1. i
INVESTIGATING WILD BEE COMMUNITY
STRUCTURE AT THE LANDSCAPE SCALE TO
EVALUATE THE ECOLOGICAL
PERFORMANCE OF A PERMACULTURE
MICRO-FARM IN A COMPARATIVE
APPROACH
ETUDE DE LA DIVERSITÉ DES COMMUNAUTÉS D’ABEILLES SAUVAGES
DANS LE BUT D’ÉVALUER LES PERFORMANCES ÉCOLOGIQUES D’UN
SYSTEM PERMACULTUREL COMPARÉ À D’AUTRES TYPES D’HABITAT
MARGAUX VILLEBRUN
TRAVAIL DE FIN D’ÉTUDES PRÉSENTÉ EN VUE DE L’OBTENTION DU DIPLÔME DE
MASTER BIOINGÉNIEUR EN SCIENCES AGRONOMIQUES
MASTER THESIS PRESENTED IN ORDER TO GET A MASTER DEGREE OF BIOENGENEER IN
AGRICULTURAL SCIENCES
ANNÉE ACADÉMIQUE 2015-2016
CO-PROMOTEURS: NICOLAS VEREECKEN (ULB), MARC DUFRÊNE (GXABT)

2. ii
© Toute reproduction du présent document, par quelque procédé que ce soit, ne peut être
réalisée qu'avec l'autorisation de l'auteur et de l'autorité académique de Gembloux Agro-Bio
Tech.
© Any reproduction of this document, by any means whatsoever, can only be achieved with
the consent of the author and Academic authority of Gembloux Agro-Bio Tech.
Le présent document n'engage que son auteur.
This document only reflects the author.

3. i
ABSTRACT (English)
Biodiversity conservation is not just a philosophic or moral issue related to ecological
conservation and diversity promotion. Indeed, the role of biodiversity for agriculture
production, landscape heritage conservation and human well-being need to be taken into
account too. However, the sensitivity of biodiversity to environmental changes shows the
importance of developing accurate tools in order to assess the state and functioning of this
biodiversity within an ecosystem. Evaluating the diversity of wild bee communities is one of
these tools. Indeed, wild bees are major pollinators of cultivated plants and wild flora; they
play a significant role for agricultural profitability, as well as ecosystem sustainability.
However, the landscape homogenization within agricultural areas has triggered losses in
feeding and nesting resources (namely because of the fragmentation of their habitats).
Nevertheless, permaculture practices appear to enhance biodiversity in a sustainable way, but
the literature lack of scientific approaches to confirm this statement.
This study is part of the second part of the research project “Organic permaculture market
gardening and economic performances1
”, which seeks to go deeper in understanding the
functioning of the Organic Farm of the Bec-Hellouin (OFBH), so as to explain how the
economic performances are related to the system’s ecological health. Furthermore, the
ultimate objective of the research project being the spin-off of such micro-farms, it is thus
meaningful to have scientific assessments of the permaculture’s benefits for agro-ecosystems,
such as their biodiversity. Therefore, the general aim of this study was to characterize and
quantify wild bees’ diversity across different type of habitats, considering local factors and
landscape influence, and to assess the economic added-value of those pollinators for some
crops of interest in market gardening.
Bee communities were sampled within 10 sites, across 4 different habitats (4 sites within the
OFBH, 2 meadows, 2 semi-natural areas and 2 conventional fields) within the French Region
of Haute-Normandie.
Firstly, wild bees’ diversity compared across the sites appears to be more important in the site
“Jardin-Clairière” (JC), which is part of the OFBH. Secondly, the classification of the
different habitat on a “biodiversity gradient” shows that wild bees’ diversity is globally higher
within the OFBH, suggesting that permaculture promote wild bees’ diversity better than the
other habitat, even semi-natural habitats. The General Linear Models (GLMs) suggested that
the functional diversity of a community is significantly negatively affected by the % of arable
land in a buffer zone of 500 m around a site, while bee abundance is significantly negatively
affected by the disruption degree of a site. The economic importance of insect pollinators was
demonstrated for the three crops studied.
In conclusion, this study assessed that permaculture systems such as the OFBH seem to be a
relevant alternative for the ecological intensification of agricultural landscapes. Indeed,
they contribute to maintain a high level of biodiversity, while ensuring high levels of food
production.
1
Maraîchage biologique permaculturel et performance économique, available at :
www.fermedubec.com/publications.aspx

4. ii
ABSTRACT (French)
La préservation de la biodiversité n'est pas juste une question philosophique ou morale liée à
l’écologie. En effet, il faut aussi prendre en compte le rôle de biodiversité dans la production
agricole, la conservation du patrimoine paysager et le bien-être humain. Cependant, la
sensibilité de la biodiversité aux changements environnementaux montre l'importance de
développer des outils précis pour évaluer l'état et le fonctionnement de cette biodiversité dans
un écosystème. L'évaluation de la diversité des communautés d'abeilles sauvages est un de ces
outils. En effet, les abeilles sauvages sont les pollinisateurs majeurs de plantes cultivées et de
la flore sauvage; elles jouent donc un rôle important dans la rentabilité des productions
agricoles et dans la pérennité des écosystèmes. Cependant, l'homogénéisation du paysage
agricole a provoqué de graves pertes en ressources alimentaires et sites de nidification
(notamment via la fragmentation de leurs habitats). Néanmoins, la permaculture semble
favoriser durablement la biodiversité, mais la littérature manque d'approches scientifiques
pour confirmer cela.
Cette étude s’inscrit dans la deuxième partie du projet de recherche « Maraîchage biologique
permaculturel et performances économique2
», qui cherche à mieux comprendre le
fonctionnement de la Ferme biologique du Bec-Hellouin (FBBH), afin d'expliquer comment
ses performances économiques sont liées à la santé écologique du système. En outre, l'objectif
final du projet de recherche étant l’essaimage des micro-fermes permaculturelles, il est donc
intéressant d'avoir une évaluation scientifique des atouts de la permaculture pour convaincre
les différents acteurs du milieu. Ainsi, le but de cette étude était, d’une part la caractérisation
et la quantification de la diversité des abeilles sauvages à travers différent types d'habitats, en
considérant l’influence de facteurs locaux et paysagers, et d’autre part, l’évaluation de
l’importance économique de ces pollinisateurs pour certaines productions d’intérêt en
maraîchage.
Les communautés d'abeilles sauvages ont été échantillonnées en Haute-Normandie (France)
dans 10 sites représentants 4 habitats différents : 4 sites dans la FBBH, 2 prairies, 2 zones
semi-naturelles et 2 cultures conventionnels.
Premièrement, la diversité des abeilles sauvages comparée à travers les sites semble être plus
importante dans le site « Jardin-Clairière » (JC), qui fait partie de la FBBH. Deuxièmement, la
classification des différents habitats sur « un gradient de biodiversité » montrent que la
diversité des abeilles sauvages est globalement plus haute dans l'OFBH, suggérant que la
permaculture promeut la diversité des abeilles sauvages mieux que les autres habitats. Les
Modèles Linéaires Généraux (GLMs) permettent de supposer que la diversité fonctionnelle
d'une communauté est négativement affectée par le % de terre arable dans une zone tampon
de 500 m autour d'un site, tandis que l'abondance des spécimens est négativement corrélée au
degré de perturbation d'un site. L'importance économique des insectes pollinisateurs a été
démontrée pour les trois récoltes étudiées.
Pour conclure, cette étude montre que les systèmes permaculturels comme la FBBH semblent
être une alternative adaptée pour l'intensification écologique de paysages agricoles. En effet,
ils contribuent au maintien durable de la biodiversité dans les systèmes agricoles, sans pour
autant limiter leur productivité, bien au contraire.
2
Maraîchage biologique permaculturel et performance économique, available at :
www.fermedubec.com/publications.aspx

5. iii
To my parents
« Il nous faudra répondre à notre véritable vocation, qui n'est pas de produire et de
consommer jusqu'à la fin de nos vies, mais d'aimer, d'admirer et de prendre soin de la vie
sous toutes ses formes. »
Pierre Rabhi
« We will have to meet our true vocation, which is not to produce and consume for the rest of
our lives, but to love, admire and care for life in all its forms. »
Pierre Rabhi

6. iv
ACKNOWLEDGMENTS
This project would not have been possible without the support of many people. Many thanks
to my adviser, Nicolas Vereecken, who managed to convey his passion for the bees, who
thoroughly trained us with inspiring workshops, spent a great amount of time identifying our
specimens, enriched our references with interesting articles and provide us with wise advice.
Also thanks to my advisor Marc Dufrêne for accepting to supervise this study, for his advice
and support.
I hereby address special thanks to Charles and Perrine Hervé-Gruyer who welcomed me at
their farm in the framework of my research programme; their generosity was unbounded, they
were always available to answer our questions, and we had very enriching and stimulating
discussions. A special thanks also the “Bec-Hellouin team”: the engineer Louise Gehin, in
charge of the research programmes, but first and foremost Louise was a charming colleague to
work with, an optimist and full of life woman and a great person; Teddy, the market gardener,
always ready to answer our questions and whose dynamism made my stay really enjoyable;
and Anne, Yvan, Hugo, Camille, Edith, Christine, Fred, the bros’ JP and JC, thank you for
welcoming and integrating me into your great team, for the nice evenings playing cards,
enjoying great music and having enriching talks.
Many thanks to Luc for putting up with me for almost 7 days a week during 5 months and for
letting me stay at his lovely thatched cottage; we were a great team and this study would have
been much more difficult without your support, help, advice and humour…
I also would like to thank the proof-readers of this report Mr. Jaloustre, Xavier and Mr.
Brook, who kindly accepted to correct my English mistakes; Timothy Weekers for showing
us Excel’s formulas in the data base; Hélène Hainaut for your help with QGIS; Lucie Perin
for sharing skills in making cross-tables; Julien Boedts and Jean-Marc Molenberg for the help
in bees’ digitization procedure; Mr. and Mrs. Lenfant and Mr and Mrs. Beuriot, who kindly
allowed us to investigate their colza field and apple orchard as sample sites; M. A. Pauly for
the identification of all these “mini-bees” that I know now are from the genus Lasioglossum;
and Geoffrey Caruso who patiently initiated us into the joys of QGIS.
Finally, I am grateful to my parents, my brothers and my friends who endured this long
process with me, always offering support and love. A special thanks to my grand-father, at
whose house I spent nice week-ends, eating delicious food, unwinding and recharging
batteries.

