Biochemical Studies on the Relationship Between Socially Parasitic ...

<strong>Biochemical</strong> Systematics and Ecology, Vol. 19, No. 3, pp. 195-206, 1991. 0305-1978/91 $3.00 + 0.00
Printed in Great Britain. © 1991 Pergamon Press DIc.
<strong>Biochemical</strong> <strong>Studies</strong> on the Relationship Between Socially
Parasitic Ants and Their Hosts
JORGEN HEINZE
Zool. Inst. II, R6ntgenring 10, 8700 WQrzburg, F.R.G.
Key Word Index--Doronomyrmex; Harpagoxenus; Leptothorax; Formicidae; Hymenoptera; social
parasites; Emery's rule; electrophoresis.
Abstract--Analysis of enzyme patterns suggests close phylogenetic relationships between socially parasitic
ants of the genera Harpagoxenus and Doronomyrmex and their Leptothorax hosts. Doronomyrmex goess-
wa/di and D. kutteri are indistinguishable in enzyme patterns from their host, Leptothorax acervorum. A
newly discovered workerless parasite from Canada, L. paraxenus, however, appears to be more closely
related to other non-parasitic species than to its host, L. sp.B (= L. canadensis?). The results are discussed
with respect to current hypotheses on the evolution of social parasites.
Introduction
Ants are among the most successful and most abundant insects on Earth. They can be
found in the most diverse habitats, and they build their nests almost everywhere: in
soil, litter, tree stumps, rock crevices, the mounds of termites and the colonies of other
ant species. Among the latter are the social parasites: ants which are no longer
capable of feeding themselves or tending their own larvae, but which instead rely on
the help of workers from other species. About three per cent of the approximately
9000 ant species are known to lead a permanent or temporary parasitic life [1]. The
freshly inseminated queens of social parasites invade the colonies of other species
which they parasitize by begging food and letting the hosts take care of their brood.
Queens of the north and central European slave-maker Harpagoxenus sub/aevis, for
example, kill or expel the host queen and the adult workers. Workers which eclose
from the conquered host brood forage, provide the parasites with food, and nurse the
Harpagoxenus larvae. The original host workers die within a year or two and have to
be replaced. Hence, Harpagoxenus workers perform slave-raids, during which they
pillage neighbouring colonies of the host species and carry away their pupae. The
queens of some workerless parasites, such as Doronornyrmex kutter/and D. goess-
wa/dl; sneak into a host nest, where they are tolerated by the resident ants. They
produce sexual offspring but none of their own workers. Whereas D. kutter/is an
inquiline, which peacefully lives alongside the fertile host queens, D. goesswa/d/'is a
host queen-intolerant parasite.
For more than a hundred years biologists have speculated on the evolutionary
origin of social parasitism. With the exception of guest ants (see below), social para-
sites are often morphologically very similar to their hosts, and in 1909 Carlo Emery
concluded that "the slave-making, temporarily and permanently parasitic ants orig-
inate from closely related forms which serve them as hosts" [2]. Today, a loose version
of "Emery's rule'--parasitic ants and their hosts are closely related--is widely
accepted. It has also been suggested that in particular the workerless parasites have
evolved intraspecifically from their host species [3-5]. Permanent social parasites are
extremely rare. Several species have been found only once and, despite extensive and
repeated searches at the typical localities, only museum material is available. The
close relationship of parasites and hosts therefore has usually been inferred from
(Received 16 November 1990)
195

196 J. HEINZE
morphological resemblance rather than detailed phylogenetic analysis [6]. Though
electrophoretic data are now commonly used to reconstruct phylogenies of social
insects [7], genetic investigations of parasites and their hosts are still comparatively
rare. In wasps and bees, it has been inferred from enzyme polymorphisms that cuckoo
bumble bees (Psithyrus spp.) are a monophyletic group which radiated secondarily to
several host species [8], and that the parasitic wasps are of polyphyletic origin [9] (but
see [10, 11]). In ants, the distribution of esterase allozymes suggests that small queens
(microgynes) in the nests of Myrmica rubra are genetically isolated from large queens
(macrogynes) and presumably belong to a separate, parasitic species [12].
