bottom) of the Luki Biosphere Reserve. The marks

indicate suspected ring boundaries.

34

Optical images of stem discs are used for the characterization of growth rings for six tropical species.

It can be concluded that this method was easy to implement on P. suaveolens, Newtonia sp. and H.

gabunense. However ring boundaries formed during periods of slow growth are difficult to

characterize. The Rubiaceae are less suitable to analyze growth rings on stem discs because of

indistinct ring boundaries, especially for A. ochroleuca.

Characterization of growth rings on stem discs gives useful information about growth ring features

on a macroscopic resolution, and to detect anomalous rings. This is not only helpful for studies on

intra-annual wood formation, but can be applied in dendrochronological research as well.

Dendrochronological studies using stem discs have been carried out successfully for instance by

Worbes (1999), Wils et al. (2009) and De Ridder et al. (2013). A drawback is that trees are

destructively sampled. Success of the characterization depends on the tree species, the individual,

endogenous factors of the stand and the site selection.

4.3. Relative intra-annual growth

4.3.1. Results per species

Polyalthia suaveolens

4 trees of the species P. suaveolens were available to determine relative intra-annual growth

dynamics. Dimensions of the trees are shown in Table 4. After drying the stem discs, they were each

sawn into 3 discs so that each thinner disc contained 11 monthly consecutive pinnings. P. suaveolens

No. 4 was selected to determine relative intra-annual growth of this species (Figure 15). Cylinders of

approximately 1.5 cm in diameter containing the pinnings were drilled from the stem disc. These

cylinders were scanned with a XCT scanner (resolution 10 µm), resulting in a three dimensional (3D)

view on the wood structure around the pinned zone (Van den Bulcke et al., 2014). Construction of

the 3D view (scanning and reconstruction) takes about 30 minutes per cylinder. Cross-sections of

each scanned cylinder of P. suaveolens (No. 4) can be seen from Figure 19 onwards.

Tree ring analysis based on density variations was suggested by Van den Bulcke et al. (2014). Growth

ring boundaries were observed distinct on XCT cross-sections due to density variations between

earlywood and latewood cells (Figure 19). The pinning canal and wound-induced features provide the

location of the latest formed xylem cells just before pinnings were executed. With the help of density

variations, relative growth measurements were performed on XCT cross-sections. Relative growth is

measured according to Seo et al. (2007), and can be seen in Table 5.

There was no observed increment for P. suaveolens No. 4 from October 21 till December 21 (Table

5). This is also observed for the pinnings performed on September 21, 2013 and December 21, 2013

of P. suaveolens No. 1. The relative growth equals 100 % for the pinnings performed on June and

35

July, 2014 of P. suaveolens No. 4 (Table 5). For P. suaveolens No. 1, 100 % relative growth was

measured at the pinning in May.

A Gompertz function was fitted to the time series of the relative growth data of P. suaveolens (No. 4).

This function is a model for intra-annual growth of this tree, and is characterized by Equation 4:

RG = 104.71 ∗ e−e3.23−0.02∗t

(4)

With RG the relative growth (%), and t the number of days since September 21, 2013. Measures of

the quality of the fit are presented in Addendum 2. The residual standard error of the fit (RSE) is 4.89.

The Gompertz model is plotted in Figure 18. The relative growth rate is plotted on the same figure,

and characterized as the derivative of the fitted Gompertz model (Equation 5):

RGR = 104.71 ∗ 0.02 ∗ e3.23−0.02∗𝑡−e3.23−0.02∗t

(5)

With RGR the relative growth rate (%) and t the number of days since September 21, 2013.

Figure 18: Relative growth data of P. suaveolens (No. 4) fitted to the Gompertz function with parameters a = 104.71, β = 3.2325 and κ = 0.022505 (left). The derivative of the Gompertz function determines relative growth rates (right).

36

Figure 19: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of P. suaveolens (No. 4). The date of the pinning is given in the upper left corner.

37

Table 5: Relative radial growth measurements (RG, %) for P. suaveolens (No. 4), X. wilwerthii (No. 9), C. paniculata (No. 13), A. ochroleuca (No. 16) and H. gabunense (No. 5). A and B (mm) are the distances respectively left and right from the pinning canal measured to obtain the increment from the onset of the growth season up to the pinning date. C and D (mm) are the distances respectively left and right from the pinning canal measured to obtain the increment from the onset of the growth season up to the harvesting date. To date the relative growth, the time (days) since the first pinning (September 21, 2013) is given.

Time P. suaveolens X. wilwerthii C. paniculata A. ochroleuca H. gab.

A B C D RG A B C D RG A B C D RG A B C D RG RG

0 0.10 0.12 1.14 1.22 9 0.00 0.00 1.35 1.39 0 0.00 0.00 1.69 1.61 0 0

30 0.00 0.00 1.37 1.41 0 0.02 0.14 0.54 0.52 15 0.00 0.00 2.02 1.83 0

61 0.00 0.00 0.72 0.64 0 0.38 0.36 3.09 2.68 13

91 0.00 0.00 3.13 2.99 0 1.27 1.31 1.95 1.76 70 0.00 0.00 0.54 0.56 0 1.04 0.98 3.5 3.38 29 0

122 0.77 0.68 2.78 2.89 26 2.10 2.26 2.10 2.26 100 0.00 0.00 0.44 0.49 0 1.48 1.46 3.30 3.03 46 0

153 0.82 0.87 1.92 1.96 44 3.88 3.69 5.47 5.55 69 0.00 0.00 0.73 0.66 0 1.57 1.61 2.83 3.03 54

181 1.70 1.71 2.89 2.66 62 3.13 3.42 4.19 4.80 73 0.31 0.27 0.78 0.73 38 1.68 1.75 2.64 2.54 66 0

212 2.26 2.10 2.41 2.25 94 3.46 3.54 4.84 5.49 68 0.55 0.62 0.96 0.89 63 1.53 1.57 1.80 1.80 86

242 2.44 2.46 2.71 2.69 91 2.15 2.20 2.46 2.20 93 0.85 0.81 0.95 0.99 86 1.73 1.68 1.93 1.91 89

273 3.00 2.93 3.00 2.93 100 1.29 1.22 1.74 1.71 73 1.36 1.35 1.43 1.46 94 1.35 1.46 1.35 1.46 100 0

303 2.68 2.74 2.68 2.74 100 - - - - -a 1.47 1.47 1.47 1.47 100 1.07 1.12 1.07 1.12 100 0

a Ring anomalies hinder the relative growth measurements.

38

Hylodendron gabunense

Four trees of H. gabunense were monthly pinned (Table 4). Six pinnings of H. gabunense No. 5 were

drilled out and scanned. Cross-section of XCT scans can be seen in Figure 21.Distinct parenchyma

bands make it easy to measure rings widths on the scans. The high number of parenchyma bands

that characterize small outer growth rings on the stem disc of H. gabunense No. 5 indicate slow

growth of this tree during last years before harvesting. This assumption was made based on the

characterization of growth rings using stem discs (Section 3.4). Based on the XCT images no growth is

observed for tree No. 5 (Table 5).