7. v
TABLE OF CONTENT
ACKNOWLEDGMENTS.........................................................................................................iv
TABLE OF CONTENT .............................................................................................................v
1. INTRODUCTION..............................................................................................................1
1.1. POLLINATION AND POLLINATING INSECT DIVERSITY : IMPORTANT
ECOSYSTEM SERVICES ........................................................................................................1
1.1.1. The ecosystem service of pollination...................................................................1
1.1.2. Plant-pollinators interaction and value.................................................................1
1.1.3. The role of bee species diversity..........................................................................4
1.2. CURRENT SITUATION: DECLINE AND DRIVERS................................................6
1.3. MEASURES AND POLICIES ......................................................................................9
1.4. PERMACULTURE AND DIVERSITY......................................................................11
1.4.1. General concepts of permaculture......................................................................11
1.4.2. The Organic Farm of the Bec-Hellouin (OFBH): a permaculture framework...14
1.5. COMMUNITY STRUCTURE AND DIVERSITY MEASURES IN THIS STUDY.15
1.5.1. Species diversity.................................................................................................15
1.5.2. Functional diversity............................................................................................ 16
1.5.3. Landscape influence on population’s diversity (Petel, 2015) ............................ 17
2. GOALS OF THE MASTER THESIS..............................................................................20
3. MATERIALS AND METHODS .....................................................................................22
3.1. DESCRIPTION OF THE STUDY AREA...................................................................22
3.2. POLLINATORS SMAPLING AND FIELD MEASURMENTS................................ 23
3.2.1. Sampling protocol .............................................................................................. 23
3.2.2. Strengths and weaknesses of the protocol.......................................................... 25
3.3. ENTOMOLOGICAL COLLECTION PREPARATION.............................................27
3.3.1. Conservation after capture..................................................................................27
3.3.2. Specimen preparation......................................................................................... 27
3.3.3. Pinning ...............................................................................................................27
3.3.4. Specimen identification......................................................................................28
3.4. DATA ANALYSIS ......................................................................................................28
3.4.1. Definition of diversity........................................................................................ 28
3.4.2. Species diversity.................................................................................................29
3.4.2.1. Sampling effort evaluation..........................................................................30
3.4.2.2. Species richness .......................................................................................... 31
3.4.3. Alpha diversity and associated indexes.............................................................. 31
3.4.3.1. Simpson’s index and classic Simpson’s index............................................31

8. vi
3.4.3.2. Shannon’s index.......................................................................................... 32
3.4.4. Piélou evenness index ........................................................................................ 32
3.4.5. Beta diversity and associated indexes ................................................................ 32
3.4.6. Species diversity comparison between sites and habitats ..................................33
3.4.7. Functional diversity............................................................................................ 33
3.4.7.1. Matrix of functional traits ...........................................................................34
3.4.7.2. Dissimilarity matrix calculation..................................................................34
3.4.7.3. Functional diversity indexes .......................................................................35
3.4.8. Seasonal diversity............................................................................................... 36
3.4.9. Verification of the spatial auto-correlation between sites..................................36
3.5. SPATIAL ANALYSIS: SITES CHARACTERISATION...........................................36
3.5.1. Local factors.......................................................................................................37
3.5.2. Landscape factors and landscape variables........................................................ 38
3.5.3. Selection of variables for the models’ creation..................................................39
3.5.4. Models selection.................................................................................................40
3.6. METHOD TO TEST THE ECONOMIC IMPORTANCE OF INSECT
POLLINATION .......................................................................................................................40
4. RESULTS......................................................................................................................... 42
4.1. SAMPLING RESULTS ............................................................................................... 42
4.1.1. Sampling effort: accumulation curves................................................................ 42
4.1.2. Repartition of the specimens between habitats ..................................................44
4.2. SPECIES DIVERSITY ................................................................................................ 47
4.2.1. Species richness and specimen abundance......................................................... 47
4.2.2. Alpha diversity ...................................................................................................50
4.2.3. Beta diversity......................................................................................................56
4.2.4. Seasonality .........................................................................................................58
4.3. MANTEL TEST...........................................................................................................58
4.4. FUNCTIONAL DIVERSITY ......................................................................................59
4.4.1. Functional richness............................................................................................. 59
4.4.2. Functional rarefaction......................................................................................... 61
4.5. SITES CHARACTERIZATION BY LOCAL AND LANDSCAPE VARIABLES ...63
4.5.1. Floristic characteristics of each site/habitat .......................................................63
4.5.2. Effects of landscape and local variables on community structure: GLMs.........63
4.6. YIELD COMPARISON WITH DIFFERENT POLLINATION TYPES....................65
5. DISCUSSION ..................................................................................................................66
5.1. SAMPLING EFFORT..................................................................................................66
5.2. COMPARISON OF THE DIVERSITY ACROSS SITES AND BETWEEN
HABITATS .............................................................................................................................. 67

9. vii
5.2.1. Diversity comparison across sites ......................................................................67
5.2.2. Diversity comparison between habitats.............................................................. 69
5.3. COMPARISON OF THE DIVERSITY AMONG THE SITES OF THE OFBH........72
5.4. ECONOMIC ANALYSIS............................................................................................ 74
6. CONCLUSION ................................................................................................................75
ACRONYMS & ABBREVIATIONS......................................................................................76
DEFINITIONS......................................................................................................................... 77
REFERENCES......................................................................................................................... 79
APPENDIX 1: SITES DESCRIPTION & CHARACTERISTICS..........................................92
APPENDIX 2: SAMPLING...................................................................................................105
APPENDIX 3: SAMPLING EFFORT & SPECIES RICHNESS..........................................111
APPENDIX 4: DIVERSITY ANALYSIS .............................................................................115
APPENDIX 5: LANDSCAPE ANALYSIS...........................................................................122
APPENDIX 6: PROTOCOL FOR THE ECONOMIC ANALYSIS .....................................129

10. 1
1. INTRODUCTION
1.1.POLLINATION AND POLLINATING INSECT DIVERSITY : IMPORTANT ECOSYSTEM SERVICES
1.1.1. The ecosystem service of pollination
In the second half of the twentieth century, humans have changed the structure of the world’s
ecosystems more rapidly than at any time in recorded human history, mainly to meet the
growing demands for food, fresh water, timber, fibre and fuel. The main changes have
affected land use, with a global expansion of croplands, pastures, plantations, and urban areas,
along with large increases in energy, water, and fertilizer consumption, accompanied by
considerable losses of biodiversity (DeFries et al., 2004; Foley et al., 2005). The scientific
world and society have raised awareness on the pervasive impacts of these changes – or
anthropocene (Crutzen, 2002) -- on humanity (G. C. Daily, 1997 Nature’s Services: Societal
Dependence on Natural Ecosystems; Millennium Ecosystem Assessment (MAE), 2005).
Consequently concerned citizens have put pressure on policy-makers and firms to take into
account the value of the world’s natural capital and ecosystems. Therefore, the relationship
between human welfare and environmental services or nature’s services was assessed and the
concept of ecosystem service has been accepted worldwide (Haines-Young & Potschin,
2009). This concept has become an important component of land management strategies,
hence the economic and social costs/benefits evaluation and the monetarization of each
ecosystem service are increasingly asked in our western societies seeking the rationalization
of our activities (Westman, 1977, Costanza, 1997; De Groot, 2002).
Ecosystem services are defined as the benefits that humans obtain from ecosystems
(Millennium Ecosystem Assessment, 2005). According to this definition, pollination is an
ecosystem function, which contributes to many ecosystem services that will be described later
(Gallai, 2009; Cardinale et al., 2012).
However, the Millennium Ecosystem Assessment (2005) evaluates the status of different
ecosystem services; according to the MAE, the regulating and supporting service of
pollination is jeopardized because ecosystem changes affect the distribution, abundance, and
effectiveness of pollinators. Several articles corroborate this statement (Corbet 1992;
Matheson et al., 1996; Allen-Wardell et al., 1998; Kearns et al., 1998; Kevan & Phillips
2001; Steffan-Dewenter et al., 2005,), while others are less alarming and put things into
perspective regarding wild pollinator conservation (Ghazoul, 2005; Kleijn, 2015). The causes
and consequences of the Anthropocene on the ecosystem service of pollination will be
described in paragraph 1.2. (Current situation: decline and drivers). The following paragraph
gives an overview of the main services offered by pollinators, especially wild bees.
1.1.2. Plant-pollinators interaction and value
Plant-pollinator relationships may be one of the most ecologically important classes of
animal-plant interaction: pollinators are involved in the sexual reproduction and therefore the
survival of ca. 87.5% of all flowering plants in temperate climates (Ollerton, et al., 2011;

11. 2
Vaissière, 2005), and they are also key to the sexual reproduction of 84% of European crops
(Williams, 1994; Klein, et al., 2007). These animal-pollinated plants generally provide pollen,
nectar and other rewards to many animal species, whose declines across large spatial scales is
expected to have knock-on effects on other species (Kearns et al., 1998).
Among animal pollinators, insects are the main actors. They include some coleopterans like
Nitidulidae on Magnolia, lepidoptera like Nymphalidae on Buddleja, and Diptera like
hoverflies, but the Hymenoptera, and more precisely the superfamily Apoidea Apiformes
(commonly called “bees”), are the most efficient pollinators. They have physical traits
designed to collect pollen: numerous “hairs” to which pollen grains adhere, and specialized
structures known as scopae or corbiculae for storing and transporting pollen on their legs
and/or abdomen. Moreover, a typical bee diet consist primarily of pollen and nectar; bees visit
flowers to feed, to rest, to look for mates and sometimes to find material with which to build a
nest (Pouvreau, 2004; Kremen, 2002; Vaissière, 2005).
The pollination of flowering plants, that is to say the transport of pollen from producing
anthers to receptor stigma, generally follows from floral visits. Pollinators act as pollen
carriers, but sometimes, only by shaking the flower, they allow pollen grains to get in contact
with the female organs in the flowers (Pouvreau, 2004).
In this study, the term “bee” will refer to any insect from the superfamily of Apoidea
Apiformes. This taxon is comprised of 7 families – 6 of which are found in Europe -- all of
them are solitary bees except for Apis mellifera (Linnaeus, 1758) which is part of the Apidea
family, as well as every specimen from the genus Bombus (Kirk & Howes, 2012) and some
species in the family Halictidae (genera Halictus and Lasioglossum) (Gibbs, 2012). By “wild
bee” we mean any non-domesticated species, that is to say all bees except Apis mellifera.
Indeed, this bee is commonly called honey bee, it is native to Europe, Asia and Africa, has a
distribution range that now encompasses virtually all regions of the world, and has been
exploited by man for many thousands years (Kirk and Howes, 2012).
Because bees are insects, they also have a substantial ecological role as staple food for
insectivores; by allowing insects to proliferate, we also allow lizards, slow worms, frogs,
newts and birds like partridges, larks, sparrows, wagtails, lapwings, and small mammals like
bats, shrews and hedgehogs to survive. Pests control is another vital ecological function that is
ensured by some insect species (Losey, 2006).
Today, the preservation of insect diversity and more precisely pollinators is not just a
philosophic or moral issue related to ecological conservation and diversity promotion. Indeed,
the ecosystem services of pollinating insect are numerous: insects do not only enable most of
the flowering plants to produce fruits and to set seed, but they also contribute to the genetic
flow between and within plant species, thus preventing the drawbacks of inbreeding
(Charlesworth, 1987; Dicenta et al., 2002; Jones & Bingham, 2010; Liu et al., 2013).