During the last few decades, several teams have intensively studied the parasite-
rich myrmicine tribe Leptothoracini. In suitable habitats, these little and inconspicuous
ants form dense populations and, in some places, parasites can regularly be collected.
Thus, Leptothorax parasites are among the best studied socially parasitic ants, and
they are the ideal species for a first biochemical approach toward Emery's rule. Electro-
phoresis has already been used in a number of leptothoracine species to estimate the
relatedness between nestmates [13-15] and to clarify the taxonomic position of
species [16, 17]. Inter- and intraspecific genetic diversity is low, and esterase or
dehydrogenase isozymes have successfully been used to characterize taxa or to distin-
guish between sibling species [17-19]. As part of a study on the systematics of the
nearctic Leptothorax "muscorum" complex, it was possible to collect data on the
variability of enzymes in a number of socially parasitic species and their hosts. A first
qualitative analysis of esterase banding patterns [19] had already supported recent
ideas for a systematic revision of the whole tribe [20, 21]. In this paper, more detailed
electrophoretic data from parasitic Leptothoracini and their hosts, especially of the
genera Leptothorax (s.str.), Doronornyrmex, and Harpagoxenus, are presented.
In temperate North America and Eurasia, the complex genus Leptothorax is
represented mainly by three subgenera: Leptothorax (s.str.) (= Mychothorax), Myrafant,
and Temnothorax. Most of 350 or more named taxa of Leptothorax are ordinary non-
parasitic species. A few are workerless parasites and one species, L. duloticus, is a
slave-maker (Table 1). The genus Doronomyrmexcomprises only workerless parasites
of L. (s.str.) (with the exception of the dubious D. pocahontas), and the three species of
Harpagoxenus available in this study are active slave-makers, with H. canadensis and
H. sublaevis enslaving colonies of Leptothorax (s.str.), but H. americanus found only in
nests of Myrafant. Finally, Formicoxenus quebecensis is a guest ant and lives in the
nests of Myrrnica alaskensis. Guest ants can be found in colonies of various ant
species, to whom they are not closely related. They beg food from their hosts but take
care of their brood themselves. The genus Formicoxenus belongs to the Leptothoracini
and has been thought to be closely attached to the subgenus Leptothorax (s.str.) [22].
Materials and Methods
Complete colonies of host species were collected during the past five years in numerous localities in North
America and Central Europe. Social parasites were collected in the following sites: Doronomyrmex kutteri:
Nyehusen, Sweden; Doronomyrmex goesswaldi and D. pacis: La Villette, France; Doronomyrrnex
pocahontas: Maligne Canyon (Jasper N. P., Alberta); Harpagoxenus canadensis: St. Sim6on (Comt~ de
Charlevoix-Est, Quebec), Tadoussac (Saguenay Co., Qua.), Rouyn-Noranda (Temiscamingue Co., Qua.),
MacKenzie Mountain (Inverness Co., Nova Scotia); /4. sublaevis: Nfirnberger Reichswald, F.R.G.; Leptothorax
paraxenus; Milton, Ontario; L. wilsoni: Moncton (Westmoreland Co., New Brunswick); Formicoxenus
quebecensis: Waswanipi (Co. de Abitibi, Qua.). Single workers, males and females were crushed individually in