The latest formed growth rings of H. gabunense No. 7 were not remarkably wider than earlier

formed rings. This is observed as the distance between parenchyma bands on the stem disc. This

suggest that growth of this tree did not decrease during last years. However, for H. gabunense No. 7

no growth was observed on XCT scans of the pinnings in September, December and March.

Although radial increment is not observed on the XCT scans, modified wound reaction cells seem to

be formed around some of the pinning canals. It can be seen on the XCT cross-section of the pinning

in January of H. gabunense No. 5 as a uniform light grey feature around the pinning canal (Figure 21).

A thin cross-section of the pinning of January was made using a microtome to characterize the

wound reaction cells. However a clear anatomical section of the latest formed cells is not easy to

provide. It can be seen from Figure 20 that the latest formed vessels are very small. This confirms

slow growth. Wound reaction cells are not visible on this cross-section.

Figure 20: Thin cross-section of the pinned zone in January of H. gabunense (No. 5). The black arrow is pointed in the direction of the latest formed cells.

39

Figure 21: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of H. gabunense (No. 5). The date of the pinning is given in the upper

left corner.

Xylopia wilwerthii

3 trees of X. wilwerthii were available to study intra-annual growth (Table 4). Growth rings of X.

wilwerthii are distinct on XCT images, analogous to P. suaveolens (both Annonaceae). Their fibre cell

wall thickness increases and the cell lumen volume decreases from earlywood to latewood. This

results in clear density variations on the XCT images. XCT scans from the pinnings of X. wilwerthii No.

9 were taken (Figure 22). Relative growth is easily measured on these images. Measurements are

presented in Table 5. It can be visually seen from Figure 23 that the Gompertz model function cannot

fit the data. ‘Negative radial growth’ is observed during some months as the relative growth

measurements do not steadily increase.

40

Figure 22: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of X. wilwerthii (No. 9). The date of the pinning is given in the upper left corner.

41

Newtonia sp.

One Newtonia sp. was monthly pinned to study intra-annual tree growth (Figure 12). Although the

studied months run from September till July, the stem contained 12 pinnings. Thus one pinning

accidentally was performed 2 times. In order to make a dated time series of relative growth the

erroneous pinning has to be detected. Therefore all 12 pinnings were scanned. Cross-sections of XCT

scans can be seen in Figure 25. Growth rings boundaries are easy to distinguish, as Newtonia sp.

(Fabaceae) can be classified into basic anatomical type 1 of Worbes (2002). Relative growth

measurements are presented in Table 6. As can be seen from Figure 24, the series of the first 8

performed pinnings show potential to fit a Gompertz function. On the contrary, relative growth of the

last pinnings is not measured, because it is not obvious whether the formation of xylem cells, which

were formed after the pinning, occurred not solely due to wounding of the cambium (wound-induced

cambial activity). The radial outgrowth on the XCT images (Figure 25, clearly seen for pinning 9 and 10)

suggest that radial increment after the pinning is due to higher (wound-induced) cambial activity.

Corynanthe paniculata

3 trees of the species C. paniculata are available to determine relative intra-annual growth dynamics

(Table 4). The pinnings of C. paniculata No. 9 were drilled out and scanned. XCT images can be seen

from Figure 26. Growth rings boundaries are clearly observed due to narrow vessel-free bands of the

latest formed xylem cells (Section 4.2). Relative growth can be easily measured with the help of XCT.

Table 5 shows relative growth measurements for tree No. 9. The onset of xylem formation for this

individual is observed around the start of March. There is no observed increment after the last

pinning, so it can be concluded that wood was formed in tree No. 9 between the end of February and

July.

Although the pinnings of October and November of C. paniculata No. 9 were not scanned, it was

assumed that there was no growth until the end of February, thus a Gompertz function could be fit to

the relative growth data, characterized by Equation 6:

RG = 100.40 ∗ e−e6−0.03∗t

(6)

With RG the relative growth (%), and t the number of days since September 21, 2013. Measures of

the quality of the fit are presented in Addendum 2. The RSE of the fit is 3.30. The sigmoid growth

curve for C. paniculata can be seen in Figure 27. The relative growth rate is plotted in Figure 27, and

characterized by Equation 7:

RGR = 100.40 ∗ 0.03 ∗ e6−0.03∗𝑡−e6−0.03∗t

(7)


42

Table 6: Relative radial growth measurements (RG, %) for Newtonia sp. (No. 12). A and B (mm) are the distances respectively left and right from the pinning canal measured to obtain the increment from the onset of the growth season up to the pinning date. C and D (mm) are the distances respectively left and right from the pinning canal measured to obtain the increment from the onset of the growth season up to the harvesting date. The consecutive pinnings are numbered from 1 to 12.

No. A B C D RG

1 0.00 0.00 1.31 1.36 0

2 0.00 0.00 2.41 2.30 0

3 0.00 2.66 0.00 2.22 0

4 0.52 0.44 3.37 2.84 15

5 1.23 1.23 4.20 4.20 29

6 1.39 1.45 3.80 3.79 37

7 1.47 1.50 3.27 3.42 44

8 0.92 0.91 1.74 1.85 51

9 - - - - -a

10 - - - - -a

11 - - - - -a

12 - - - - -a

a Relative growth is not measured due to uncertainty about ‘wound-induced’ cambial activity (described for other topical species by Couralet et al., 2010a).

Figure 23: Relative growth data of X. wilwerthii.

Figure 24: Relative growth data of Newtonia sp. Only 8 of the 12 pinnings are measured, and thus a Gompertz function could not be fitted.

43

Figure 25: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of Newtonia sp. (No. 12). The presumable date of the pinning is given in the upper left corner. Only the first 8 pinnings are assumed to be monthly,

due to an erroneous pinning between pinning 9 and 12.

44

Figure 26: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of C. paniculata (No. 13). The date of the pinning is given in the upper left corner.

Figure 27: Relative growth data of C. paniculata (No. 13) fitted to the Gompertz function with parameters a = 100.40, β = 6.0006 and κ = 0.032495 (left). The derivative of the Gompertz function determines relative growth rates (right).

45

Aidia ochroleuca

Three trees of the species A. ochroleuca were monthly pinned (Table 4). A. ochroleuca No. 16 was

used to determine relative intra-annual growth. 11 pinnings were sampled and scanned. XCT images

are illustrated in Figure 28. Similar to C. paniculata, vessel density decreases to the end of the growth

rings (Section 4.2). Therefore growth ring boundaries are sufficiently visible to measure relative

growth. Table 5 shows the relative growth measurement for A. ochroleuca No. 16. There is no radial

increment observed before October 21 and after June 21.