12. 3
In addition, many studies have estimated the economic importance of bees. Almost 150 crops,
representing 84% of European cultures, are directly dependent on the activity of pollinators
(Klein et al., 2007; Gallai et al., 2009).
Recently the monetary value of pollination services in the world was evaluated to be reaching
153 to 285 billion euros per year (Gallai et al., 2009). In Europe, the value of plant production
relying on pollinating insects is estimated to be 16.2 billion euros, of which between 2.3 and
5.3 billion euros worth was in France in 2010 (Gallai et al., 2009; MEEM3
, 2016) and 1,27
billion euros worth in the United Kingdom (Breeze et al., 2011). These figures show the
vulnerability of European productivity and food supply to ecosystem changes.
In France, the South of the country depends more on pollinators than northern regions, as
illustrated in Figure 1. In the Eure – the French department where this study took place -
pollination service worth was 32.1 billion euros in 2010 (red circle on the map, Figure 1).
Figure 1: Value of the pollination service for each department of France (2010) (source: MEEM, 2016)
The economic evaluation of pollination is based on the calculation of the difference of
monetary value between a fruit resulting from insect pollination and a fruit produced from
auto-fertilization. However the method was recently reviewed in an article assessing some
knowledge gaps which could influence the previous estimations (Hanley et al., 2014)
For example Garratt et al., (2014) showed that the number of seeds per apple for Cox and
Gala was significantly higher in open pollinated fruits than pollinator excluded fruits. Other
3
MEEM: Ministère de l’Environnement, de l’Energie et de la Mer.

13. 4
studies on strawberries showed that bee pollination could substantially increase the income
per seedling (Andersson et al., 2012; Wietzke et al., 2016). Furthermore, the benefit from
wild bees’ pollination in organic strawberry production, namely Osmia bicornis and Osmia
cornuta, was demonstrated last year in a study conducted in Brussels-Region. These results
show a possible alternative to the use of commercial Bombus like B. terrestris in agriculture
(Pont, 2015).
However, the previous monetary evaluation does not take into account the fact that bee
pollination can also improve other important aspects of crop production such as crop
nutritional qualities and shelf life (Klatt et al., 2014). Indeed, fruits produced from
entomophilous pollination can show higher contents in ashes and vitamins (Eilers et al.,
2011).
1.1.3. The role of bee species diversity
First of all, the great bee species richness contributes to maintain the flora richness: In
France, about 1 200 bee species have been recorded, while the planet counts about 20 000
different species (Ascher & Pickering, 2016), of which 85% are solitary bees (Pouvreau,
2004; O’Toole, 2012). That is each nest is the work of a single female working alone, and the
nectar collected is not converted into honey but only mixed with pollen and deposited in
brood cells to feed the larvae.
The fact that females of many species of solitary bees specialise in their source of pollen is
one of many aspects of the importance of preserving the diversity of bee species. Indeed,
those oligolectic species usually gather pollen on a particular family or genus of flowering
plants, and are more efficient compared to generalists’ activity on the same plant type
(Larsson, 2005). For example, Eucera (Apidae) visit only legumes (Fabaceae), the hyper-
specialised bee Andrena florea (Fabricius, 1793) (Andrenidae) collects pollen only on
Bryonia, the mining bee Colletes hederae (Schmidt & Westrich, 1993) (Colletidae) is only
active on ivy (Hedera helix), and the mining bee Andrena hattorfiana (Fabricius, 1775)
(Andrenidae) is specialised on some endangered species of Dipsacaceae (Larsson, 2005;
O’Toole, 2012). Likewise, the species Mellita dimidiate (Fabricius, 1976) (Mellitidae) and
Bombus ruderarius (Müller 1776) (Apidae) are specialised on the sainfoin (Onobrychis
species), a perennial legume which was largely grown as fodder when animals where still
used as draught power. Since the end of the second World War, the sainfoin have been
replaced by monoculture of alfalfa, sunflower and corn fields; besides both species
aforementioned are now protected species at a regional scale (Meriguet, 2004). However, the
sainfoin might experience a revival given its richness in tannins (interesting for naturally
fighting against gastrointestinal parasites in animal gut), its hardiness and not-bloating
properties and the nectar and pollen quality which made “Gâtinais” honey’s fame (Abeille de
France, 2013; Brinkhaus et al., 2015).
Secondly, recent studies showed that the number of different species interacting with a crop
could influence yields positively. According to Brosi & Briggs (2013), if one bee species is

14. 5
removed from an ecosystem, the fructification rate could decrease by up to 32%. Indeed,
different species have different foraging methods, some being more efficient than others.
Thus, it is not the abundance but the specific and functional diversity which determine yields
(Pywell et al., 2015). This conclusion was generalised at the world scale with a study
conducted on 600 farm plots, 41 cropping systems, annual and perennial, monocultures, and
with different densities of wild pollinators and Apis mellifera (Garibaldi et al., 2013).
Greenleaf & Kremen (2006) illustrate this conclusion, showing that sunflowers’ pollination
efficiency increases with the behavioural interactions based on the competition between wild
bees and honey bees and the abundance and species richness of wild bees. Likewise, Brittain
et al., (2013) showed that in almond orchards honey bees tend to transfer pollen between the
same varieties due to a behavioural fidelity, whereas wild bees allow crossings between
varieties. Yet some varieties are self-incompatible; therefore almonds’ yield increases when
wild bees pollinate along with honey bees.
In France, many agricultural productions depend on pollinating insects, especially fruit
species (apples, pears, all kind of berries…), arable crops (rape, sunflowers, field beans…)
and vegetable crops (tomatoes, melons, courgettes…) (Ministère de l’Ecologie du
Développement durable et de l’Energie & OPIE, 2015). Pollinating insects also have a
significant role in the production of seed crops in France, especially for hybrid oil seeds,
forage legumes (clover and alfalfa), most of garden crop seeds (carrots, cabbages, onions,
leaks, radishes…) and flower crop seeds (marigolds, wallflowers…). All in all, these seed
crops account for 2.5 to 3 million hectares of arable land, representing a turnover of 7 billion
euros in France (Plan National d’Action, 2016; from a data compilation issued from
FranceAgriMer, CTIFL and GNIS).
In addition, when the species richness is enhanced, the resilience of a system increases.
Indeed, the ecosystem function is covered by many species, which can interact and replace
one another, instead of being reliant upon one single pollinator species. Moreover, diseases,
pests and viruses recorded in honey bee hives are characteristics of intensive animal breeding
(Garibaldi et al., 2014).
Finally, it is crucial to maintain a sufficient diversity of pollinators; otherwise the
consequences could be irreversible and get only worse. Indeed, we saw that less diversity
would mean less productivity; to compensate this lack of productivity, more land would be
needed for crop production at the expense of natural areas, and thus biodiversity is likely to
continue to decline (Garibaldi et al., 2014). However, diversity protection should not compete
with food production given that we expect that demand for agricultural products could double
in the coming decades, putting enormous pressure on agriculture to produce more (Godfray et
al., 2010; UNPD, 2011). That is why, international conservation organisations should partner
with agricultural programmes in order to promote ‘land sparing’ or ‘land sharing’, depending
on the local context (Fischer et al., 2008; Baudron & Giller, 2014). Furthermore,
inappropriate policies for biodiversity conservation, such as pastures converted into forests,
can lead to a reverse trend: less bee species richness because of a confined environment
(Rollin, 2013).

15. 6
It should be noted that other pollinating insects also play a significant role for pollination
service (Orford et al., 2015; Rader et al., 2016). Indeed, non-bee pollinators have been
generally overlooked because of poor knowledge on their taxonomy, difficulties to follow
them (some flies and small wasps move quickly) or researchers’ choice not to collect them
based on the erroneous assumption that they were not relevant to pollination (Meier et al.,
2006; Pape et al., 2009). Nevertheless, it has been demonstrated that non-bee insects, despite
being less effective than bees per flower visit, perform more visits; thus both factors
compensated each other, resulting in these insects’ provision of a unique benefit regarding
fruit set (Rader et al., 2016). For instance, within the hoverfly family (Diptera: Syrphidae),
adults usually are generalist flower visitors. Furthermore, at least some hoverfly species are
able to use resources from highly disturbed habitats, including agricultural fields, making
their crop pollination service more robust to changes in land use (Winfree et al., 2011;
Raymond et al., 2014). For all these reasons, specimens from the Syrphidae family were also
collected but they were not taken into account in the dataset of this study because their
identification process is running. They shall be included in the next studies on the area.
1.2.CURRENT SITUATION: DECLINE AND DRIVERS
Which bees are declining?
In recent years the decline of bees has been largely reported in studies worldwide (Williams,
2005; Fitzpatrick et al., 2007; Colla et Packer, 2008; Patiny et al., 2009; Cameron et al., 2011;
Burkle et al., 2013; Goulson et al., 2008 and 2015; Nieto et al., 2014). In Europe, wild bees’
populations are significantly collapsing, particularly species specialised in their source of
pollen, species with a reduced geographical distribution and species with a slower life cycle
(Biesmeijer et al., 2006). For example Osmia dalmatica (Westrich, 1996) feeds exclusively
on Dipsacaceae and Bombus gerstaeckeri (Morawitz, 1881) is a particular species known for
its scarcity and oligolecty on species of the genus Aconitum (Ranunculaceae) (Michez et al.,
2013). According to Biesmeijer and his team (2006), more than 50% of the sites sampled in
Britain and the Netherlands saw a decrease in diversity and abundance of wild bees and their
functionally linked plants. Even the cuckoo bees seem to be affected although they are part of
the upper trophic level. On the other hand, Carvalheiro et al (2013) showed that in North-
West Europe, the massive species richness loss occurred before 1990, whereas such negative
trends have slowed down recently for several taxa, thanks to increasing public investment in
conservation.
The ecology and behaviours of more than 50% of European bees have not been studied.
Consequently there is not enough data available to activate protection status. Our study
contributes directly to fill this knowledge gap. Nevertheless, according to Nieto & colleagues
(2014), 9.2% of identified wild bee species are considered to be in danger.
The following part aims to report studies which seek to identify general trends in the response
of bees to human impacts. However, geographic patterns of bee decline and diversity are not
understood sufficiently well to ensure that such generalisations are valid because their

