Apis mellifera ruttneri, a new honey bee subspecies from Malta

Abstract

Endemic honey bees of the island nation of Malta are described as a distinct geographic race, Apis mellifera ruttneri, based on discriminant morphological analysis. Mitochondrial DNA and behavioral characteristics support a closer relationship of A m ruttneri to A m intermissa of North Africa than to European subspecies, similar to the situation with endemic island honey bees of Sicily (A m sicula). These findings suggest a shared evolutionary history among bees populating the islands of the central Mediterranean region. Recent importations of non-native honey bee subspecies present a clear threat to conservation of this unique honey bee of limited distribution. The subspecies is named after Professor Friedrich Ruttner, who has contributed so much to the understanding of intra-specific taxonomy in the honey bee.

Full text

Original
article
Apis
mellifera
ruttneri,
a
new
honey
bee
subspecies
from
Malta
WS
Sheppard
MC
Arias
A
Grech
MD
Meixner
1
1
Department
of Entomology,
166
FSHN
Building,
Washington
State
University,
Pullman,
WA
99164-6382,
USA;
2
Department
of Biology,
University
of São
Paulo,
São
Paulo,
SP,
Brasil;
3
Xehda
Ghasel,
Triq
il-Ferrovija,
Attard,
BZN
02,
Malta;
4
Institut für
Bienenkunde,
2
Karl
von
Frisch
Weg,
61440
Oberursel,
Germany
(Received
17
July
1997;
accepted
6
August
1997)
Summary —
Endemic
honey
bees
of
the
island
nation
of
Malta
are
described
as
a
distinct
geo-
graphic
race,
Apis
mellifera
ruttneri,
based
on
discriminant
morphological
analysis.
Mitochondrial
DNA
and
behavioral
characteristics
support
a
closer
relationship
of A m
ruttneri
to
A
m
intermissa
of
North
Africa
than
to
European
subspecies,
similar to
the
situation
with
endemic
island
honey
bees
of
Sicily
(A
m
sicula).
These
findings
suggest
a
shared
evolutionary
history
among
bees
populating
the
islands
of the
central
Mediterranean
region.
Recent
importations
of
non-native
honey
bee
subspecies
present
a
clear
threat to
conservation
of this
unique
honey
bee
of limited
distribution.
The
subspecies
is
named
after
Professor
Friedrich
Ruttner,
who
has
contributed
so
much
to
the
understanding
of
intra-specific
taxonomy
in
the
honey
bee.
Apis
mellifera
ruttneri
/
taxonomy
/
subspecies
/
Malta
/
evolution
INTRODUCTION
Within
the
endemic
range
of
the
honey
bee,
Apis
mellifera
L,
behavioral
and
morpho-
logical
variation
provides
the basis
for
sub-
specific
classification.
Phylogenetic
rela-
tionships
based
on
this
classification
have
been
used
to
form
hypotheses
about
the
ori-
gins
and
subsequent
dispersion
of
particular
races
or
groups
(Ruttner,
1988).
In
some
cases,
alternative
hypotheses
have
been
pro-
posed
based
on
molecular
data
(Cornuet
and
Garnery,
1991;
Garnery
et
al,
1992).
How-
ever,
one
interesting
commonality
has
been
the
consistency
with
which
molecular
and
morphological
data
support
a
phylogeo-
*
Correspondence
and
reprints
Tel: (1) 509
335
5180;
fax: (1) 509 335
1009; e-mail:
shepp@mail.wsu.edu