A Gompertz function was fit to the data presented in Table 5. The relative growth during the growing

season 2013-2014 for A. ochroleuca (No. 16) is characterized by Equation 8:

RG = 111.97 ∗ e−e1.54−0.01∗t

(8)

With RG the relative growth (%), and t the number of days since September 21, 2013.

Figure 28: Cross-sections taken at the height of the pinnings in the reconstructed XCT volumes of A. ochroleuca (No. 16). The date of the pinning is given in the upper left corner.

46

Measures of the quality of the fit are presented in Addendum 2. The RSE of the fit is 3.75. The

sigmoid growth model is well fitted to the relative intra-annual growth data (Figure 29). The relative

growth rate is plotted in Figure 29, and characterized by Equation 9:

RGR = 111.97 ∗ 0.01 ∗ e1.54−0.01∗𝑡−e1.54−0.01∗t

(9)


Figure 29: Relative growth data of A. ochroleuca (No. 16) fitted to the Gompertz function with parameters a = 111.97, β = 1.5361 and κ = 0.012746 (left). The derivative of the Gompertz function determines relative growth rates (right).

4.3.2. General discussion

The pinning method

The pinning method was successfully applied for most pinnings. However, some needles did not

perforate the cambium, so a wound-induced reaction had not occurred. Consequently, a reliable

time series of pinnings cannot be made for some trees. This was particularly the case for P.

suaveolens and H. gabunense, which are species with a hard and/or thick bark. To make sure that all

pinnings reach the cambium, removing the bark before inserting a needle can be considered. To

determine wood formation after the pinning date, it is also interesting to remove the needle after

pinning. This way the formation of newly formed xylem cells can enclose the pinning canal. This was

observed for Pinus teada L. by Yoshimura et al. (1981).

The use of XCT to determine relative intra-annual growth

Cambial pinnings were performed to determine the amount of radial xylem growth over a period of

one growing season. They split up the growing season into small fragments (Seo et al., 2007), in this

case months (Section 3.3). Due to wounding with a thin needle, typical wound-induced wood

structures are formed (Larson, 1994). A digital volume of the pinned zone is obtained using XCT. For

47

all the studied species (P. suaveolens, H. gabunense, X. wilwerthii, Newtonia sp., C. paniculata, and A.

ochroleuca), XCT images show distinguishable wound-induced structures. Also growth ring

boundaries of all six studied species are visible on XCT images, so ring widths of dried wood can be

measured. In general, XCT scans are useful to determine intra-annual xylem growth following the

protocol in Section 3.5.

The use of XCT has several clear advantages. Firstly, the digital volume enables scrolling through the

pinned zone in the 3 dimensions, and select the most suitable section in order to measure relative

growth. Unlike making thin cross-sections with a microtome, the use of the XCT technique will not

remove any interesting features. Similarly, the wound-induced reaction can be characterized for the

complete zone. Secondly, the technique is less time consuming, and has a larger probability to obtain

reliable data, compared with the preparation of cross-sections using a microtome. The construction

of a digital volume of one pinned zone takes about 30 minutes (Section 4.3.1).

Nevertheless, XCT images are sometimes insufficient to determine radial growth, e.g. to count radial

rows of xylem cells in order to detect little radial increment, e.g. pinning 9 till 12 of Newtonia sp. No.

12, or to make sure that H. gabunense No. 5 did not grew (Section 4.3.1). More detailed anatomical

structures (e.g. on a cellular level) cannot be characterized using the available XCT images. In order to

prevent this drawback, higher resolution scans can be made, but this will increase the time of

reconstruction of the volume. Thin cross-section, that are made using a microtome, provide a more

detailed anatomical study of the pinned zones.

Few growth ring boundaries are not visible on the XCT images, and ring anomalies cannot be

detected. Therefore characterization of growth rings on digitized stem discs is very complementary

(Section 4.2). Ring widths can be measured and compared between digitized stem discs and XCT, so

anomalies and non-visible rings can be detected.

Intra-annual growth measurements

For the species P. suaveolens, C. paniculata and A. ochroleuca, a reliable time series of relative

growth measurements from the first until the last pinning could be established. The onset and

cessation of radial xylem growth for the observed individuals was observed during the period of 11

months (Table 5). Therefore, 11 monthly pinnings are sufficient to describe intra-annual wood

formation of the observed trees (P. suaveolens No. 4, C. paniculata No. 9, and A. ochroleuca No. 16).

The onset and cessation of radial xylem growth of P. suaveolens was observed for 2 trees (No. 1 and

No. 4). The onset of growth started after the end of December in the 2 trees. The cessation of tree

No. 1 was observed in May, while this was observed in June (one month later) for No. 4 (Table 5).

Individuals of the same species this show different intra-annual growth dynamics. The onset for C.

48

paniculata No. 13 and A. suaveolens No. 16 was observed in March and October respectively. Hence

the latter species and P. suaveolens started to grow at different times of the growing season.

Similarly, the cessation of C. paniculata occurred in July, while it occurred in June for A. ochroleuca.

As can be seen from Table 7, maximum xylem growth differs among species (April for P. suaveolens

and A. ochroleuca, and March for C. paniculata). It can be concluded that the onset and cessation of

radial xylem formation, as well as maximum xylem growth varies from one species to another and

among trees of the same species, according to Couralet et al. (2010a).

Table 7: Monthly (from the 21st of the previous month to the 20st of the current month) relative radial growth rates (%) for P. suaveolens No. 4 (POL), X. wilwerthii No. 9 (XYL), Newtonia sp. No. 12 (NEW), C. paniculata No. 13 (COR) and A. ochroleuca No. 16 (AID), based on the relative growth measurements on XCT images.

Month POL XYL NEW COR AID

Sept. 0 9 0 0 0

Oct. 0 6 0 0 0

Nov. 0 -15 0 0 13

Dec. 0 70 15 0 16

Jan. 26 30 14 0 17

Feb. 18 -31 8 0 8

Mar. 18 4 7 38 12

Apr. 32 -5 7 25 20

May -3 25 - 23 3

June 9 -20 - 8 11

July 0 -73 - 6 0

Due to an erroneous pinning in Newtonia sp., a reliable time series of this species is not available

(Table 6). However, the first 8 pinnings show a consistent series of relative growth data. Therefore, it

is assumed that the first 8 pinnings can be linked to the months September 2013 till April 2014. The

studied tree of Newtonia sp. started to grow in December (Table 7). Because XCT does not give

reliable results for the latest performed pinnings due to radial outgrowth after pinnings, cessation of

growth cannot be observed via XCT. The number of possible (non-wound induced) formed xylem

cells after the pinnings will be small, because they cannot be distinguished on the XCT images.

Therefore, thin cross-sections can allow for additional information. Mäkinen et al. (2008) determined

radial xylem growth by counting radial rows of xylem cells. This technique can by implemented by

counting radial rows of xylem cells from the position of the cambium when pinned to the last formed

cell on the radial outgrowth on the one hand, and next to the outgrowth on the other hand, in order

to test the assumption that new xylem cells are not formed after pinning. Therefore, the diameter of

49

the cylinders containing the pinning has to be larger, such that cells next to the radial outgrowth can

be counted.