16. 7
datasets is often geographically limited, with the majority of data arising from North America
and Western Europe (Archer et al., 2014; Mayer et al., 2011). Yet a recent study conducted
by De Palma and colleagues (2016) showed that species’ responses to land-use changes vary
among regions. Therefore they suggest that geographically-restricted models might be
inadequate to support broad conclusions, and bee conservation strategies shouldn’t rely
indiscriminately on such models (De Palma et al., 2016).
Why are they declining?
One characteristic of insect populations is that they fluctuate widely from year to year
(Varley, 1970). However there is strong evidence that decline in bee populations has become
more severe since the Second World War (Biesmeijer et al., 2006; Burkle et al., 2013;
Carvalheiro et al., 2013; Ollerton, 2014). Scientific consensus is forming that there are
certainly several factors, depending on the species and local circumstances, working together
on this phenomenon, especially on interaction networks between pollinators and plants
(Burkle et al., 2013).
Climate change is frequently mentioned as a cause of bee decline, but it remains difficult to
quantify because this driver has direct and indirect effect on bees’ populations (Rasmont et
al., 2015). Increases in temperature can affect directly communities, while the environmental
changes have a snowball effect on flora and the wild bees with which they are associated
(Burkle et al., 2013). Indeed, when there are differences in the climatic niches of bees and
their host plants, future climate changes could lead to spatial mismatches between areas
suitable each one (Rasmont et al., 2015). However the tendency is not only towards a decline
but also towards a new mapping of the populations; e.g. some species became invasive
because of hotter temperatures in Europe, like Colletes hederae (Schmidt and Westrich, 1993)
which crossed the English Channel and is now colonising northern France and Belgium
(Dellicour et al., 2014).
Nieto and colleagues (2014) mentioned the following factors as main threats for bees:
Agricultural intensification changed the landscape dramatically after the Second
World War. Indeed, in order to meet food requirements, ‘improvements’ such as
hedgerow removal, drainage of wetland, ploughing of ancient pasture, moorland and
heathland, were made, heavily supported by European subsidies (Kirk & Howes,
2012; Biesmeijer et al., 2006; Burkle et al., 2013; Carvalheiro et al., 2013).
Furthermore the use of pesticides and chemical fertilisers along with the production
shift from ‘traditional’ fodder crops (despite that heir known to be equivalent if not
superior in quality compared to imported soybean cake (de Visser, 2014) towards
maize and soybean models, led to a decrease in the availability of forage for all bees, a
removal of nesting sites for many species and the lethal intoxication of bees with
insecticides such as pyrethroids, then replaced by neonicotinoids (Rollin, 2013;
Rundlöf et al., 2015; Cariveau et al., 2015; David et al., 2016).

17. 8
Thus, land use changes have resulted in a vicious circle: With the decline of bees,
farmers growing insect-pollinated crops shifted towards crops that do not need
pollinators, but which provide less foraging flowers, exacerbating the bees decline.
For wild flowers, fewer bees means plants setting less seed, which in turn triggers a
decline in wild flowers, leading to less forage for bees, thus aggravating bees losses
(Kirk & Howes, 2012).
Urbanisation is often seen as a competitor of bee conservation because urban sprawl
would rarefy nesting and foraging resources, fragment the habitat and modify the local
microclimate (Collins et al., 2000; McKinney, 2002; Johnson & Klemens, 2005;
Biesmeijer et al. 2006; Potts et al., 2010; Nieto et al., 2014; Wastian, 2016).
However, according to literature, the effects of urbanisation on biodiversity remain
unpredictable (McDonnel & Hahs, 2008 & 2013; McKinney, 2008). Indeed, for
several reasons (e.g. the diversity of ecological niches, hotter temperatures, alternative
habitats, etc), towns can be considered as biodiversity hotspots (Araujo, 2003; Hope et
al., 2003; Kühn et al., 2004). Nevertheless, specific richness seems to be declining
(McKinney, 2006). On the contrary, specimen abundance tends to increase, probably
because of the apparition of new species, specialists of urban areas. But other sources
imply that even bees abundance appears to be going down in towns (Hernandez et al.,
2009; Winfree et al., 2011).
Honey bees’ domestication has triggered many consequences on wild bee
communities. First of all, intensive breeding has reduced the resilience of hive
populations which have become more sensitive to pests and diseases. Among these,
the American and European foulbrood is a bacteria treated using antibiotics or the
destruction of infected colonies. The same goes for the parasitic mite (Acarapis
woodi), the gut parasite (Nosema apis), the new species (Nosema ceranae), and the
most serious one, a mite called varroa (Varroa destructor). All are responsible for
massive losses among honey bee colonies (Kirk & Howes, 2012; Rollin, 2013). Above
all, recent studies have shown that viruses and parasites are transmissible from
domesticated colonies to wild bees (Graystock et al., 2014; Fürst et al., 2014;
McMahon et al., 2015; Tehel et al., 2016).
Moreover, it was demonstrated that domesticated bees and bumblebee colonies are an
important source of competition for wild bee populations (Dupont. et al., 2004; Paini
& Roberts, 2004; Roubik & Villanueva-Guttiérez 2009; Shavit et al., 2009; Vereecken
et al., 2015). Indeed, a resources overlap was observed for honey bees and wild bees.
Yet, a hive contains about 30 000 individuals which need to gather huge quantities of
pollen and nectar to ensure the colony survival and meet the beekeepers’ and
consumers’ demand (Vereecken et al., 2015). To illustrate the competition impact,
Cane et al. (2016) published recently a study indicating that the resources consumed
by one single beehive could feed up to 330 000 wild bees. This phenomenon of
competition has a greater impact within poor sites such as urban areas or landscape of
monocultures (Vereecken et al., 2015).

18. 9
Figure 2 below summarises the relative importance of the different drivers for the decline of
bee populations.
Figure 2 Relative importance of drivers for wild bees decline in Europe (Nieto et al., 2014).
1.3.MEASURES AND POLICIES
Policy makers are starting to realise the importance of bees for societal well-being including
food security and ecosystem preservation. Therefore the conservation of bees has become an
issue of paramount importance (Garibaldi et al., 2014). That is why, in Europe, the project
Status and trend of European Pollinators (STEP, http://step-project.net/) was launched from
2010 to 2015 by Dr Simon Potts of Reading University (UK), in order to reveal the causes
and also the consequences of the decline in bee populations. The measures proposed aim at:
- Avoiding pesticides known for their negative impact on bees;
- Reducing the use of herbicides that eliminate flowering plants;
- Keeping the perennial flowered fallow lands;
- Integrating flowering crops among rotations (e.g. colza, lucerne, sainfoin, sunflower)
- Editing a red list which describes the evaluation criteria for the risk of European
species’ extinction (Nieto et al., 2014)4
The Common Agricultural Policy (CAP) of the European Union (EU) also includes new
mandatory (CAP pillar I) and incentivised (CAP pillar II) guidelines targeting pollinators,
such as the diversification of crops at the farm level, the development of wildlife-friendly
zones called ‘Ecological Focus Areas’ (EFA) accounting for 3.5 to 5% of farmland, and the
introduction of permanent meadows or pastures. These measures are part of the “Habitats
Fauna and Flora” directive (92/43/CEE), their application started in 2014 and should be
generalised in 2020. Farmers have different options to meet the EFA requirements: the
creation of buffer strips, leaving land fallow, planting catch crops, green cover, nitrogen-
4
One single taxon of bumblebee from Normandy is mentioned as vulnerable on this red list: Bombus muscorum
(Linnaeus, 1758) (Sagot & Mouquet, 2016).