graphic
association
of
honey
bee
subspecies
from
Mediterranean
island
sources
to
a
sub-
species
of
northern
Africa,
A
m
intermissa,
or
perhaps
to
Iberian
honey
bees
with
African
origins
(Badino
et
al,
1985;
Gar-
nery
et
al,
1993;
Arias
and
Sheppard,
1996).
Morphometric
studies
demonstrate
clear
distinctiveness
of several island
subspecies,
including
A m
sicula
of
Sicily
and
A m
adami
of
Crete,
and
suggest
that
isolation
and
reduced
gene
flow
was
followed
by
insular
adaptation
and
differentiation.
Fur-
ther,
fossil
evidence
supports
a
shared
evo-
lutionary
history
for the
mammalian
fauna
of
Sicily
and
Malta,
predating
the
Pleistocene.
On
the
basis
of
these
factors,
we
investi-
gated
the
honey
bees
of
Malta
compared
to
their
closest
neighboring
subspecies
of
Africa,
Sicily
and
Italy.
MATERIAL
AND
METHODS
Samples
of adult
honey
bees
were
collected
from
15
colonies
in
eight
apiaries
throughout
Malta
and
preserved
in
liquid
nitrogen.
A
subset
of
each
sample
was
transferred
to
70%
ethanol
for
morphometric
analysis.
Fifteen
worker
bees
per
sample
were
dissected
and
measured
for
39
mor-
phometric
characters
according
to
Ruttner
et
al
(1978).
Wing
venation
angles
and
characters
of
size
(except
length
of
the
tergites)
were
mea-
sured
with
a
CCD
camera
and
an
on-screen
mea-
suring
system
(Meixner,
1994).
Length
of
ter-
gites
and
characters
of
pigmentation
and
pilosity
were
measured
with
a
microscope
and
an
ocu-
lar
micrometer.
The
statistical
analysis
of
the
data
was
performed
with
SPSS/PC
(1988),
using
the
programs
for
cluster
analysis
and
discriminant
analysis.
All
characters
measured
were
included
in
the
analyses.
Reference
colony
samples
of A
m
carnica
(31),
ligustica
(25),
adami
(24),
inter-
missa
(11)
and
sicula
(17)
from
the
collection
of
the
Institut
für
Bienenkunde,
Oberursel,
were
included
in
the
statistical
analyses.
Characterization
of
mitochondrial
DNA
in
the
Maltese
samples
was
based
on
the
fragment
profile
produced
by
EcoR1
restriction
enzyme
digestion.
Extraction
and
non-Southern
blot
visu-
alization
methods
were
published
previously
(Sheppard
and
McPheron,
1990;
Schiff
et
al,
1994).
Voucher
specimens
of A
m
ruttneri
were
deposited
at
the
MT
James
Museum,
Washington
State
University
and
the
Institut
für
Bienenkunde,
Oberursel.
RESULTS
A
cluster
analysis
of
the
morphological
data,
including
the
Malta
bees
and
all
the
refer-
ence
samples,
identified
six
different
groups
that
could
be
confirmed
by
discriminant
analysis.
In
figure 1,
discriminant
factor
1
(accounting
for
45%
of
the
variation)
is
plot-
ted
against
factor
2
(30%).
The
bees
of
Crete,
and
those
of
Italian/Carniolan
origin
are
clearly
very
distinct
from
the
bees
of
North
Africa,
Sicily
and
Malta,
which
clus-
ter
closer
together.
While
this
figure
shows
strong
relations
of
the
Maltese
bees
to
the
A
m
sicula
/A
m
intermissa
group,
it
does
not
provide
much
resolution
among
these
races.
However,
’contamination’
of
the
Malta
sam-
ples
with
bees
imported
from
Italy
can
be
ruled
out
by
these
results.
A
discriminant
analysis
including
only
the
Maltese
honey
bees,
A
m
sicula,
A
m
intermissa
and
A
m
ligustica
provides
better
resolution
of
this
cluster
(fig
2).
Samples
of
A
m
ligustica
were
included
in
this
analy-
sis
to
further
confirm
that
the
samples
col-
lected
in
Malta
are
distinct
from
this
group.
Here,
too,
factor
1 (67%
of
the
variance)
is
plotted
against
factor
2
(27%).
Figure
3
shows
a
detailed
analysis
of A m
intermissa,
A
m
sicula
and
the
honey
bees
from
Malta.
Factor
1 (78%
of
the
variance)
is
plotted
against
factor
2
(22%).
The
char-
acters
contributing
most
to
the
discrimina-
tion
of
these
races
are
’dark
stripe
on
ter-
gum
4’,
length
of
forewing,
width
of
wax
mirrors,
length
and
width
of
metatarsus
and
three
characters
of
wing
venation.
Analy-
sis
of
mtDNA
with
EcoR
1 restriction
enzyme
revealed
that
the
haplotypes
of Mal-
tese
bees
were
typical
of
those
found
in
both
A
m
sicula
and
A
m
intermissa
(Smith
et
al,
1991;
WSS,
unpublished
data).