For X. wilwerthii ‘negative radial growth’ is observed during some months as the relative growth

measurements do not steadily increase (Figure 23, Table 7), which is caused by clear eccentric

growth, and a large number of ring anomalies (Figure 11). Seo et al. (2007) pointed out the necessity

to account potential eccentricity of the stem when analyzing cambial pinnings. As concluded for the

characterization of growth rings of X. wilwerthii, irregularity of the stem impedes intra-annual growth

research by using cambial pinnings. Even though anomalous rings are detected and characterized,

and relative growth is unambiguously measured, a representative time series cannot be provided. A

higher degree of knowledge on formation of anomalous rings in space and time is necessary.

Couralet et al. (2010a) did not manage to determine a specific growth trend for X. wilwerthii, C.

paniculata and A. ochroleuca by using cambial marks and stem discs. This was caused by irregularities

in the wood. Despite the efforts of Couralet et al. (2010a), a combination of stem discs, cambial

marks and XCT was able to observe clear growth trends for individuals of C. paniculata and A.

ochroleuca.

For H. gabunense, characterization of the rings widths of the most recent growing seasons showed a

decreasing yearly radial increment. Higher light competition within the stand could be the cause of

the decreasing radial increment, as endogenous factors could be limiting for tree growth (Cook,

1992; Speer, 2010). Also no increment is observed on XCT images of H. gabunense No. 5 and No. 7.

However, modified wound reaction cells seem to be formed around some of the pinning canals.

Microscopic analyses could not confirm the presence of those cells. Because no growth is observed,

wound-induced cambial activity is assumed. Similarly, the formation of wound reaction wood all year

round was already observed by Couralet et al. (2010a). This could strengthen the hypothesis that

wound reaction and radial growth are independent from each other, which is a requisite to heal a

wound event though no cambial growth occurs.

The Gompertz function as an intra-annual growth model

Similar to Deslauriers (2003), Rossi et al. (2003), and Mäkinen et al. (2008), a Gompertz function was

fitted to tree growth data, and can be used as a model for tree growth. The Gompertz function can

be fitted to the growth-time series of P. suaveolens No. 4, C. paniculata No. 13 and A. suaveolens No.

16. As can be seen in Table 8, the best fit is found for the series of C. paniculata. Intra-annual growth

can thus be modelled as a sigmoid growing curve. This sigmoid growth trend was already observed in

Figure 4 (Détienne and Mariaux, 1977).

50

Table 8: Residual standard errors (RSE) for the Gompertz function fitted to the relative growth measurements.

Species RSE

Polyalthia suaveolens 4.98

Corynanthe paniculata 3.30

Aidia ochroleuca 3.75

4.4. Anatomical features around the pinning zone

XCT digital volumes easily provide a 3D view of the pinning zones. In this way, the wound-induced

tissue can be visualized, and its volume can be measured. These measurements are useful in order to

determine where around the pinning canal a thin cross-section should be made, so required

structures can be seen. Based on an exploration of XCT wood volumes, thin cross-sections were

made using a microtome (Section 3.6). These sections are used to obtain information that cannot be

detected with XCT, and to get a more anatomical view of the wound-induced features. Anatomical

descriptions of pinned zones by Verheyden et al. (2004), Seo et al. (2007) and Couralet et al. (2010a)

are used to determine different zones on the cross-section of the pinning zones. Features around the

pinned zones can be seen on cross-section taken above the pinning canal (Figure 30 to Figure 32).

According to Couralet et al. (2010a), it can be seen that these features are variable in size and

structure depending on species and time of wounding.

4.5. Climate-growth relationships

Figure 33 illustrates the average monthly precipitation from 1948 till 2006, and average monthly

solar irradiance, relative humidity and temperature from September 2013 till July 2014 of the Luki

Biosphere Reserve. In order to compare with the reliable relative growth time series (temporal

resolution of one month) the meteorological measurements were rescaled to monthly data.

However, relevant climatic events would not be taken into account due to rescaling.

From June to September the monthly precipitation is less than 50 mm, this period defines a distinct

dry period (Figure 33). A smaller intermediate dry period is observed around January. For the growth

season 2013-2014, the air temperature of September is 21.6°C, rises to a maximum of 26.1°C in April,

and decreases to 20.5°C in July. The relative air humidity was more or less uniform during the whole

year, due to a thick, low-level, non-precipitating cloud layer during the dry season (Pendje and Baya

ki, 1992). Solar irradiance shows a bimodal curve, with peaks in the first and second part of the wet

season, which is divided due to the intermediate dry period.

51

Figure 30: Thin cross-sections of the pinned zone in December (top) and January (bottom) of P. suaveolens No. 4. Growth rings (green arrow), the positon of the cambium at the time of the pinning (yellow arrow), the wound-reaction cells

(WR), and the earlier formed wood cells that contain deposits due to the wounding (DEP).

WR

WR

DEP

DEP

52

Figure 31: Thin cross-sections of the pinned zone in March of X. wilwerthii No. 9 (top), in September of Newtonia sp. No. 12 (middle), and in February of A. ochroleuca No. 16 (bottom). Growth rings (green arrow), the positon of the cambium at the time of the pinning (yellow arrow), the wound-reaction cells (WR), and the earlier formed wood cells that contain

deposits due to the wounding (DEP).

WR

WR

WR

DEP

DEP

DEP

53

Figure 32: Thin cross-sections of the pinned zone in February of Newtonia sp. No. 12 (top), and in April of C. paniculata No. 13 (bottom). Growth rings (green arrow), the positon of the cambium at the time of the pinning (yellow arrow), the

wound-reaction cells (WR), and the earlier formed wood cells that contain deposits due to the wounding (DEP).

WR

WR

DEP

DEP

54

Figure 33: Time series of the monthly relative growth rate (%) observed for P. suaveolens No. 4, C. paniculata No. 13, and A. ochroleuca No. 16. for three studied species (1 tree per species) in the Luki reserve (top). Average monthly

precipitation from 1948 till 2006 (Prec.), and average monthly solar irradiance (SR), relative humidity (RH) and air temperature (Air T.) from September 2013 till July 2014 (averages from the 21st of the previous month to the 20st of the

current month) were measured at the meteorological station of Luki (bottom).

0%

10%

20%

30%

40%

Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July

0%

10%

20%

30%

40%


0%

10%

20%

30%

40%


0

5

10

15

20

25

30

0

50

100

150

200

250

300

350

400


Air

T. (

°C)

Pre

c. (

mm

) /

SR (

W/m

²) /

R

H (

%)

Month

Prec. (mm) RH (%) SR (W/m²) Air T. (°C)

Polyalthia suaveolens

Corynanthe paniculata

Aidia ochroleuca

55

The onset of radial growth in P. suaveolens No. 4 occurred at the same time when the intermediate

dry period (January) is observed (Figure 33). Maximum growth was observed at the end of the

second peak of solar irradiance. In less than a month after the start of the dry season (June) growth

stopped. Radial growth in C. paniculata No. 13 started 2 months later, when solar irradiance peaked

(Figure 33). So the first 5 months of the wet season, no growth was observed. For A. ochroleuca No.