19. 10
fixing crops, and/or hedges. Each option is an Agri-Environment Scheme (AES) developed as
part of the EU Rural Development Programmes (RDPs) which pays farmers to provide a
range of environmental services (Gadoum et al., 2007). However, Kleijn et al., (2006)
demonstrated that AES resulted in positive effects for only half of the studied species.
Nevertheless, very few studies focus on the efficiency of one specific AES. Instead, most
studies are conducted on the whole set of measures (Knop et al., 2006; Albrecht et al., 2007).
In a nutshell, the AES efficiency depends on the targeted species and the landscape context
(Henry et al., 2012). They were shown to be more efficient when the landscape is simple and
poorly diversified (Batary et al., 2011; Tscharntke et al., 2005).
One frequently used option is the establishment of sown wildflower strips on farmland, which
can in some cases promote bee abundance and species diversity (Carvell et al, 2004; Marshall
et al., 2006; Scheper et al., 2013). However, other studies have shown the limits of its
efficiency (Rollin, 2013; Uyttenbroeck et al., 2016). Indeed flower strips are often made of
annual plants, thus the composition of mixes is very dynamic, fluctuating from one year to the
next, and only generalist insects can cope with these interannual changes in resources
provided (Rollin, 2013). These insect are common species of bumblebees like B. pascuorum
and B. hortorum (Pywell et al., 2006; Carvell et al., 2007), whereas many oligolectic species,
such as Eucera nigrescens (Pérez, 1879), Trachusa byssina (Panzer, 1798), Megachile
ericetorum (Lepeletier, 1841) and Hoplitis tridentata (Dufour and Perris, 1840), directly rely
on legumes. Even some polylectic species rely on legumes, like Osmia aurulenta (Panzer,
1799), Andrena ovatula (Kirby, 1802) and Anthidium punctatum (Latreille, 1809) (Michez et
al., 2013). Another issue with flowered- strips is that after a few years the resources dwindle
and it is necessary to re-sow the seed mixes (Dicks et al., 2010).
Concerning the efficiency of grasslands’ extensive management - meaning late mowing,
pasture management instead of mowing for silage, limited number of cuttings and decrease in
the use of fertilisers and pesticides - different studies showed important variations in their
effect on bees’ abundance and diversity: On the one hand, Kohler et al. (2007) and Potts et al.
(2009) found no impact from these AES, while Albrecht et al. (2007) and Knop et al. (2006)
saw more abundance and diversity when meadows are under extensive management. On the
other hand, Kearns & Oliveras, (2009) saw that bees’ diversity and abundance was negatively
correlated to the stocking density, whereas Kruess & Tscharntke (2002) demonstrated that a
lower stocking density could enhance bees’ abundance but it had no effect on species’
diversity. To conclude, the impact of extensive practices regarding grasslands seems quite
random and closely linked to local conditions. For instance, Orford et al., (2016) showed that
a modest increase in plant diversity with legumes and forbs in conventional grasslands could
improve pollinator functional diversity, richness and abundance.
The decrease in the use of fertilisers and pesticides was shown to have very little influence on
wild bees’ diversity. Instead, they need to be completely eliminated before a significant
difference is observed (Goulson et al., 2002; Kleijn & Sutherland, 2003; Kleijn et al., 2001,
2006).

20. 11
In France, policy makers and politicians are also acknowledging the ecological and economic
value of pollinating insects. The Ministry of Ecology and Sustainable Development launched
the project of a National Action Plan (Plan National d’Action, PNA) for the conservation of
bees and wild pollinating insects (Plan National d’Action, 2016). The PNA is based on 5
action points:
- Decrease significantly the use of pesticides;
- Increase floral resources in green, farmed and natural areas
- Mobilise farmers, forest, natural areas and green space managers, and experts of the
environment;
- Reinforce scientific knowledge of the ecology and biology of wild pollinating insects;
- Raise awareness among a wider public and train actors from agricultural and
environmental professions.
The French government also participated in the European project Urbanbees through the
elaboration of a guide for the ecological management of urban areas in order to promote wild
bees and nature within towns (Coupey et al., 2014).
Finally, society and people in general are also becoming conscious of the importance of
preserving biodiversity. Indeed, the danger to which bees are exposed has been largely
mediatised. Unfortunately “bees’ decline” can be an argument for companies’ marketing
strategies with a view to generate lucrative business, by the sale of beehives and offering
apicultural services to firms and private individuals (personal remark). Yet, knowing the
competitive interactions between honey bees and wild bees, the species diversity of the latter
is then jeopardised, especially within poor and artificialized environments like urban areas
(Vereecken et al., 2015).
1.4.PERMACULTURE AND DIVERSITY
Within the context of this study, namely the trade-off between biodiversity conservation and
food production, permaculture seems to be a possible alternative. This is what we intend to
demonstrate in the following paragraph, through literature and one case study: the Organic
Farm of the Bec-Hellouin (OFBH), in France (Normandy), where the study was conducted.
1.4.1. General concepts of permaculture
In the past 50 years, the negative environmental and social impact of industrial agriculture,
urbanisation, and non-renewable resources extraction and depletion has given rise to great
concern among citizens and the scientific community. In response to these critics, different
movements and disciplines with a focus on sustainability have emerged, and permaculture is
one of them (De Steiguer 2006; Hawken 2007).
The concept of permaculture was first coined by the Australian David Holmgren, and then a
student and his professor Bill Mollison took it up in the late 70’s. The word “permaculture”
originally referred to permanent agriculture (King, 1911), but it was rapidly expanded to
permanent culture as it was understood that social perspectives were a key aspect to develop
truly sustainable systems (Smith, 2002; Mollison & Holmgren, 1978). However, this report

21. 12
will focus on those aspects of permaculture relevant to agriculture and biodiversity
conservation.
Bill Mollison gave the following definition of permaculture:
“[Permaculture] is the conscious design and maintenance of agriculturally productive
ecosystems which have the diversity, stability, and resilience of natural ecosystems. It
is the harmonious integration of landscape and people providing their food, energy,
shelter, and other material and non-material needs in a sustainable way” (Mollison,
1988).
In other words, permaculture philosophy could be summed up as follows; it consists of:
- Working with nature, using or drawing one’s inspiration from natural guilds;
- Observing before acting;
- Looking at systems in all their functions, rather than asking only one yield of them;
- Allowing systems to demonstrate their own evolutions by enabling self-organisation
and working with natural succession and biomimicry concepts (Mollison, 1988).
In brief, permaculture could be seen now as a worldwide-known agroecological movement,
with a unique approach to system design. However, unlike agroecology and despite general
interest, permaculture has limited representation in the scientific literature, and lacks a clear
definition, sometimes resulting in oversimplified claims (Fergusson & Lovell, 2013).
Permaculture is also considered as applied ecoliteracy (Orr, 1992). Indeed, permaculture
designs systems in such a way that they do not interfere with nature’s inherent ability to
sustain life. To do so, the first step is to understand ecosystems; and this understanding is
called ecological literacy or ecoliteracy (Capra, 1997).
In practice, permaculture is based on different design principles which are applicable in any
climate and at any scale. They are drawn from various disciplines: ecology, energy
conservation, landscape design and environmental science. However, practical techniques
within a principle should be closely linked to local conditions and culture (Mollison, 1988).
Table 1 summarises ten of those principles and their brief description is drawn from Bill
Mollison’s publication (1988).

22. 13
Table 1 Permaculture principles and their applications at the OFBH.
Permaculture
principles
Description Application examples at the OFBH
Relative location
of elements in the
system
Making connections between elements
through an appropriate design. Every
element is placed in relationship to another
so that they assist each other: the needs of
one element are fulfilled by the yields of
another one.
The gardens are located under the Bec
River’s level so that irrigation relies mostly
on gravity rather than on a pump.
Each element
performs many
functions
Each element in the system should be
chosen and placed so that it performs as
many functions as possible. Then the
system resilience is enhanced.
Ponds are used for irrigation, aquatic crops,
they represent habitats for functional
biodiversity and they buffer temperature
variations, thus creating a microclimate.
Each important
function is
supported by many
elements
Important basic needs such as water, food
and energy should be ensured in two or
more ways.
Food production relies on an incredible
variety of fruits and vegetables, including
grains and perennial wheat.
Efficient energy
planning
The system should be designed in zones
and sectors, according to local factors
(maintenance and monitoring intensity
required, market access, slope, local
climate, specific soil conditions, etc.)
A different zone is assigned to each site of
the farm mostly according to the level and
the intensity of management required
(bedding, seedling, unweeding, harvesting,
etc). See the zonal planning of the OFBH in
Appendix 1.
Using biological
resources
Plants and animals are used to provide
fuel, fertilisers, tillage, pests control,
nutrient recycling, habitat enhancement,
soil aeration, erosion control, etc.
The chickens’ scratching and digging
behaviours are used to keep the soil aerated.
Their eating habits contribute to control
pests (e.g. they eat slugs’ eggs).
Energy cycling Using incoming natural energies as well as
those generated on-site to ensure a
complete energy cycle and thus lessen
external inputs.
One of the OFBH project is to install a
turbine in the Bec-River in order to generate
electricity.
Small-scale
intensive systems
A permaculture system is turned for hand-
tools on small sites and modest fuel-users
on larger sites. Small-scale being the most
efficient.
Intensity is reached with plants stacking
and time stacking.
The farm makes a point not to be
mechanised; it is an efficient small-scale
intensive system thanks to hand-labour.
Accelerating
succession and
evolution
Using what is already growing,
introducing plants that are adapted to local
environment, raising organic levels
artificially (mulch, green manure, etc.).
One of the farm’s practices is the cultivation
on mounds. It is a way of accelerating
fertilisation by adding layers of horse
manure, compost, residues, etc.; being
partly recycled within the farm.
Edge effects An edge is the interface between two
mediums. A system with complex edges is
more productive: wherever a boundary
exists, resources from two different
ecologies can be used.
The OFBH is a combination of different
gardens which all have their specificity in
terms of management, ecology and design,
thus creating productive edges: e.g. the
“Island-Garden” surrounded by water,
protected by the food-forest.

23. 14
To conclude, instead of being energy- or capital-intensive, permaculture design is
imagination-intensive. But above all, it requires a multidisciplinary knowledge (experimental
as well as scientific) in order to think in terms of systems with inter-connexions between
elements, for the purpose of saving energy and obtaining yields from every particular niche
(Mollison, 1988).
1.4.2. The Organic Farm of the Bec-Hellouin (OFBH): a permaculture framework
In France, 30 % of farms are not inherited through family succession, and this number is
constantly increasing (Morel & Léger, 2016). The production and marketing methods used by
these new farmers appear to be contributing to a viable agro-ecological transition, the results
of which are supposed to provide more sustainability and resilience in the agricultural
landscape. Among them, 15 % are turning to vegetable production as this has the advantage
of requiring less land surface compared to other production such as crops, livestock or
viticulture. An increasing number of these “new generation” growers are opting for organic
practices, on very small scale farms, with high diversity and short marketing channels (Morel
& Léger, 2015). These micro-farm projects are being carried out by people who often have
strong environmental and social aspirations and who are inspired by practices little known in
the traditional French agricultural world, such as permaculture, bio-intensive gardening or
natural agriculture (Hervé-Gruyer, 2015). The economic viability of these atypical initiatives
is of great interest to both the project leaders, who often request references, and the
professionals who support those (Morel & Léger, 2016). The vivability could be defined here
as the capacity of market gardeners to generate a sufficient income to reward themselves
while maintaining an acceptable workload (Morel et al., 2015).
Perrine and Charles-Hervé Gruyer are two of those new farmers who decided to quit their
previous careers (she was an international lawyer and he was sailor) to create the Organic
Farm of the Bec-Hellouin (OFBH) back in 2004, in Haute-Normandie, France (49°13'24.9"N
0°43'42.5"E). Unlike its conventional neighbours, the farm took a different trajectory, to the
point that it is now a reference in alternative agricultural practices and attracts scientists and
visitors from around the world (Ecole de Permaculture du Bec-Hellouin (EPBH) et al., 2015).
It is not only a place of high productivity, but it also contributes to scientific researches and
education. Indeed, since 2011, the OFBH is one of the experimental sites for a research
program leaded by the National Institute for Agricultural Research (INRA) and
AgroParisTech. Moreover, renowned trainings in permaculture and market gardening now
take place in the eco-centre of the farm.
The farm extend across 6 hectare, of which about 4500 m² are cultivated, with more than 800
varieties of fruits and vegetables grown in its gardens, in complete respect of the environment.
This large diversity of plants are grown under permaculture principles and intensive organic
gardening techniques inspired from the experimentations of John Jeavons (1974) and Eliot
Coleman (1995) (EPBH et al., 2015). The production system is a diversified market
gardening system. Vegetables are produced organically and sold in baskets to consumers or to
local restaurants and cooperatives. Fruits can be transformed into cider, compotes, apple juice
and jams, which are partly sold at the farm’s shop.