40 ill running buffer with 15% glycerol and 0.01% bromothymol blue. Proteins were separated in 12.5-cm-long
7.5% polyacrylamide gels (gel buffers: 0.47 M Tris-HCI, pH 8.8 and 0325 M Tris-HCI, pH 8.0 for phospho-
glucose isomerase (PGI); running buffer 036 M glycine, 0.025 M Tris, pH 8.3) at 10°C with a current of approxi-
mately 10-20 mA for 3 h, and on 7.5- or 13-cm-long cellulose acetate plates (Cellogel, Milano, and Helena
Laboratories, Beaumont, Texas; gel buffer and running buffer: 0.01 M sodium phosphate/citrate, pH 6.4) with a
constant voltage of 200 or 350 V for 1.5 h. Dehydrogenases were stained using the following reagents: 2 mg

BIOCHEMICAL STUDIES ON PARASITIC ANTS AND HOSTS 197
TABLE 1. RANGE OF THE NON-PARASITIC SPECIES OF LEPTOTHORAX (S.STR.) AND OF THE SOCIALLY PARASITIC SPECIES OF
LEPTOTHORAX, HARPAGOXENUS AND DORONOMYRMEX, BASED ON [1 ] AND AUTHOR'S OBSERVATIONS. SPECIMENS OF THE
SPECIES PRINTED IN BOLD LETTERS WERE EXAMINED IN THIS STUDY
Non-parasitic Typical habitat Known range
L. acervotum Alpine and boreal Holarctic
(Fabricius, 1973) coniferous forests
occasionally in deciduous forests
L. muscorum Alpine and boreal Palaearctic
(Nylander, 1846) coniferous forests
L. gredleri Light pine stands, rose and Central Europe
(Mayr, 1855) blackthorn thickets
L. scarnni Alpine coniferous forests Caucasus, Pontus
(Ruzsky, 1905)
L. craesipilis Pine and cottonwood forests U.S. Rocky Mts
(Wheeler, 1917)
L tetractus Alpine and boreal Nearctic
(Francoeur, 1986} coniferous forests
L spagnico/us Spruce bogs Central Quebec
(Francoeur, 1986)
L. sp.A Open alpine and boreal Eastern North America
coniferous forests
L. sp.B Boreal coniferous forests, Eastern North America
occasionally in deciduous forests
L. sp.C. Alpine coniferous forests Canadian Rocky Mts
L. sp.D Alpine coniferous forests Canadian Rocky Mrs
Parasite Host species Known range Type of parasitism
L. faberi L. sp.D Jasper N. P., AIta. Inquiline
(Buschinger, 1982) (type locality)
L. wilsoni L sp.B. Quebec, New Brunswick, Workerless, queen-intolerant?
(Heinze, 1989) New Hampshire
L. pataxenus
(Heinze and Alloway, in L sp.B Southern Ontario and Workerless, queen-intolerant?
prep.) Quebec
D. geesswa/di L. acervorurn Alps, S. Sweden Workerless, queen-intolerant
(Kutter, 1967)
D. kutteri L. acervorum Alps, S. Sweden, Inquiline
(Buschinger, 1966) Central Germany
D. pacis L. acervorum Alps Inquiline
(Kutter, 1950)
D. pocahontas L. sp.C Maligne Canyon, Alta. ?
(Buschinger, 1979) (type locality)
N. cenaden$is L. spp.A, B Eastern North America Slave-maker
(M. R. Smith, 1939)
H. subleevis L. acervorum, Central and Northern Slave-maker
(Nylander, 1849) L. muscorum, Europe
L gred/eri
Parasites of species belonging to the subgenus Leptothorax (MyrafantJ
H. arnericanus L. ambiguus, Eastern North America Slave-maker
(Emery, 1895) L. curvispinosus,
L. Iongispinosus
L. duloticus L. ambiguus, Eastern United States Slave-maker
(Wesson, 1937) L. curvispinosus,
L. Iongispinosus
L. minutissimu$ L. curvispinosus Eastern United States Inquiline
(M. R. Smith, 1942)
NAD or NADP, 2 mg NBT, 0.2 mg PMS, and approx. 10 rng of the substrate dissolved in 5 ml of staining buffer
(0.2 M Tris-HCI, pH 7.0, for lactate dehydrogenase (LDH), 0.5 M Tris-HCI, pH 8.0, for the other dehydro-
genases).