16, radial growth covered the complete wet season (Figure 33). 2 peaks were observed: the first at

the end of the first half of the wet season (January), and the second after 3 months of highest solar

irradiation (April).

Couralet et al. (2010b) stated that annual radial stem growth (based on a dendrochronological study)

is positively related to the amount of precipitation during the early wet season for A. ochroleuca, and

to precipitation at the end of the wet season for C. paniculata. The current intra-annual study of

these species confirms this statement, as most of the intra-annual growth of A. ochroleuca occurred

during the first half of the wet season, and C. paniculata started to grow at the beginning of the

second half of the wet season. As a conclusion for those two species, a high yearly radial xylem

increment can be a response to high amounts of monthly precipitation from October till January for

A. ochroleuca, and to high amounts of precipitation from February till May for C. paniculata.

Differences between those two species could be caused by a difference in their root system (Couralet

et al., 2010b. A. ochroleuca can benefit from the first rains that penetrate directly into the soil and

are taken up by the shallow root system of the species. On the contrary, C. paniculata starts growing

when precipitation water reaches deeper soil levels, so that their pivot-like root system can uptake

the water.

4.6. Phenology-growth relationships

Phenological events (the presence of old and new leaves, flowering, defoliation and dissemination)

were monthly observed for the 18 pinned trees every 21st of the month from January 2014 till

September 2014 (Addendum 3). In addition to the recent data, a historical phenological study in the

Luki Biosphere Reserve from 1948 till 1957 is available for the six studied species. The historical

observations can be seen from Figure 34 till Figure 37, and from Addendum 4.

Both in the recent (Addendum 3) and historical data (Figure 34), defoliation did not occur in the

evergreen species (Section 3.2). For P. suaveolens, flowering events were not observed for all the

recently studied trees. However, 2 trees of P. suaveolens had fruits in September, similar to 30 % of

the historical observed trees. Both the recent and historical data sets show dissemination in January

and February. It can be seen in Figure 33 that P. suaveolens No. 4 started to grow in January, at the

same time that dissemination occurred. During the growing period of tree No. 4, flowering was

56

observed in a maximum number of ‘historical’ trees, while fruiting occurred mostly at the onset of

the wet season, where no radial increment for P. suaveolens occurred.

Similar to P. suaveolens, historical flowering was observed during the growing season for Newtonia

sp. No. 12 (onset of growth in December) (Table 7), and maximum fruiting occurred in the dry

season, when growth was not observed (Figure 35).

For C. paniculata No. 13, dissemination was recently observed in January and February (Addendum

3). Dissemination occurred for most of the historical trees around November. The onset of xylem

formation that is observed for C. paniculata No. 13 occurred in March. In this month the tree showed

maximum growth (Figure 33). It could be linked to the historical increase in the number of observed

trees for fruiting and the maximum number of trees for flowering (Figure 36).

Relative intra-annual growth was observed from November till June for A. ochroleuca No. 16 (Figure

33). As can be seen from the historical data in Figure 37, November corresponds to the start of an

increasing number of observations for fruiting and the start of a decreasing number of observations

for flowering. The recent phenological observations show that fruiting occurred in February-March,

and dissemination occurred in April-May (Addendum 3). Likewise, in the historical data a high

number of fruiting and dissemination events were observed for these months. Couralet et al. (2010b)

suggested that flowering months for C. paniculata and A. ochroleuca (both Rubiaceae) are also the

months where a maximum correlation between their monthly rainfall and tree ring width was

observed. The obtained relative growth-time series in this study possibly showed that in these

months (November for A. ochroleuca, and March, for C. paniculata), where the amount of monthly

rainfall seems to influence the total yearly growth of the trees, radial growth started.

Based on the available phenological observations and measured relative growth-time series (Section

4.3.1), it can be concluded that for P. suaveolens there could be a link between the onset of radial

growth and the increasing number of observations of dissemination. The highest numbers of

flowering observations could be associated to growth periods for P. suaveolens and Newtonia sp.,

and to the onset of growth for the Rubiaceae. Similarly, the lowest numbers of fruiting observations

occur in the growing season of for P. suaveolens and Newtonia sp., while an increase in of the

numbers of fruiting could be linked to the onset of growth for the Rubiaceae. Assumptions cannot be

made for H. gabunense and X. wilwerthii, due to the lack of a reliable growth-time series.

57

Figure 34: Average monthly percentages of the observed trees of Polyalthia suaveolens to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 30 trees was observed

for P. suaveolens.

Figure 35: Average monthly percentages of the observed trees of Newtonia sp. to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 19 trees was observed for

Newtonia sp.

0%

5%

10%

15%

20%

25%

30%

35%

% o

f o

bse

rved

tre

es

Flowering Fruiting Defoliation Dissemination

0%

5%

10%

15%

20%

25%

30%

% o

f o

bse

rved

tre

es


58

Figure 36: Average monthly percentages of the observed trees of Corynanthe paniculata to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 76 trees was observed

for C. paniculata.

Figure 37: Average monthly percentages of the observed trees of Aidia ochroleuca to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 17 trees was observed for A.

ochroleuca.

0%

10%

20%

30%

40%

50%

% o

f o

bse

rved

tre

es


0%

10%

20%

30%

40%

50%

60%

% o

f o

bse

rved

tre

es


59

5. Conclusion

Combining growth ring and high-resolution pinning data leads to fundamental knowledge on tropical

tree growth. For the species P. suaveolens, C. paniculata and A. ochroleuca, a reliable time series of

relative growth measurements for one growing season was established. Relative growth was

measured in order to remove the growth signal due to eccentricity. A sigmoid Gompertz curve was

fitted to the growth-time series, and is used as a model for relative intra-annual growth of the tropical

species. It can be concluded that the onset and cessation of radial xylem formation, as well as

maximum xylem growth varies from one species to another, and among trees of the same species,

according to Couralet et al. (2010a).

XCT scans are useful to determine relative intra-annual xylem growth. The digital volume enables

scrolling through the pinned zone in the 3 dimensions, and select the most suitable section in order to

measure relative growth. Unlike making thin cross-sections with a microtome, the use of the XCT

technique will not remove any interesting features. Similarly, the wound-induced reaction can be

characterized for the complete zone. Analyses of cambial pinnings using XCT considerably reduces the

time spent in the laboratory. In this way the pinning technique can compete with other intra-annual

growth determination techniques, e.g. sanded surfaces and micro-sections. More rapidly analysis

enables to increase the number of individuals to be studied, so the knowledge gap on growth

dynamics in tropical forests can be filled. Although, in order to obtain a clear view on more detailed

anatomical structures (e.g. on a cellular level) higher resolution scans can be made, but this will

increase the processing time of reconstruction of the volume. Another possibility is making thin cross-

section with a microtome, which provide a more detailed anatomical study of the pinned zones. XCT

volumes provide a detailed indication where thin cross-sections can be made. Furthermore, the

characterization of growth rings on digitized stem discs is complementary to XCT analysis, when

growth ring boundaries are not visible on the XCT images and/or ring anomalies cannot be detected.

Climate-growth relationships were described for the species with a reliable growth-time series (P.

suaveolens, C. paniculata and A. ochroleuca). Most of the intra-annual growth of A. ochroleuca

occurred during the first half of the wet season, and C. paniculata started to grow at the beginning of

the second half of the wet season, according to Couralet et al. (2010b). Maximum growth was

observed at the end of the second peak of solar irradiance. The onset of radial growth of P.

suaveolens is possibly linked to dissemination. Growth in P. suaveolens and Newtonia sp., and the

onset of growth of the Rubiaceae possibly associate with flowering. Similarly, fruiting possibly occurs

in the growing season of for P. suaveolens and Newtonia sp., while an increase in of the numbers of

fruiting observations could be linked to the onset of growth for the Rubiaceae.

60

6. Prospective research

In the Nkula park of the Luki Biosphere Reserve, individuals of the tree species Terminalia superba

were pinned during the same period as the 18 studied species (Section 3.3). In addition 4 pinned T.

superba were equipped with each three point dendrometers. The dendrometers measure radial stem

increment at high temporal resolution. The combination of tree ring analysis, wood anatomy and XCT

on cambial pinnings of stem discs, as described in this work, could be performed for these four T.

superba. Moreover, dendrometer data could add high temporal data to study intra-annual wood

formation more in detail.

The dendrochronological principal of the aggregated tree model (Section 2.1.2) states that tree

growth depends on multiple limiting factors. The climate signal could be determined using cambial

pinnings, dendrometers and meteorological data. To determine the endogenous signal within the

stand such as the influence of light-competitive trees, techniques to reconstruct the shape of the

tree and his environment are introduced (Section 6.2 to 6.5).

6.1. Dendrometer data

During the pinning period, dendrometers (Ecomatik, Germany) measured at high temporal resolution

(30 minutes intervals) variations in stem diameter. On four T. superba, 3 point dendrometers were

installed on stem on the same height, and the distance between two dendrometers was 120 degrees.

Thus variations at different positions around the tree circumference could be compared.

Figure 38: Dendrometer (Ecomatik, Germany) installed on T. superba.

61

6.2. Non-destructive visualization of the stem contour

3 methods were used to visualize to contour of the stem at the height of the dendrometers. First, 2

wires made of iron and copper were tested. The wires enwrapped the tree just below the

dendrometers. Beaten to the tree with a hammer, the wires had to be shaped like the contour of the

tree. To be sure the wires were shaped, they were left hanging for about three days. Despite of this

method, the stem shape could not be reconstructed.

Trigonometry was used in the second method. Just below the dendrometers, an arbitrary number of

points were marked on the contour of the stem (in this case 16 or 18 points). Below each

dendrometer, a point was indicated on the stem. The distances between two dendrometers were

measured with a tree caliper. An equal number of points was marked between two dendrometers.

One reference point was chosen between two dendrometers. The distances from this reference

point to the others points of this section of the contour were measured with the clamp. This was

repeated for a second point. The same protocol was followed for the two other sections of the

contour. The data were implemented in Excel and based on trigonometry, a 2D-plot visualizes the

marked stem points. A polygon was fitted, and the stem contour was reconstructed.

Finally, a 3D-reconstruction of the stem was made (Section 6.4).

Figure 39: Visualization of the stem contour at the height of the dendrometers. The 3 large dots represent the position of the dendrometers.

-10

0

10

20

30

40

50

-30 -20 -10 0 10 20 30

Dis

tan

ce (

cm)

Distance (cm)

62

6.3. Alternative crown projection method

To study the variation of radial tree growth within the stem, the dendrometers were placed at the

same height. Crown projections were made to receive more information about this variation. The

crowns of the surrounding trees that could influence the growth of the dendrometer tree were also

mapped. The method is based on the principles of the plumb method of Pardé and Bouchon (1988).

Because there wasn't a plumb and mirror system present in the lab of the Luki reserve, some

creativity was required.

A Yardage Pro 500 Laser Rangefinder (Bushnell Outdoor Products, United States of America) was

installed on a tripod. The tripod was placed under the tip of a branch of the tree. The tip must

represent a point on the contour of the crown. After placing the tripod, the ocular of the Yardage Pro

500 Laser was leveled (Figure 40). The tip of the branch had to be visible in the center of the ocular,

otherwise the tripod had to be moved and the leveling had to be repeated (Figure 41). When the tip

was visible in the center of the ocular, a wooden stick was stabbed into the ground at the position of

the tripod. Wooden sticks were used for projecting the tree crown on the ground surface. The sticks

visualized the contour of the crown. They were numbered sequentially. A Yardage Pro Tour Laser

Rangefinder (Bushnell Outdoor Products, United States of America) was used to measure the height

of the tip. The height was measured perpendicular to the ground surface at the position of the stick.

Figure 40: Leveling the ocular of the Yardage Pro 500 Laser Rangefinder on the tripod

Figure 41: Checking the visibility of the tip of the branch through the ocular

The next position of the tripod forms an imaginary line segment with the previous position. The line

segment touches the tips of the branches between those two positions. The method used for the

previous position, was repeated. This procedure was repeated until the starting position had been

reached.

63

After crown projection, a position around the tree from which the most projection points were

visible, was chosen. The tripod was placed on this position. A compass was installed leveled on the

tripod. A laser (Draper Tools LTD, United Kingdom) was positioned on top of the compass. The laser

rotated manually from zero to 360 degrees (Figure 42). Each time the laser hit a stick, the number of

the stick and the angle was written down. The angle between the north and the tree was also

measured. The distance between the position of the compass and the object was measured with the

measuring tape. The measured angles and distances were implemented in a mathematical software

program, GeoGebra (International GeoGebra Institute, Austria). The projection of the crown could as

such be visualized (Figure 43).

Figure 42: Rotating the laser (Draper Tools LTD, United Kingdom) on top of the compass. The compass was installed on a tripod. The laser had to go through the center of the compass.

Figure 43: Method of the visualization of the crown projection in GeoGebra (International GeoGebra Institute, Austria) using the measurements (angles and distances).

64

One compass position wasn't able to visualize all projection points, so an additional position had to

be used. The angles and distances for the missed projection points, were measured from the new

compass point. The distance and angle between the compass points was also measured. Additional

compass points had to be used until all projection points were observed.