24. 15
Finally, the OFBH’s farming system is an example of how ecological intensification can be
put into practice (De Liedekerke de Pailhe, 2014). Therefore, several research projects have
been conducted on the farm to examine natural and effective farming practices that contribute
to the regeneration of the biosphere (EPBH et al., 2015; De Liedekerke de Pailhe, 2014;
Morel et al., 2015; Morel & Léger, 2014).
The major strength of this system is the diversified production, resulting in a high level of
planned (cultivated fruits, vegetables and reared animals) and unplanned (wild flora and fauna
such as insects, birds, etc.) biodiversity. Therefore, it is very likely that the farm’s economic
performance showed in the last study (EPBH et al., 2015) could be explained by its ecological
performances, namely in terms of biodiversity conservation. Yet, as we explained above, wild
bees’ diversity is an interesting indicator of the state and health of a given environment.
Therefore, this report contributes directly to the evaluation of the ecological performance of
the farm. The following paragraph aims to explain the different aspects of diversity that this
study is referring to.
1.5.COMMUNITY STRUCTURE AND DIVERSITY MEASURES IN THIS STUDY
It is clear that biodiversity is closely linked to the functioning of ecosystems (e.g. Cardinale et
al., 2006). However, it remains to be specified what is exactly covered by this concept of
biodiversity. The following aspects of this notion will be analysed in this report.
1.5.1. Species diversity
First of all the term species should be defined. In ecology, a species is a collection of
individuals, whose phenotypic and behavioural traits determine where and when they can live
and how they interact with individuals from other species (McGill et al., 2006). Species were
also defined as a set of individuals potentially able to interbreed and create a viable and fertile
offspring, within natural conditions (Mayr, 1942).
According to Cardinale et al. (2002), interspecific facilitation between species - such as the
delivery of resources to other individuals through biophysical interactions - allows enhanced
ecosystem functioning. Likewise, Baselga (2010) demonstrated that species evenness within
an ecosystem had many impacts on the latter’s functioning, namely its resilience and stability
when facing climate change or invasions from exotic species, diseases and pests.
Consequently, species diversity - which considers both the number of different species
present in the community (species richness) and their relative abundance (species evenness) -
has become one of the most basic parameters to assess environmental conditions (Cairns et
al., 1993). Indeed, the possibility to summarise both sets of information in a single value
using a diversity index, is of significant appeal and usefulness to ecologists.
However, several recognised drawbacks of this routine diversity measure should be taken into
account (e.g. Hurlbert, 1971; Iknaya et al., 2014). First of all, empirical experiments do not
always show a relationship between diversity and environmental stress (Connell, 1978;

25. 16
Bagousse-Pinguet et al., 2014). Indeed, too many species within an ecosystem could
jeopardise the system’s stability, because of inter-specie competition. For now, no
scientifically relevant causes have been found to explain this fact (Loreau & Behera, 1999;
Steiner et al., 2005; Naeem, 2009). Secondly, species evenness is not always correlated to the
resilience or stability of an environment facing toxic stress (Kimbro & Groshold, 2006;
Wilsey & Stirling, 2007; Scrosati & Heaven, 2007). Finally and above all, one key issue is
that measures of species richness are limited by the inherent difficulty to detect the presence
of rare species.
Nevertheless, in order to mitigate those limits from interfering with the results’ robustness,
the functional diversity of each community structure was also evaluated.
1.5.2. Functional diversity
In order to understand better ecosystem functioning and assess more accurately their state,
evolution and health, different approaches could be considered. On the one hand, the
taxonomic approach appears to remain only descriptive, thus it does not allow comparing
mediums drastically different in their species composition (McGill et al., 2006). On the other
hand, the functional approach has been widely used since its development in the 1990’s
(Tilman et al., 1997; Mouillot 2013; Swenson 2014; Cadotte et al., 2011). It consists in
studying ecosystem functioning and levels of disturbance through their functional diversity
(Mouillot et al., 2013). It is based on the quantity, differentiation and diversity of functional
traits within a given natural community. In other words, the comparison of community
structure, according to species’ functional traits, is based on several principles (Alaerts,
2015):
- The use of species’ ecological/behavioural traits as independent variables;
- The measurement of those traits’ distribution among species;
- The study of the traits evolution and variation according to changes over time and
environmental parameters respectively.
A functional trait can consist in any morphological, biochemical, physiological,
organizational or behavioural characteristic expressed by an organisms’ phenotype (Goudard
et al., 2007; Cadotte et al., 2011; Naeem 2009). According to Shipley’s definition (2009),
traits are also “properties possessed by all (or most) [species] irrespective of geography or
taxonomy”. Therefore, “trait-based community ecology can be generalized” (Shipley, 2009).
For example, functional traits determine where a species can live (Lavorel et al., 1997), how
efficient it will be as a competitor or predator (Davies et al., 2007), or how it will store
nutrients (Hillebrand et al., 2008).
Thus, interests of the functional approach are numerous. Indeed, a community can rapidly
respond to environmental changes and natural selection will especially affect the individuals’
traits. Therefore, traits determine how species affect their environment and respond to the
latter (Naeem, 2009; Diaz et al., 2013).

26. 17
Furthermore, the Functional Diversity index can be directly related to an ecosystem service
or ecological function. For example, in their pioneering study, Tilman (1997) showed that
more functional diversity, as well as a specific functional composition, would allow the
production of more biomass in a given ecosystem. Nevertheless, in order to identify traits
linked to relevant ecological services or functions, great care should be taken to choose traits
connected to their environment (Violle et al., 2007).
In addition, functional diversity of animals predict ecosystem functioning better than species-
based indices (Garland & Carter, 1994; Gagic et al., 2015). This statement has also been
demonstrated within bees’ communities. Indeed, different studies have shown that the quality
and efficiency of pollination services were influenced by species’ functional traits and by the
diversity of those traits, allowing a functional complementarity (Williams et al., 2010; Fründ
et al., 2013; Martins et al., 2015). However, the stability of the system is ensured only when
functional traits are present at a sufficient level in order to maintain resilience (Fischer et al.,
2016)
Finally, the ultimate goal of the functional approach is to explore a group of organisms
diversified in terms of ecology by identifying some traits which make sense from a functional
point of view, that is to say that they must be related to ecosystem services or functions.
Nevertheless, the structure of communities is the result of biotic and abiotic filters, thus the
local community can be completely different from the regional one (LeRoy Poff, 1997;
Sydenham et al., 2015). For example, landscape parameters influence the persistence of
species characterized by particular traits (Martins et al., 2015). That is why the landscape
influence on community structure of bees will be analysed afterwards.
1.5.3. Landscape influence on population’s diversity (Petel, 2015)
The relative importance of habitat and landscape factors is still debated in the contemporary
literature (Goddard, 2010). However it was clearly demonstrated that landscape context had a
significant influence on biodiversity, ecosystem functioning and ecosystem services
(Rosenzweig, 1995; Hanski, 1999). Moreover, if a site presents a particular originality, it
might contribute more to the sectorial diversity since different species have different habitat
requirements (Blondel, 1980). The habitat quality might remain the main factor explaining
species presence or absence, but landscape context surrounding the site explains species
diversity too (Jonsen & Fahrig, 1997; Theobald, Miller, & Hobbs, 1997; Thies & Tscharntke,
1999; Hanski, 1999; Loreau et al., 2003; Burel et al., 2004).
Therefore, both spatial scales – local factors and landscape context - should be considered
when talking about factors affecting communities’ structure within a site (Kotliar & Wiens,
1990):
i. The habitat quality regarding species’ specific needs, defined by local factors: the size
and local conditions of the site (flora, nesting places, microclimate…). According to

27. 18
Hopfenmüller et al., (2014), the main local factors influencing bee populations’
richness and viability are the size and quality of the habitat. Indeed, it was
demonstrated that the site area is positively correlated to insect species richness
(MacArthur & Wilson, 1967). However, some studies showed that the habitat quality
could play a more prominent role than the size on those populations (Franzen &
Nilson, 2010).
For wild bees, the notion of habitat quality includes both the feeding and the nesting
resources quality, which are often species-specific (Westrich, 1996). Both types of
resources depend largely on the flora present on the site and on the permanence of
availability of these resources from year to year. On the one hand, plants offer pollen,
nectar, sometimes oil and perfumes to bees, but also resin, fibres and petals or pieces
of leaves necessary for building their nests (Simpson & Neff, 1981; Michener, 2007).
That is why a high floral diversity is essential given species specialization concerning
their foraging and nesting behaviours. On the other hand, floral resource abundance is
also a key element in order to answer wild bees’ protein needs (ensured by pollen
consumption) and carbohydrate needs (ensured by nectar collection). Depending on
the bees species and flora characteristics, sometimes from 7 000 up to 11 000 flowers
are required for the development of one single larva (Müller et al., 2006).
Regarding nesting sites, the requirements vary between species. The majority – about
70% - of solitary bees nest in the ground (Cane, 1991), but they may also nest in
hollow stems, in holes bored in wood, soft stone, the walls of houses or even empty
snail shells. Thus it is valuable to maintain microhabitats such as patches of bare soil,
preferably well drained and bathed in sunlight, namely for ground-nesting species
(Linsley, 1958). Bumblebees have a range of preferred nest sites, such as unmanaged
grasslands, the base of hedgerows and fields and woodland edges (Kirk & Howes,
2012). At the OFBH, some nests of Chelostoma florisomne were observed in hollow
stems constituting the thatched roof of some of the farm buildings (see illustrations in
Annex 1).
Considering the influence of those local factors on the structure and diversity of bee
populations, a permaculture system, moreover practicing organic gardening, appears
to provide a suitable habitat to promote pollinating insects for the following reasons:
- Habitat diversity increases nesting resources, in turn increasing the opportunity
for wild bees to colonise the area (Cane et al. 2006; Banaszak-Cibicka et al.,
2007). Furthermore, conditions for bees are enhanced by high proportions of
non-crop habitat in the landscape, while wasps are favoured by connecting
corridors, which are themselves reinforced by hedge density and organic fields
(Holzschuh et al., 2010);
- No agrochemicals are used in organic farming, yet they widely impact bees’
survival rate (Potts et al., 2010);