Description
of
Maltese
bees
The
honey
bees
of
Malta
are
similar
in
size
to
A
m
sicula
and
A
m
intermissa,
but
have
shorter
legs
and
wings;
the
wings
are
also
much
narrower.
The
abdomen
of
the
bees
found
in
Malta
is
considerably
wider
(ster-
num
index
[SI]
=
77.74)
than
in
its
relative,
A m
intermissa
(81.52).
Thus,
A
m
ruttneri
is
the
’broadest’
subspecies
of
A
mellifera,
even
broader
than
A
m
mellifera
(SI
=
78.61)
(Ruttner
1992).
While
the
pigmentation
of
the
bees
does
not
differ
much
from
A
m
intermissa
or
A m
sicula,
they
have
consid-
erably
longer
hair
on
the
abdomen.
Whereas
the
cubital
distances
of A m
ruttneri
differ
greatly
from
A
m
sicula
and
A
m
intermissa,
the
cubital
index
is
similar
to
A
m
sicula,
but
different
from
A
m
intermissa.
Table
I
shows
the
means
and
standard
deviations
for
some
characters
for
A
m
ligustica,
A m
sicula,
A
m
intermissa
and
the
Maltese
sam-
ples.
DISCUSSION
Morphometric
analysis
of
the
honey
bees
of
Malta
supports
their
classification
as
a
distinct
subspecies.
They
are
clearly
differ-
entiated
from
the
closest
neighboring
sub-
species
A
m
intermissa,
A
m
sicula
and
A m
ligustica.
We
propose
to
name
the
sub-
species
Apis
mellifera
ruttneri
in
honor
of
Professor
Friedrich
Ruttner,
who
has
con-
tributed
so
much
to
the
science
of
honey
bee
taxonomy.
The
mitochondrial
haplotype
found
in
Maltese
honey
bees
is
typical
of
African
and
Sicilian
subspecies.
Previous
studies
demonstrated
a
phylogenetic
link
between
A
m
sicula
and
African
honey
bees
(Badino

et
al,
1985;
Garnery
et
al,
1993).
The
shared
mitochondrial
heritage
between
A m
sicula
and
A m
ruttneri
probably
reflects
common
elements
in
the
origins
of
these
subspecies
from
the
African
lineage.
The
present
day
occurrence
of
honey
bee
populations
with
African
mtDNA
lineages
in
Iberia
(Smith
et
al,
1991)
and
the
Mediterranean
islands
must
also
be
interpreted
in
light
of
exten-
sive
refugia
formation
and
bottlenecks
that
occurred
repeatedly
during
the
eight
glacial-interglacial
cycles
known
to
have
occurred
in
Europe
during
the
past
800
000
years
(Kukla,
1977).
Fossil
remains
of
mammals
of
African
origin,
such
as
hippopotamus
and
elephant,
are
known
from
Pleistocene
formations
in
both
Sicily
and
Malta
(Potts
and
Behrens-
meyer,
1992;
Zammit-Maempel,
1985).
Extensive
adaptations
for
insular
conditions
in
these
animals,
such
as
an
overall
reduction
in
size
to
’pygmy’
status,
suggest
that
their
occupation
of
Sicily
and
Malta
was
of
con-
siderable
duration,
predating
the
Pleistocene.
Whether
the
movement
of
honey
bees
of
the
’African’
branch
to
Iberia
and
the
Mediterranean
islands
reflects
dispersal
pro-
cesses
similar
to
those
of
African
mammals
remains
unanswered.
Preliminary
evidence
drawn
from
mitochondrial
DNA
supports
a
recent
(Pleistocene)
origin
for
honey
bee
subspecies
(Cornuet
and
Garnery,
1991;
Arias
and
Sheppard,
1996),
as
hypothesized
by
Ruttner
(1988).
Resolution
of
the
issue
should
be
improved
through
calibration
of
the
rates
of
sequence
divergence
from
addi-
tional
molecules.
Behaviorally,
A m
ruttneri
appears
well-
adapted
for
the
seasonally
xeric
conditions
of
Malta.
They
are
able
to
find
food
and
water
during
the
dry
period
from
April
through
September
and
normally
rear
brood
throughout
the
year
(AG,
unpublished
data).
Other
characteristics
of A m
ruttneri
sup-
port
the
close
association
with
A
m
inter-
missa
and
A
m
sicula,
including
the
pro-