The height of the crown circumference was measured, although only at projection points. These

values could provide only a simple image of the crown height, and could be used in the investigation

of the influence of surrounding trees. Figure 44 gives an example of the crown projection.

The use of optical methods induced some difficulties. First, the tip of the branches of the

dendrometer tree wasn't always visible. The view from the ground surface was impeded by

suppressed trees, shrubs and the sun. Secondly, the laser beam was hindered to reach the sticks by

the vegetation, which necessitated removal of the vegetation between laser and sticks.

Figure 44: The result of the alternative crown projection method for a T. superba.

65

6.4. 3D-reconstruction of the stem

Van den Bulcke et al. (2013) represented an inexpensive tool-chain for total tree assessment, starting

with the reconstruction of the stem of the standing tree. The studied species were birch (Betula spp.)

and willow (Salix spp.), both European hardwood species. Due to the flexibility, the tool-chain can

also be used in the tropics (Van den Bulcke et al., 2013). For the dendrometer trees in Luki (see

Dendrometer), a 3D-reconstruction of the stem is presented, based on the protocol of Van den

Bulcke et al. (2013). Data derived from this method can be used for studies on tree growth as

presented in this thesis. The information is supplementary to data of the dendrometers (Section 6.1)

and the measured crown parameter (Section 6.3).

For the stem reconstruction, the methodology of photogrammetry is applied. To shoot images, the

required tools are a tripod, camera, laser, a stick and measuring ropes (Van den Bulcke et al., 2013).

All images of the dendrometer tree in the valley of park Nkula were taken with a Nikon Coolpix

AW110 (Nikon Europe B.V.). The camera was mounted on a tripod. A laser (Draper Tools LTD, United

Kingdom) was positioned on the tripod to tilt the camera at the chosen angle. Images were shoot at

an angle of zero, 50 and 80 degrees with the horizontal plane. The camera was tilted at arbitrary

angle to visualize the tree at a moderate distance with maximal usage of the field view (Van den

Bulcke et al. 2013).

The same procedure was repeated for an arbitrary number of 16 positions to make sure that enough

data were collected. The angle between successive positions was 22.5 degrees. The images from

different viewpoints were taken at similar distances so they had not to be resized according to the

first image (Van den Bulcke et al., 2013). The distance between the position of the tripod and the

tree was five meters. To shoot from the correct position, a stick and 2 measuring ropes were used.

This emplacement is shown in Figure 45. The removal of the vegetation from the under floor around

the dendrometer tree was needed. The vegetation impeded the correct determination of the

shooting positions and disturbed the image of the dendrometer tree.

The off-line calibration and further steps of protocol were performed in the laboratory. Off-line

calibration of the camera was necessary in order to correct for lens distortions and tilt. A grid with

fixed distances between the nodes was made. The grid was photographed at the same tilt angles of

the camera with the horizontal plane at which field images were taken. Van den Bulcke et al. (2013)

represented more detailed information and further steps of the tool-chain. The result is presented in

Figure 46. A drawback of the method is that concave structures couldn't be visualized as inward

curving surfaces (Van den Bulcke et al., 2013). The visualization of the big buttresses of Terminalia

superba create some difficulties.

66

Figure 45: The determination of the correct shooting position by means of 2 measuring ropes and a stick.

Figure 46: 3D reconstruction of the stem, following the protocol of Van den Bulcke et al. (2013).

67

6.5. Plot design and measurements around the studied tree

To gather more information on the environment of the dendrometer trees, plots were installed. A

circular plot was set with the dendrometer tree as the center. All trees in direct ‘competition’ with

the dendrometer tree in terms of sunlight interception had to be taken into account. The distances

between the dendrometer tree and his light competitors were measured with a measuring tape. The

radius of the circular plot was equal to the greatest measured distance. In the plot, all trees with a

circumference greater than 20 centimeters were numbered. The competitors were marked with a

letter. The suppressed trees were marked with a Roman numeral. The distances between the

dendrometer tree and the numbered trees were measured with a measuring tape.

Two parameters of the suppressed trees were measured: the DBH and the height to the top. To

measure the DBH, a tapeline was used. When buttresses were too large, the DBH could not be

measured. As an alternative, the diameter just above the buttresses was taken. Each height was

measured tree times, using a Yardage Pro Tour Laser Rangefinder (Bushnell Outdoor Products,

United States of America). When the plot was inclined, a Blume Leiss was used to measure the

height. The Blume Leiss can immediately correct for the slope. The same protocol was used for the

light competitive trees. Moreover, the height to the first branch of the crown was measured.

For each tree, the species and phenological state were determined by a local botanist. To visualize

the plot in a 2D-figure with a mathematical software program, the relative positions of the trees

need to be known. A compass and measuring tape were used to obtain the data, following the

protocol that is presented in Section 6.3.

When the ground surface of the plot wasn't horizontal, the slope was determined with a Blume Leiss.

The Blume Leiss and the Yardage Pro Tour Laser Rangefinder are optical instruments. The same

remark as in Section 6.3 had to be made. The vegetation of the forest floor had to be removed, to

allow proper measurements of height and distance.

68

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Addenda

Addendum 1: Code to fit the Gompertz function in R (R Core Team, 2015).

#FitForPolyalthiaSuaveolens

time <- c(0, 30, 61, 91, 122, 153, 181, 212, 242, 273, 303, 334, 365)

Pol <- c(0, 0, 0, 0, 26, 44, 62, 94, 91, 100, 100, NA, NA)

Xyl <- c(9, 15, 0, 70, 100, 69, 73, 68, 93, 73, NA, NA, NA)

New <- c(0, 0, 0, 15, 29, 37, 44, 51, 46, 34, 92, 81, NA)

Cor <- c(0, 0, 0, 0, 0, 0, 38, 63, 86, 94, 100, NA, NA)

Aid <- c(0, 0, 13, 29, 46, 54, 66, 86, 89, 100, 100, NA, NA)

growth.data <- data.frame(time, Pol, Xyl, New, Cor, Aid)

growth.data

model.Pol <- nls(Pol~a*exp(-exp(b-k*time)), data=growth.data,start=list(a=100,b=10,k=0.1))

nls(Pol~a*exp(-exp(b-k*time)), data=growth.data,start=list(a=100,b=10,k=0.1))

summary(model.Pol)

coef(model.Pol)

plot(time, Pol,xlab="Time (Days since September 21, 2013)", ylab="Relative growth (%)")

plot.x <- 1:365

plot.y <- coef(model.Pol)[1]*exp(-exp(coef(model.Pol)[2]-coef(model.Pol)[3]*plot.x))

lines(plot.x, plot.y)

legend("topleft",legend=c("Measurements","Fitted Gompertz curve"),lty=c(NA,1),pch=c(1,NA))

par(mar=c(5,5,2,5))

plot(time, Pol,xlab="Time (Days since September 21, 2013)", ylab="Relative Growth (%)")

plot.x <- 1:365

plot.y <- coef(model.Pol)[1]*exp(-exp(coef(model.Pol)[2]-coef(model.Pol)[3]*plot.x))

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plot.d.y <- coef(model.Pol)[1]*coef(model.Pol)[3]*exp(-exp(coef(model.Pol)[2]-

coef(model.Pol)[3]*plot.x)+coef(model.Pol)[2]-coef(model.Pol)[3]*plot.x)

lines(plot.x, plot.y)

par(new=T)

plot(plot.x, plot.d.y, lty=2,type="l",ylab="",xlab="", axes=F)

legend("topleft",legend=c("Measurements","Fitted Gompertz curve", "Gompertz

derivative"),lty=c(NA,1, 2),pch=c(1,NA, NA), cex=0.55)

axis(side=4)

mtext(side=4, line=3, "Relative growth rate (%)")

timeres <- c(0, 30, 61, 91, 122, 153, 181, 212, 242, 273, 303)

plot(timeres, resid(model.Pol),xlab="Time (Days since September 21, 2013)", ylab="Residuals (%)")

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Addendum 2: Measurements for the quality of the fitted Gompertz function.

Figure 1: Residuals of the Gompertz function fit to the relative growth data of P. suaveolens.

Figure 2: Residuals of the Gompertz function fit to the relative growth data of C. paniculata.

Figure 3: Residuals of the Gompertz function fit to the relative growth data of A. ochroleuca.

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Table 1: Parameters and measures for the quality of the Gompertz function fit to the relative growth data of three species, with RSS the residual sum of squares, df the degrees of freedom and RSE the residual standard error.

Species a b k RSS df RSE

Polyalthia suaveolens 104,7090538 3,2325288 0,02250205 191,3 8 4,89

Corynanthe paniculata 100.3967783 6.0006176 0.0324905 83,4 8 3,30

Aidia ochroleuca 111.97071037 1.53610411 0.01274656 112,5 8 3,75

Table 2: Measures for the quality of the parameters of the Gompertz function fit of three species.

Species a b k

Std. Error t value Pr(>|t|) Std. Error t value Pr(>|t|) Std. Error t value Pr(>|t|)

Polyalthia suaveolens 4.840 21.636 2.19e-08 4.851e-01 6.663 0.000159 3.493e-03 6.442 0.000200

Corynanthe paniculata 3.280 30.604 1.41e-09 6.944e-01 8,61 2.50e-05 3.834e-03 8,474 2.88e-05

Aidia ochroleuca 6.156 18,19 8.58e-08 1.445e-01 10,63 5.37e-06 1.542e-03 8,265 3.45e-05

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Addendum 3: Meteorological data of the Luki station

Table 1: Average monthly solar irradiance (SR), relative humidity and temperature from September 2013 till July 2014 (averages from the 21st of the previous month to the 20st of the current month) were measured at the meteorological station of Luki.

Month Air T. (°C) RH (%) PAR (uE) SR (W/m²)

Sept. 20 21,62 86,19

Oct. 23,35 85,45 112,94 237,11

Nov. 24,68 88,10 133,04 283,60

Dec. 24,54 91,71 134,05 282,72

Jan. 24,91 91,79 134,31 271,80

Feb. 25,58 88,36 174,49 359,77

Mar. 25,88 88,37 171,93 355,19

Apr. 26,15 87,16 173,25 358,95

May 25,56 90,26 147,33 304,92

June 23,86 91,58 105,14 209,48

July 20,45 91,30 89,11 165,81

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Addendum 4: Phenological observations of the pinned trees from January till September 2014.

Table 1: Observations (X) of the presence of old leaves (OL) and/or new leaves (NL) on the pinned trees every 21st of the month from January 2014 till September 2014. Leaves were considered old when they had been present since the previous growth season, and new when they had sprouted during the current growth season.

No. Species January February March April May June July August September

OL NL OL NL OL NL OL NL OL NL OL NL OL NL OL NL OL NL

1 Polyalthia suaveolens X X X X X X X X X X X X X

2 P. suaveolens X X X X X X X X X X X X X



5 Hylodendron gabunense X X X X X X X X X

6 H. gabunense X X X X X X X X X



9 Xylopia wilwerthii X X X X X X X X X X X X X

10 X. wilwerthii X X X X X X X X X X X X X

11 X. wilwerthii X X X X X X X X X X X X X

12 Newtonia sp. X X X X X X X X X X X X X

13 Corynanthe paniculata X X X X X X X X X X X X X

14 C. paniculata X X X X X X X X X X X X X

15 C. paniculata X X X X X X X X X X X X X

16 Aidia ochroleuca X X X X X X X X X X X X

17 A. ochroleuca X X X X X X X X X X X X

18 A. ochroleuca X X X X X X X X X X X X

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Table 2: Observations of flowering (FL) and fruiting (FR° events on the pinned trees every 21st of the month from January 2014 till September 2014. The observation of the phenological event is indicated with an ‘X’.


FL FR FL FR FL FR FL FR FL FR FL FR FL FR FL FR FL FR

1 Polyalthia suaveolens X

2 P. suaveolens

3 P. suaveolens X

4 P. suaveolens

5 Hylodendron gabunense

6 H. gabunense

7 H. gabunense X

8 H. gabunense

9 Xylopia wilwerthii X X X

10 X. wilwerthii X X X

11 X. wilwerthii X X X

12 Newtonia sp.

13 Corynanthe paniculata

14 C. paniculata

15 C. paniculata

16 Aidia ochroleuca X X

17 A. ochroleuca

18 A. ochroleuca

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Table 3: Observations of defoliation (DEF) and dissemination (DISS) events on the pinned trees every 21st of the month from January 2014 till September 2014. The observation of the phenological event is indicated with an ‘X’.


DEF DISS DEF DISS DEF DISS DEF DISS DEF DISS DEF DISS DEF DISS DEF DISS DEF DISS

1 Polyalthia suaveolens X X

2 P. suaveolens X X

3 P. suaveolens X X

4 P. suaveolens X X

5 Hylodendron gabunense X

6 H. gabunense X

7 H. gabunense X

8 H. gabunense X

9 Xylopia wilwerthii X X

10 X. wilwerthii X X

11 X. wilwerthii X X

12 Newtonia sp.

13 Corynanthe paniculata X X X

14 C. paniculata X

15 C. paniculata X

16 Aidia ochroleuca X X

17 A. ochroleuca

18 A. ochroleuca

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Addendum 5: Historical phenological observations in the Luki Biosphere reserve (1948-

1957)

Figure 1: Average monthly percentages of the observed trees of Hylodendron gabunense to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 70 trees was observed

for H. gabunense.

Figure 2: Average monthly percentages of the observed trees of Xylopia wilwerthii to which phenological events were observed in the forest Reserve of Luki from January 1948 till December 1957. An average of 82 trees was observed for X.

wilwerthii.

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