28. 19
- Microclimate creation (with ponds, hedges, windbreaks, etc.) help to buffer
temperatures, thus conditions are more suitable for wild bees which are
generally thermophiles (Collins et al., 2000);
- Many other practices such as hand weeding, no tillage, mulching, crop
diversity, Conservation Biological Control, cover crops and green manure
contribute to maintain the diversity of pollinators and they might even enhance
it (The Xerces Society for Invertebrate Conservation, 2009).
ii. Landscape factors influencing the structure of wild bee communities are the
landscape’s layout and composition as well as connectivity between habitats
(Hopfenmüller et al., 2014). Those landscape factors are quantified through landscape
indices also called landscape variables, which will be developed in Chapter 3
“Materials and Methods”. They are based on the characteristics of measurable patches
such as their surface area, their perimeter or their abundance. A patch is a homogenous
entity in the landscape which differs within its environment (Forman, 1995).
Landscape composition is determined by the different land-uses and their relative
abundance (Farina, 2000). According to different studies, this parameter affects
pollinator communities, especially those of wild solitary bees (Steffan-Dewenter et al.,
2002; Dauber et al., 2003; Arhné, 2009).
Landscape layout describes the spatial configuration of patches (Farina, 2000). A
more complex configuration with many different land-uses seems to impact positively
the richness of wild bee communities (Hopfenmüller, 2014).
Connectivity is the landscape’s ability to enhance or impede the movement of
organisms between habitats (Taylor et al., 1993). A few studies tested the effect of
connectivity on wild bee communities, but none of them detected significant influence
(e.g. Meneses et al., 2010; Jauker et al., 2013).
As explained above, permaculture practices allow a notable spatial heterogeneity, with
different land-uses and connectivity. In addition, the landscape surrounding the
different sites used in our study is fairly diversified and could have a significant
impact on our results. Therefore it is necessary to study the impact of landscape
factors on the diversity of wild bee communities. The results will be of particular
interest since no such scientific studies about the impact of permaculture on the
diversity of bees have been done before (Ferguson, 2014).

29. 20
2. GOALS OF THE MASTER THESIS
This study is part of the second section of the research project “Organic permaculture market
gardening and economic performances5
”, leaded by the research institute Sylva6
and the dual
research unit SAD-APT (Sciences Action Développement – Activités Produits Territoire)
comprising the French National Institute for Agronomic Research (INRA) and the University
AgroParisTech, at the OFBH (the Organic Farm of the Bec-Hellouin) and other experimental
farms within the French territory. The first objective of this research project was to
demonstrate the economic efficiency of vegetable micro-farms, conceived according to
permaculture principles. The first report’s conclusion showed the economic viability of such
farms, and the hypothesis was logically assumed that the economic success of the OFBH was
closely linked to its ecological performances, namely the local regeneration and upgrading of
a range of ecosystem services such as biodiversity. Therefore, the second section of the
research project seeks to go deeper in the understanding of the OFBH’s functioning so as to
explain how the economic performances are related to the system’s ecological health.
Furthermore, the ultimate objective being the spin-off of such micro-farms, it is thus
meaningful to have scientific assessments of the permaculture’s benefits for agro-ecosystems,
such as their biodiversity.
Yet, as stated in the introduction, the study of communities’ structure of wild bees within an
ecosystem appears to be a reliable indicator of its ecological health. Moreover, the importance
of wild bees’ role has been demonstrated both ecologically (feed for insectivores, auxiliaries,
maintenance of floral diversity, etc.) and economically (pollination service, yield and quality
improvements, etc.). Therefore, these pollinators are insects of interest which are well worth
discovering deeper.
Therefore, the global aim of this study was to characterize and quantify wild bees’
diversity, considering local factors and landscape influence, and to assess the economic
added-value of those pollinators for some crops of interest in market gardening.
Specifically, the following sub-objectives were considered in this study:
- Analyse both the species and functional diversity of wild pollinators within the farm,
at three different spatial scales (site, habitat and landscape) and evaluate the potential
of each site compared to one another. At the farm level, this study seek namely to
appraise the influence of the floral composition, the permaculture design and the
disturbance rate (soil preparation, harvests, etc.) of a site on its wild bee community.
- Assess the global performance of permaculture for bee conservation and enhancement,
by comparing the OFBH with different types of habitats (conventional fields of arable
5
Maraîchage biologique permaculturel et performance économique, available at :
www.fermedubec.com/publications.aspx
6
Research institute of the farm which leads different research programs within the farm, in partnership with
other organisms. These programs aim at promoting permaculture and expanding the concept of micro-farms.

30. 21
crops, semi-natural habitats and grasslands), in order to position the farm on a
“biodiversity gradient”.
- Evaluate the yield difference when comparing harvests issued from insect pollination
versus auto-fertilisation or wind-pollination. The crops of interest selected are fava
beans, courgettes and blackcurrants.
- From there, suggest arrangements and adjustments to favour the unplanned
biodiversity of wild pollinators all year long into the OFBH’s gardens.

31. 22
3. MATERIALS AND METHODS
This part of the report was inspired from two Master Theses conducted in 2015 in Belgium,
about bees’ diversity. Under the leadership of Professor N. Vereecken and after comparison
with other techniques, the same protocol was followed to sample bees and analyse results.
Species diversity and landscape influence analysis was drawn from T. Petel’s report (2015)
while functional diversity was drawn from R. Alaerts’ report (2005).
3.1.DESCRIPTION OF THE STUDY AREA
The study zone is located in the Eure, a department of 6040 km² in Normandy, which is a
North-West Region of France. According to the French official website of meteorology
www.meteofrance.com, the area is characterized by an oceanic climate with rather cold
winters, the mean temperatures being around 3°C to 4 °C in January, and fresh and wet
summers with mean temperatures around 16°C to 17 °C in July. Precipitation is relatively
abundant with 123 rainy days per year, being about 820 mm of rain per year.
The Organic Farm of the Bec-Hellouin (OFBH) is situated in the Risle Valley, which is listed
as a Natura 2000 site, according to the ‘Inventaire National du Patrimoine Naturel’ (INPN
2016). This designation indicates the protected statue of the valley’s wetlands because they
contribute significantly to the maintenance or restoration of a natural habitat type or of
threatened, rare or vulnerable species. These areas are called Sites of Community Importance
(SCI); they are defined by different criteria in the European Commission Habitats Directive
(92/43/EEC), which are detailed in Appendix 1 Sites characteristics and selection criteria.
In all, 10 sampling sites were selected, within 4 different habitats; Table 2 summarises their
characteristics, their description and the reasons for the selection of each site are detailed in
Appendix 1.

32. 23
Table 2 Habitats and sites name, description, location and area.
Habitat type Habitats description Site name
Site characteristics
Latitude
Longitude
Surface (m²)
The Organic
Farm of the
Bec-Hellouin
(OFBH)
(Hab1)
Situated in a valley
classified "Natura 2000",
that is to say a rather
preserved zone,
surrounded with meadows
and woodlands. The farm
is under organic practices
and permacultural
gardening.
1. Pommiers (Po)
49°13'18.66"N
0°43'51.43"E
650
2. Ile-Jardin (IJ)
49°13'19.90"N
0°43'51.95"E
430
3. Mandala (Ma)
49°13'20.43"N
0°43'49.26"E
585
4. Jardin-Clairière (JC)
49°13'18.13"N
0°43'52.68"E
1 000
Meadow
(Hab2)
Located in the same valley
as the OFBH, nearby the
farm.
5. Herbage-Pâturé (HP)
49°13'53.17"N
0°43'26.43"E
18 600
6. Herbage-Fauché (HF)
49°13'15.11"N
0°43'56.54"E
20 094
Arable crop
(Hab3)
Two cultivated areas
located on the Vièvre's
plateau, surrounded by
different types of land use
(meadows, arable crops,
wooded countryside).
7. Colza (Co)
49°13'24.56"N
0°43'42.61"E
20 700
8. Verger (Ve)
49°15'35.17"N
0°38'44.03"E
129 900
Wilderness
(Hab4)
Two uncultivated areas,
one is located in the same
valley as the OFBH while
the other one is farer on
the Vièvre's plateau.
9. Friche-Plateau (FP)
49°15'18.48"N
0°37'45.91"E
43 400
10. Friche-Vallée (FV)
49°14'43.07"N
0°37'20.24"E
3 700
3.2.POLLINATORS SMAPLING AND FIELD MEASURMENTS
3.2.1. Sampling protocol
In a few countries, some Hymenoptera are subject to statutes of protection, therefore
beforestarting any sampling activity, it was necessary to inquire if any capture and killing
authorisations were required (Bellmann, 1999). However, in France, no Hymenoptera are
subject to protection statutes (OPIE, 2012). In the Eure department only 4 insect species are
protected: Cerambyx cerdo (Linnaeus, 1758), Masculinea arion (Linnaeus, 1758),
Osmoderma eremita (Scopoli, 1763) and Coenagrion mercuriale (Charpentier, 1840) (INPN,
2016). These species were very unlikely to be trapped with our sampling methods, therefore
no authorisation was necessary.
A team of two master students worked on the study and the 10 sites were shared between
them. Each site was always sampled by both of them alternatively during one sampling day;
one student sampled in the morning or the afternoon and vice versa. Each site was sampled
twice a month, from the second fortnight of March until the end of June 2016, except for the
sites of the OFBH whose sampling started in the first fortnight of March. A sampling day