duction
of
large
numbers
of
queen
cells
prior
to
swarming
(up
to
80).
Maltese
bees
also
show
an
ability
to
defend
against
local
predacious
wasps,
a
behavior
reported
for
A m
sicula
(Ruttner,
1992).
This
behavior
is
not
found
in
Italian
stocks
imported
to
Malta
(AG,
unpublished
data).
Maltese
bees
exhibit
increased
defensiveness
during
peri-
ods
of
low
humidity
and
absconding
behav-
ior
during
prolonged
dearth
periods.
Although
the
origin
of A
mellifera
is
likely
to
be
further
east,
Ruttner
(1988)
con-
siders
the
Mediterranean
region
to
be
the
center
of
diversity
for
the
species
based
on
the
large
number
of
subspecies
in
the
cir-
cum-Mediterranean
region.
The
distinct
insular
honey
bees of
Sicily,
Crete,
Cyprus
and
Malta
appear
to
support
this
contention.
Unfortunately,
the
potential
for
loss
of
this
germplasm
is
high,
given
the
limited
geo-
graphic
ranges
of
these
subspecies,
the
loss
of
feral
populations
to
introduced
parasitic
mites,
and
the
importation
of
non-native
stocks.
Further
analysis
of
variation
in
the
honey
bee
using
molecular
genetic
approaches
is
crucial
to
establish
the
extent
of
the
species
diversity
and
to
identify
endemic
populations
that
most
critically
require
attention
for
conservation.
ACKNOWLEDGMENTS
We
are
indebted
to
numerous
beekeepers
of
Malta
for
allowing
us
to
sample
their
colonies.
Agnes
Mohr
assisted
in
morphometric
analysis.
We
thank
the
Ministry
of
Food,
Agriculture
and
Fisheries
of
Malta
for
providing
logistical
support
for
collecting
activities.
Résumé —
Apis
mellifera
ruttneri,
une
nouvelle
race
d’abeille
mellifère
à
Malte.
Les
abeilles
endémiques
de
l’île
de
Malte
sont
décrites
comme
une
race
géographique
distincte,
A
m
ruttueri,
sur
la
base
d’une
analyse
morphologique
discriminante.
Les
abeilles
de
Malte
sont
semblables
en
taille
à
A
m
sicula
et
à
A
m
intermissa,
mais
pos-
sèdent
des
pattes
et
des
ailes
plus
courtes ;
les
ailes
sont
également
beaucoup
plus
étroites.
L’abdomen
des
abeilles
trouvées
à
Malte
est
beaucoup
plus
large
(index
du
sternum
(SI)
=
77,74)
que
celui
de
la
race
apparentée
A
m
intermissa
(SI
=
81,52).
Ainsi
A
m
ruttneri
est
la
sous-espèce
d’A
mellifera
la
plus
large ;
elle
est
même
plus
large
que
A m
mellifera
(SI
=
78,61)
(Ruttner,
1992).
Le
tableau
I
donne
les
moyennes
et
les
déviations
standard
de
cer-
tains
caractères
pour
A
m
ligustica,
A
m
sicula,
A
m
intermissa
et
pour
les
échan-
tillons
maltais.
L’haplotype
mitochondrial
trouvé
chez
l’abeille
maltaise
est
typique
des
sous-espèces
africaine
et
sicilienne.
Les
caractéristiques
de
l’ADN
et
celles
du
com-
portement
plaident
en
faveur
d’une
relation
plus
étroite
d’A
m
ruttneri
avec
A m
inter-
missa
d’Afrique
du
Nord
qu’avec
les
sous-
espèces
européennes,
situation
semblable
à
celle
des
abeilles
endémiques
de
Sicile
(A
m
sicula).
Ces
résultats
suggèrent
que
les
abeilles
qui
ont
peuplé
les
îles
de
la
Médi-
terranée
centrale
ont
connu
un
développe-
ment
commun
au
cours
de
l’évolution.
La
sous-espèce
est
nommée
ruttneri
en
hom-
mage
au
professeur
F
Ruttner
qui
a
tant
contribué
à
la
connaissance
de
la
taxinomie
infraspécifique
de
l’abeille
mellifère.
Mal-
heureusement,
en
raison
de
l’étendue
res-
treinte
de
la
race,
de
la
perte
causée
aux
populations
locales
par
l’acarien
Varroa
jacobsoni
et
de
l’importation
de
souches
de
races
étrangères,
la
situation
de
cette
race
est
en
grand
danger.
La
probabilité
de
la
voir
disparaitre
est
très
élevée.
Apis
mellifera
ruttneri
/
taxonomie
/
race
/
Malte
/
évolution
Zusammenfassung
— Apis
mellifera
rutt-
neri,
eine
neue
Honigbienenrasse
von
Malta.
Endemische
Honigbienen
der
Insel
Malta
werden
auf
Grund
der
morphologi-
schen
Diskriminanzanalyse
als
eine
eigene

geographische
Rasse,
A m
ruttneri,
beschrie-
ben.
Ihre
Größe
ist
der der
A m
sicula
und
A
m
intermissa
ähnlich,
sie
haben
aber
kür-
zere
Beine
und
Flügel;
außerdem
sind
die
Flügel
viel
schmaler.
Der
Hinterleib
der
Bie-
nen
aus
Malta
ist
deutlich
breiter
(Sternum
Index
(SI)
=
77,74)
als
bei
der
verwandten
Unterart
A
m
intermissa
(SI
=
81,52).
A
m
ruttneri
ist
demnach
die
breiteste
Rasse
von
A
mellifera,
sogar
noch
breiter
als
A
m
mel-
lifera
(SI
=78,61)
(Ruttner
1992).
Tabelle
I
zeigt
die
Mittelwerte
und
Standardabwei-
chungen
einiger
Merkmale
von
A
m
ligu-
stica,
A
m
sicula,
A
m
intermissa
und
den
maltesischen
Proben.
Der
Haplotyp
der
Mit-
ochondrien
der
Malta-Bienen
ist
typisch
für
die
afrikanischen
und
sizilianischen
Ras-
sen.
DNA
und
Verhaltensmerkmale
spre-
chen
für
eine
engere
Verwandtschaft
von
A
m
ruttneri
zur
nordafrikanischen
A
m
intermissa
als
zu
europäischen
Rassen,
ähn-
lich
wie
bei
der
ebenfalls
endemischen
sizi-
lianischen
Honigbiene
(A
m
sicula).
Diese
Ergebnisse
lassen
auf
eine
gemeinsame
Ent-
wicklung
der
Bienen
der
zentralen Inseln
des
Mittelmeers
im
Verlauf
der
Evolution
schließen.
Diese
Rasse
ist
nach
Friedrich
Ruttner
benannt,
der
so
viel
zum
Verständ-
nis
der
intraspezifischen
Taxonomie
der
Honigbienen
beigetragen
hat.
Durch
die
begrenzte
geographische
Verbreitung
die-
ser
Rasse,
durch
die
Verluste
der
Wildpo-
pulation
durch
die
eingeführte
parasitische
Milbe
und
durch
die
Importation
von
Zucht-
stämmen
fremder
Bienenrassen
muß
der
Bestand
dieser
einzigartigen
Bienenpopu-
lation
leider
als
sehr
gefährdet
gelten.
Die
Wahrscheinlichkeit
des
vollständigen
Ver-
lustes
ist
sehr
hoch.
Apis
mellifera
ruttneri
/
Taxonomie
/
Geo-
graphische
Rasse
/
Malta
/
Evolution
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