198 J. HEINZE
Results and Discussion
Genera~patterns of enzyme variabi/ity
Though individual Leptothoraxants weighed less than 1 mg, it was possible to stain up
to six or more different enzyme systems in a single, crudely homogenized adult. The
following enzymes gave reproducible, though often weak stains in females, males and
workers: ADH (alcohol dehydrogenase, EC 1.1.1.1), 0¢-GPDH (0¢-glycerophosphate
dehydrogenase, EC 1.1.1.8), LDH (lactate dehydrogenase, EC 1.1.1.27), ~-HBDH (~-
hydroxybutyrate dehydrogenase, EC 1.1.1.30), MDH-1 and MDH-2 (malate dehydro-
genase, EC 1.1.1.37), ME (malic enzyme, EC 1.1.1.40), IDH (isocitrate dehydrogenase, EC
1.1.1.42), PGD (6-phosphogluconate dehydrogenase, EC 1.1.1.44), G6PDH (gtucose-6-
phosphate dehydrogenase, EC 1.1.1.49), AO (aldehyde oxidase, EC 1.2.1.5), XDH
(xanthine dehydrogenase EC 1.2.3.2), TO (tetrazolium oxidase or superoxide dis-
mutase, EC 1.15.1.1), and PGI (phosphoglucose isomerase, EC 5.3.1.9). Some other
enzymes, which could be easily stained in a Drosophi/a standard, gave no results in
leptothoracine ants (e.g. glutamate-oxalacetate transaminase, EC 2.6.1.1, hexokinase,
EC 2.7.1.1, alkaline phosphatase, EC 3.1.3.1), or were clearly visible only in larvae (leucine
aminopeptidase, EC 3.4.11.1) or pupae (unspecific esterases, EC 3.1.1.1, especially Ester-
ase-7 [19]). IDH and PGD could not be stained in polyacrylamide gels with a gel buffer
of pH 8.0 or 8.8, but on cellulose acetate gels and ultrathin polyacrylamide gels for IEF.
As has also been reported for esterases [19], enzyme patterns of Leptothorax (s.str.)
and its parasites are rather uniform compared with those of Myrafant. Whereas in 26
Myrafant and Temnothorax from North Africa and Europe, 12 electromorphs of IDH
have been separated in starch gels [16], in the present study only two tDH banding
patterns could be found in 20 taxa of Leptothorax(s.str.) and its parasites (Table 2). The
apparent difference in genetic variability might be caused in part by the use of different
separation techniques, but ultrathin layer IEF showed similar results: only two IDH
electromorph patterns in Leptothorax (s.str.), Harpagoxenus sub/aevis, H. canadensis,
and Doronomyrmex, but at least five different patterns in eight Myrafant species.
Similar results were obtained with other enzymes, e.g. TO, with four different electro-
morphs in eight Myrafantand only one in all Leptothorax (s.str.) (data not presented).
In Leptothorax (s.str.) and its parasites, XDH, ADH, LDH, AO, TO, G6PDH appear to
be uniformly monomorphic throughout the species. MDH-2 and ME were found to be
polymorphic in the non-parasitic species, and they are also variable at least in
Harpagoxenus sublaevis [15]. Of MDH-1, IDH and PGD, two or more electromorphs
have been separated, and the different taxa were more or less fixed for one of them
each [17]. Thus, specimens of L. acervorum from Sweden, Germany, France, Japan
and Alaska all have a fast migrating MDH-1 with an approximate isoelectric point of
7.2, but more than 95% of all L. sp.B ( -- L. canadensis?) from New England and Eastern
Canada have a stow electromorph (pl = 6.6) (Table 2). Similarly, the European Lepto-
thorax (s.str.) and the nearctic species L. crass/pi/is and L. sp.A all have a fast migrating
IDH, whereas L. sp.B. and L. retractus exclusively have a slow electromorph. Addition-
ally, very rare allozymes have been detected only when large numbers of colonies
from a single site are examined. Thus, in 80 colonies of L. muscorum from the
Reichswald population in Southern Germany, workers from two colonies had hetero-
zygote, three-banded MDH-patterns with a previously unknown, very fast migrating
allozyme, and in one of 30 colonies of L. sp.B from the Toronto region, workers had an
aberrant heterozygous banding pattern. PGI is variable in most species, and here again
most electromorphs have been observed in some species but not in others.
Harpagoxenus
Harpagoxenus sublaevis and H. canadensis are indistinguishable from each other in
all studied enzymes, and all their electromorphs are also present either in one of their
hosts (L. acervorum, L. muscorum and L. gredleriin H. sublaevis and L. spp. A and B in

BIOCHEMICAL STUDIES ON PARASITIC ANTS AND HOSTS 199
TABLE 2. FREQUENCY OF DIFFERENT ELECTROMORPHS IN POPULATIONS OF LEPTOTHORAX (S.STR.), DORONOMYRMEX,
AND HARPAGOXENUS
L. acervorum
Reichswald, D
Oberleinach, D
Babenhausen, D
Nyehusen, S
La Villette, F
Gran Sasso, I
Hokkaido, J
Denali, N. P., Alas.
L. muscorum
Reichswald, D
Nyehusen, S
La Villette, F
Akkus, TR
L. gred/eri
Sommerhausen, D
L. retractus
Rouyn-Norenda, Que.
Maligne C.; Alta.
Kananaskis, Alta.
L. crassipilis
Curecanti, Colo.
L. sp.A
Tadoussac, Qu@.
Rouyn-Noranda, Qu@.
Mt. Monadnock, N. H.
L. sp.B
Tadoussac, Qu@
York, Ont.
Milton, Ont.
Moncton, N. B.
Mt, Monadnock, N. H.
L. sp.C
Maligne C., Alta.
Banff, Alta.
L. sp.D
Maligne Lake, Alta.
L. paraxenus
Milton, Ont.
L. wilsoni
Moncton, N. B.
D. pocahontas
Maligne, C., Alta.
D. pacis
La Villette, F
D. goessvvaldi
La Villette, F
D. kutteri
Nyehusen, S
H. sublaevis
Reichswald, D
FL canadensis
St. Sim@on, Qu@.
Tadoussac, Que.
F. quebecensis
Waswanipi, Qua.
MDH-1 IDH PGD ~I
v s f x s f v s f x u v s m f x
X -- -- X -- X X -- × X X
X -- -- X -- X X
X -- -- X -- X X -- X X X X
X -- -- X -- X X X X X
X -- -- X -- X X
x -- -- x x -- -- x -- X
x -- -- x -- X X
x -- -- x -- X X
X * -- X -- X * -- X X
X -- -- X -- X X
X -- -- X -- X X X
X -- -- X -- X X
X -- -- X -- X X
X -- X -- X -- X
X -- X -- X -- X
X -- X -- X -- -- X X
X -- -- X -- X X
X -- -- X -- )4 -- X X
X -- -- X -- X -- X X X
X -- -- X -- X X
-- X X -- X -- X X --
-- X X -- X -- X X --
-- X X -- X -- X X --
-- X X -- X -- X --
-- X X -- X -- X X --
X -- * X -- X X
X -- -- X -- X X
-- X X -- X -- X
X -- -- X -- X X
X --
X -- -- X X X
X -- -- X
X -- -- X -- X X X --
X -- -- X -- X X
X -- )'< -- X -- X
X -- X -- X -- X
X -- X -- X -- X
X X -- X X
Electromorphs are characterized by their different electrophoretic velocities (u = extremely slow, v : very slow, s = slow,
m = medium, f = fast, x = very fast). More detailed data on enzyme patterns and collecting sites of non-parasitic Leptothorex
(s.str.) have been published elsewhere [17]. (* Elect~morph is very rare.)

200 J. HEINZE
H. canadensis) or other species belonging to the subgenus Leptothorax (s.str.). From
the present data it is not possible to confirm a closer relationship between H. sublaevis
and H. canadensis to L. acervorum which had been suggested on morphological
criteria and esterase banding patterns [19]. In fact, the only Leptothorax (s.str.) species
which is similar to Harpagoxenus in all studied enzymes, L. retractus, is the only
species which is not parasitized by the slave-makers. It appears likely that Harpa-
goxenus diverged at an early stage of speciation from the ancestors of Leptothorax
(s.str.) and radiated secondarily over several host species.
Harpagoxenus americanus, on the other hand, differs in all enzymes which could be
stained in the little material available--MDH, IDH, ME, PGD, TO and esterases--from
its presumed congeners and from Leptothorax (s.str.). It shares some electromorphs
with its hosts, L. (Myrafant) Iongispinosus, L. (114.) ambiguus, and L. (M.) curvispinosus
and other North American Myrafant, e.g. MDH-1 (Fig. 1). Though the data base is
small, it supports the idea that H. americanus is not closely related to the other species
of Harpagoxenus and to Leptothorax (s.str.) but instead attached to Myrafant. Given the
comparatively high genetic variability in the only extensively studied North American
Myrafant, L. (M.) Iongispinosus [14], and the little knowledge we have of other nearctic
species, it is not yet possible to decide whether H. americanus is closer to one of its
hosts than to any other non-parasitic species. Nevertheless, the data corroborate
recent proposals to split the genus Harpagoxenus. It had been concluded from charac-
teristics such as sexual behaviour, host specificity, and ecological needs that H.
americanus does not belong to H. sublaevis and H. canadensis [20, 23]. Though a
formal revision has not yet been published, several authors have de facto transferred
H. americanus to its own taxon, Protomognathus [1, 4].
Doronomyrmex
The European workerless parasites Doronomyrmex goesswald/; D. kutter~ and D.
pacis are morphologically similar to their common host, L. acervorum. In all four
species, antennae and legs are covered with abundant suberect hairs, which are lack-
ing in the other non-parasitic palaearctic Leptothorax (s.str.). D. kutteri and D. goess-
wa/di had originally been described as species of Leptothorax. In the laboratory, it was
possible to cross D. pacis or D. goesswaldi males with D. kutteri females and viable
hybrid offspring were produced [20, 24]. Therefore, and because of common
karyological and morphological characters, it was concluded that the three species
belong to one single genus.
All enzyme electromorphs seen in the palaearctic Doronomyrmexare also common
in L. acervorum, with the exception of the very slowly migrating PGI-electromorph of
D. pacis, which has as yet been found only in a single I. acervorum nest from central
Italy (Table 2). D. goesswaldiand D. kutteriare also indistinguishable from their host in
esterase patterns (no data available for D. pacis) [19]. In one of three D. goesswa/di
colonies, a female had a heterozygous PGI banding pattern. This was the 9nly hetero-
zygous banding pattern observed in a workerless parasite. Though only few nests
from single populations could be studied, it is likely that a more extensive search will
confirm low heterozygosity levels in these species. Workerless parasites are rare and
females mate near or within the nest, thus the inbreeding coefficient should be high.
Though at least D. goesswaldiand D. kutteri have electromorphs identical to their
host, L. acervorum, the data do not suffice to decide whether Doronomyrmex evolved
directly from the host species, as the strong version of Emery's rule and the models of
some authors predict [5, 25, 26]. The two species are also biochemically very similar to
a second, not parasitized species, L. muscorum, from which they differ in their ester-
ase banding pattern [19]. The genetics of this complex set of 20 bands are not clearly
understood and for an estimation of phylogeny this system so far is not appropriate.
Two more L eptothorax (s.str.) species differ little from Doronomyrmex: L. gredler/has

O
Q
-I-
FIG. 1. MALATE DEHYDROGENASE (MDH) PATTERNS OF ADULT LEPTOTHORACINE ANTS, SEPARATED BY tEF ON 0.2 MM THIN POLYACRYLAMIDE GELS (pH RANGE 4-9, APPROX. 4500 Vh). a-c: L.
(Myrafant) ambiguus (a from Shawinigan, Quebec, b and c from Burlington, Vt.), d: L. (M.) curvispinosus (Cambridge, Mass.), e: Flarpagoxenus americanus (Renselaerville, N. Y.), f-h: Harpagoxenus
canadensis (f and g from Rouyn-Noranda, Que., h from Bale Ste. Catherine, Que.), i: L. (M.) rugatulus (Ogden, Ut.), j-n: L. sp.A (from La Baie, Shawinigan, Ashuapmushuan, Matagami, and Tadoussac, all in
Quebec), o: L. sp.B (Trois Pistoles, Que.), p: L. gredleri(Sommerhausen, Germany), q: L. sp.A (Bar Harbor, Me), r: Formicoxenus quebecensis (Waswanipi, Que.), s: hybrid of L. spp.A and B (Chapais, Que.), t
and u: L. muscorum (Nyehusen, Sweden, and La Villette, France) v: L. retractus (Aiguebelle, Que.), w: L. acervorum (Pipplinger Au, Germany). The allozymes of the more cathodal MDH-2 could not be
separated in this pH gradient. Arrows indicate the position of tetrazolium oxidase (TO) electromorphs, visible only as lighter bands against the background of the gel, which is slightly violet due to reduced
tetrazolium salts The lines between lanes i and j, and between r and s indicate the position of water-soluble, coloured pl marker proteins, which were lost during the histochemical staining.