33. 24
started at 9 a.m. and ended around 5 p.m. This time range is known to be sufficient to cover
the whole community of wild bees. It was demonstrated that there was no significant
difference in wild bee diversity and abundance between pan-captures lasting 24 hours and
pan-captures only during office hours (Geroff et al., 2014; Gezon et al., 2015). It is important
to note that this capture intensity neither affects the regeneration of wild bee populations, nor
the abundance, diversity and composition of their functional groups (Gezon et al., 2015).
As far as possible, the sampling days were during the warmest and sunniest periods, since
bees are poikilothermic organisms, meaning that they are active only when temperatures
match their comfort zone (Willmer & Stone, 2004). The best conditions being “good weather”
with the highest daily temperature above 15°C, an overall cloudless sky and winds under 15
km/h (Willmer & Stone, 2004 ; Ahrne et al., 2009; Fortel et al., 2014). Table 23 in Appendix
2 details the meteorological conditions and gives general comments for each sampling day.
In order to maximise the sampling effort, two different sampling methods were chosen: One
active trapping method with a net, and one passive trapping method with coloured pans. The
coloured pans method is standardised for bee capture (Westphal et al., 2008). The pans are
small soup bowls 16 cm in diameter and 5 cm deep (reference BP137
), painted in yellow
(801802), blue (801806) or white (801801) with an ultraviolet rays reflecting paint from the
brand « Rocol TOP traceur de chantier ®» and half-filled with soapy water. The soap8
lowers
the surface tension of the water, causing the insects to sink directly to the bottom of the bowl
(Westphal et al., 2008). The soapy water should be colourless and odourless to avoid any
influence on traps’ attractiveness. The colour combination was chosen to maximise captures,
as different bee species and sexes are attracted by different colours (Heneberg & Bogusch,
2014; Westphal et al. 2008).
Within each site, the UV-bright pans were allocated to places judged as the most attractive
and complementing each other. Moreover, pans-trapping is known to be easily influenced by
the environment (Fortel et al., 2014; Roulston et al., 2007). Therefore, three triplets of three
pans in different colours were repeated within each site, being nine pans per site in total. Pans
within a triplet is spaced 3 to 5 meters apart to avoid interferences (Figure 3), and the space
between each triplet within a site was maximised as far as possible by laying them along a
diagonal (Droege, 2002; Geroff et al., 2014; LeBuhn et al., 2003).
7
http://www.peiffer.eu/bol-en-plastique-blanc
8
Liquid soap, Froggy ecological, vaisselle sensitive vitamin

34. 25
Figure 3 Disposition of a triplet of coloured pans in the 'Island-Garden' (site IJ) - 14th
March 2016
To complement the pan-trapping method, some captures were realised with a net. Indeed, the
pans are not efficient to capture some taxa (Cane et al., 2000; Fortel et al., 2014; Roulston et
al., 2007). Accordingly each site was trawled with a net for about one and a half hour in the
morning and again in the afternoon.
Specimens collected with a net were put into sample bottles of 60 mL, filled with a 70%
alcohol concentrated solution, until their pinning and identification in a laboratory with
binoculars.
3.2.2. Strengths and weaknesses of the protocol
Two complementary trapping methods should be used; one active and the other passive. Net-
trapping compensates most of the pan-trapping biases, but this method depends strongly on
the trapper’s skill, while pan-trapping efficiency depends on the user’s ability to assess the
ideal spot to place the bowls for an optimum capture (Cane et al., 2000). However bias with
the net capture was minimal since both students were rookies at the beginning of the study.
In addition, on the one hand, the passive sampling allows for continuous sampling over an
appreciable time lapse, but might require more logistics and be influenced by environmental
hazards like wind, heavy rain or animals (e.g. sheep, horses and donkey in the farm’s pasture).
On the other hand, the active sampling allows for observing, identifying and filtering the
captures, but it is more time-consuming and the efficiency is dependent on the sampler.
Many other capture techniques are used in entomology and the following are the most
common ones: the “malaise trap”, “glass barrier” and “nest trap” are passive methods, while
“pooter” and bare hands captures are active methods. They all have advantages and
drawbacks which are described in Appendix 2.2., Table 24 (Roth & Couturier, 1963,
Hosking, 1979; Westphal et al, 2008).
However, given the time, budgetary and logistic constraints, the combination of both chosen
methods are considered to be the most efficient in Europe. This statement was demonstrated
in a study conducted in different European habitats and biogeographical regions by Westphal

35. 26
et al (2008). The results of this study concerning the efficiency of different trapping methods
are shown in Figure 4. Pan trapping appears to be the most efficient method, followed by
transects with a net. The pan traps have the advantage of being easy to install, they give the
possibility to sample different sites at the same time, they can be combined with another
method, and they provide high capture rates in a short time lapse (Heneberg & Bogusch,
2014). The net is probably the best way to capture a large spectre of insects in different micro-
habitats (flowers, ground, nest, and even flying insects).
Nonetheless, some biases need to be discussed concerning the pan traps. On the one hand, it
was demonstrated that they could underestimate the abundance of wild bee species of larger
size (mainly Bombus ssp and Apis mellifera) and species from the Colletes genus. On the
other hand, these traps could overestimate populations from the Halictidae family (Grundel et
al., 2011; Roulston et al., 2007).
Furthermore, pan trapping efficiency decreases as the percentage of floral cover rises in a site.
Indeed, bees find flowers more attractive than coloured pans. This phenomenon triggers a
dilution effect on wild bees’ populations. Moreover, the relative attractiveness of the pan
colours can also be compromised by the main colour of the floral population of the site
(Grundel et al., 2011; Roulston et al., 2007).
Net captures are known to overestimate slower moving specimens, less dynamic and lower
flying. For example, the capture of insects visiting trees was much more difficult and less
efficient than that of those foraging on the herbaceous stratum. Finally, given the natural
human curiosity, the sampler was more prone to capture a new specimen from a different
species, which until then had not been captured, rather than another common specimen
already massively counted in his vials (personal observations).
TNP: trap nests with paper
TNR: Trap nest with reed internodes Nests
OP: Observation plots (OP),
ST: Standardized transect walks
VT: Variable transect walks,
PT: Pan traps Pans
Values are means ± SE.
Linear mixed-effects models: F5,87 = 90.30, P <
0.0001; N = 96 observations.
Figure 4 Numbers of detected bee species among the methods that were tested in semi-natural
habitats (Westphal et al., 2008).
Net

36. 27
3.3.ENTOMOLOGICAL COLLECTION PREPARATION
3.3.1. Conservation after capture
It was almost imperative to capture and kill the specimens in order to create a collection for
the following reasons:
- Wild bees iconography in the literature is rather poor;
- Species diversity is very high in some areas and not so well known (e.g. the
Mediterranean Region in France);
- Species identification requires a binocular examination, whereas field tests permit the
identification of only the (sub-)genus;
Furthermore, the capture of bees aimed to:
- Generate a reliable, scientifically robust dataset;
- Obtain an inventory of the local and regional diversity;
- Create an entomological collection of reference which could be used for research,
teaching and natural heritage assessment;
- Improve our knowledge on species and our strategies for bee conservation.
After capture, the specimens were transferred into vials filled with a solution of 70% ethyl
alcohol. It keeps them moist and ensures a rapid death. However, alcohol conservation can
degrade the bees’ hair; therefore a rinsing step was necessary before pinning.
3.3.2. Specimen preparation
For the specimen’s preparation, a part of the protocol of Mouret and his team was followed,
inspired by a protocol developed at the USDA Bee Biology and Systematics Laboratory
(Mouret et al., 2007; LeBuhn et al., 2003). The bees and hover flies were removed from the
alcohol and placed in a fine meshed strainer, amply watered with tap water in a wash bottle to
rinse the alcohol and then carefully dried with paper towel. When the insect was completely
dry, namely the hair and wings, the pinning began.
3.3.3. Pinning
For the pinning, an entomologic pin (n°1, Ento Sphinx®) was planted in the middle of the
thorax, perpendicular to the body axis. The pin’s tip was then pushed into a soft holder, such
as polystyrene. The insect was positioned between the upper two-thirds of the pin while the
labels were in the lower third. This method is described in the protocol of LeBuhn et al.
(2003).
In order to ease the identification process, the insects were prepared so as to expose every
useful part of the body:
- The wings were placed in a delta form, as far as possible, to be able to see the wings’
cells;
- The legs have to be well apparent, especially the hind legs;

37. 28
- The tergites and sternites;
- The antennas to be able to count each segment to determine the specimen’s gender;
- The male genitalia;
- The hair aspect and colour;
- The mandibles have to be opened and the tong rolled out to ease some genus
identification.
3.3.4. Specimen identification
Most of the time, in order to identify the species from the subclade Aculeata of the
Hymenoptera order, it is necessary to kill, prepare and pin the insects. Indeed, some
identification criteria are microscopic and require binocular observation, such as the small
holes on the surface of some segments, the form of the eye-spots, or the length of the
segments of the antennas (Bellmann, 1999).
Currently, there is no recent publication concerning the whole Aculeata subclade (Bellmann,
1999). Thus diverse references were used for the identifications: The “Key of the genera of
British bees, Graham A. Collins” was used to identify the genus and the keys listed in
Appendix 2.3 were referred to, in order to determine the species.
Species determination was mostly done by Professor Nicolas Vereecken.At the beginning of
the study, a few specimens were identified by us at the OFBH, but our lack of time and
experience in that field were important impediments to the progress of the study. Finally, only
the most common specimens were determined by us: some species from the Bombus genus,
Apis mellifera (Linnaeus, 1758), Andrena cineraria (Linnaeus, 1758), Andrena fulva
(Warncke et al., 1974) and Osmia cornuta (Latreille, 1805).
Once identified, three labels were placed at the bottom of the pin. Essential information about
the place where the insect was captured, its geographic coordinates, the capture’s date, the
trapping method, the name of the sampler, the identification number (ID) of the insect and its
genus and species names was written on each label (Appendix 2.4).
At the same time, a database was constituted in Excel®, listing all the information above and
more, such as the type of habitat. This database was analysed at a later stage, when all the data
set was encoded.
3.4.DATA ANALYSIS
For each index and parameter, equations and calculation details are given in Appendix 4.1.
3.4.1. Definition of diversity
The analysis of the structure of wild bee communities is based on the analysis of the diversity
within and between populations. First of all, it is wise to define these terms: