First incidence of inquilinism in gall

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First incidence of inquilinism in gall-forming psyllids, with a
description of the new inquiline species (Insecta, Hemiptera,
Psylloidea, Psyllidae, Spondyliaspidinae)
Blackwell Science, Ltd
MAN-MIAO YANG, CHARLES MITTER & DOUGLASS R. MILLER
Accepted: 28 December 2000
Yang, M.-M., Mitter, C. & Miller, D. R. (2001). First incidence of inquilinism in gall-forming
psyllids, with a description of the new inquiline species ( Insecta, Hemiptera, Psylloidea, Psyllidae,
Spondyliaspidinae). — Zoologica Scripta, 30, 97–113.
The two largest lineages of holometabolous gall-forming insects, cynipid wasps and cecidomyiid
flies, have given rise to numerous obligate inquilines, species which are unable to form galls
themselves and survive by inhabiting galls formed by other species. In contrast, only a single
obligate inquiline, an aphid, is known in the sternorrhynchous Hemiptera, the hemimetabolan
lineage in which gall-forming is best developed. We describe the first known gall inquiline in
psyllids (Sternorrhyncha, Psylloidea), Pachypsylla cohabitans Yang & Riemann sp. n. All other
members of this genus produce closed galls on hackberries, Celtis spp. (Ulmaceae). Newly
hatched nymphs of P. cohabitans feed next to nymphs of several species of leaf gall-makers,
becoming incorporated into the gall as the stationary nymphs are gradually enveloped by leaf
tissue. In the mature gall, the inquilines occupy separate, lateral cells surrounding a central cell
containing a single gall-maker. Pachypsylla cohabitans is similar in morphology to leaf-gallers,
but differs in nymphal and adult colour, allozyme frequency, especially in the malic enzyme, and
in adult phenology. Laboratory-reared progeny of side-cell females, when caged alone,
never form galls, while progeny of centre-cell individuals alone only form galls comprising
single individuals. Multiple-cell galls are formed only when adults of side-cell and centre-cell
individuals are caged together. Experimental removal of centre-cell nymphs in early stages of
gall initiation leads to smaller galls or death of side-cell individuals. We conclude that the
side-cell individual is an obligate inquiline that is incapable of forming a gall on its own but is
derived from a leaf-galling ancestor. We speculate on selective forces that might favour this
evolutionary transition.
Man-Miao Yang, Department of Entomology, National Chung Hsing University, Taichung 40227,
Taiwan. E-mail: mmyang@dragon.nchu.edu.tw
Charles Mitter, Maryland Center for Systematic Entomology, Department of Entomology, University
of Maryland, College Park, MD 20742, USA. E-mail: cm42@umail.umd.edu
Douglass R. Miller, Systematic Entomology Laboratory, Beltsville Agricultural Research Center,
PSI, United States Department of Agriculture, Beltsville, MD 20705, USA. E-mail:
dmiller@sel.barc.usda.gov
Introduction
Plant-feeding insects in diverse lineages have evolved the
ability to induce galls, which are abnormal plant structures
that typically provide both shelter and enhanced nutrition to
the gall-dweller (Dreger-Jauffret & Shorthouse 1992). In
turn, other insects, themselves incapable of inducing a gall,
have evolved specific adaptations for exploiting the shelter
and nutrition that insect galls make available. Such species
are called inquilines. The term ‘inquiline’ comes from the
Latin inquilinus, meaning temporary inhabitant or guest (Brown
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
1956), and has been used in diverse ways. For the purposes of
this study, the term will be restricted to gall inhabitants that
feed on gall tissues, without directly damaging the gall-inducer,
but are unable to induce galls independently (Skuhravá et al.
1984; Shorthouse & West 1986; Shorthouse 1991; Wiebes-Rijks
& Shorthouse 1992). Quite often, the inquiline habit seems
to have evolved within the gall-forming lineage itself. In the
two holometabolous insect groups where gall-forming is best
developed, the cecidomyiid flies (Skuhravá et al. 1984) and
the cynipid wasps (Roskam 1992), numerous inquilines have
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Psyllid gall inquiline • M.-M. Yang et al.
evolved, constituting up to 6 – 8% of the total species diversity
of the gall-forming lineage. In contrast, in the hemimetabolous
insect lineage in which gall-forming is best developed, the
Sternorrhyncha ( Hemiptera), only a single inquiline species,
an aphid (Aphidoidea), has been described. In the other two
gall-forming sternorrhynchan superfamilies, Psylloidea and
Coccoidea, inquilinism is unknown.
In this paper, we report the first instance of a gall inquiline
species in the Psylloidea, which includes about 350 described
gall-forming species (Mani 1964; Hodkinson 1984; DregerJauffret & Shorthouse 1992). We demonstrate that this
species, which we describe in the genus Pachypsylla, is incapable
of inducing a gall itself; instead, the newly hatched nymphs
seek out and feed next to the nymphs of a leaf gall-forming
sibling species, becoming enclosed in the developing gall as
it grows around the true gall-former. To place this finding in
context, we briefly review the incidence of obligate inquilinism
across gall-forming groups, and discuss the hypotheses that
have been proposed to explain inquiline evolution.
Background: occurrence of multiple individuals in
Pachypsylla leaf galls
Psyllids of the North American genus Pachypsylla feed on
hackberry trees (Celtis) in the subgenus Euceltis (Ulmaceae).
They produce a variety of closed gall types on different parts
of the host, including petioles, buds, twigs and leaves. The
gallers on each plant part comprise a distinct monophyletic
group, containing one or more species (Yang 1995). Adult
morphology within each group is homogeneous, but gall
morphology is variable.
Within the leaf gall-forming complex, a variety of gall
morphologies have been recognized and assigned descriptive
names, such as the ‘star’ gall, ‘blister’ gall or ‘nipple’ gall. There
has been much debate about whether these morphological
types represent different species (Riley 1876, 1883, 1884,
1890; Cockerell 1910; Crawford 1914; Tuthill 1943; Riemann
1961; Moser 1965; review in Yang & Mitter 1994). A recent
re-examination of this complex using morphological, allozyme
and life history evidence ( Yang 1995) strongly favours the
interpretation of multiple species. The leaf gall-makers treated
in this paper include: the hairy nipple gall-maker, P. celtidismamma
Riley, 1876; the glabrous nipple gall-maker, P. celtidisglobula
Riley, 1890; the star gall-maker, P. celtidisasterisca Riley, 1890;
and the blister gall-maker, P. celtidisvesicula Riley, 1884.
The number of nymphs within a single hackberry psyllid
gall varies from 1 to 17 and probably more, depending on the
gall type and population. Twig galls, formed by P. celtidisinteneris
Mally, 1894, are usually monothalamous, i.e. containing only
one individual per gall. Petiole gallers (P. venusta Osten-Sacken),
bud gallers (P. celtidisgemma Riley) and some leaf-inhabiting
species, such as the P. celtidismamma complex, often produce
galls that are polythalamous, i.e. containing multiple individuals;
98
in these, each nymph is enclosed in a separate cell. In the
petiole and bud gallers, all individuals in the gall are similar
in appearance, with no obvious differences related to cell
position. In contrast, within multiple-cell leaf galls, one can
generally distinguish between a centre-cell individual,
presumably the one that initiated the gall, and one or more
‘side-cells’. Individuals in the two cell positions differ in
colour and other traits.
Moser (1965) observed multiple cells in hairy nipple galls
(P. celtidismamma) on C. occidentalis leaves in New York, USA.
He termed the side-cells ‘marginal galls’, and hypothesized
that they represented individuals of the blister gall, the only
other co-occurring leaf gall type, whose galls had been overgrown by and incorporated into those of an adjacent nipple galler.
Riemann (1961), in contrast, concluded that side-cell individuals found in several different leaf gall types in Texas, USA,
represent a single undescribed species that is not conspecific
with any of the leaf gall-formers. He hypothesized that the
side-cell species is an inquiline that is unable to induce a gall
independently, and is incorporated into a gall by feeding next
to a true gall-maker during gall initiation in the spring. Two
variants of Riemann’s hypothesis also seemed initially plausible:
each leaf gall type could have a separate inquiline species;
alternatively, inquilinism could be facultative, and the inquilines
conspecific with the host gall-former.
To test these hypotheses, centre-cell vs. side-cell nymphs from
the two types of nipple galls occurring in Maryland, USA, were
compared with each other and with those from single-celled
galls of other co-occurring leaf gall types, using allozyme,
morphological and life history characters. A rearing experiment
was conducted to test the ability of side-cell individuals to
induce galls. Dependency of the side-cell individuals on the
centre-cell individual was further investigated by removing
the centre-cell individual at different stages of gall initiation.
Materials and methods
Samples for electrophoretic and morphological studies
Nymphs were collected in the autumn of 1992 and 1993 from
the hairy nipple gall and glabrous nipple gall, plus two other
types of galls that commonly co-occur with these, at three
sites in Maryland, USA. Site ‘CMt’ was the Poplar Grove
campsite in Catoctin Mountain Park, Frederick County. At
this site, at about 450 m elevation, both adult trees and seedlings of the host plant, C. occidentalis, were abundant, and
collections were made from several trees. The common psyllid
gallers were those inducing the hairy nipple gall and the blister
gall. Site ‘NAL’ consisted of a single tree of C. tenuifolia, about
12 m high, in a small copse surrounded by open lawn, next
to the National Agricultural Library (NAL) in Beltsville,
Prince George’s County, about 85 km south-east of the
‘CMt’ site. Few hackberry seedlings occur near this tree but
there is another hackberry 3 m high about 500 m away. The
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M.-M. Yang et al. • Psyllid gall inquiline
‘BB’ site is a residential neighbourhood on Branchville Road
in Berwyn, Prince George’s County, about 5 km from the NAL
site. The BB population contains many hackberries, from
small seedlings to tall trees. Collections were made from
several individuals of C. tenuifolia. The abundant gallers were
those inducing the glabrous nipple gall and the star gall.
Each gall collected was dissected, and in the case of polythalamous galls, which occurred in all gall types except the
blister gall, the side-cell nymphs were separated from the
centre-cell nymph. All collected nymphs were either frozen
at – 80 °C for later electrophoretic and morphological
studies, or were reared in small glass vials to obtain associated
adults for morphological comparisons. Three to 21 nymphs
from each cell position in each locality were analysed by
electrophoresis. Five to 30 individuals of each category were
examined morphologically. All material was collected by the
first author (MMY ), except the North Dakota samples which
were collected by J. Riemann. Most material is deposited in
the psyllid collection of the National Museum of Natural
History, Smithsonian Institution, which is located at Beltsville,
Maryland, USA ( USNM). A pair of adult males and females
plus two nymphal paratypes are deposited in each of the
following institutions: The Natural History Museum, London
( BMNH); Naturhistorisches Museum, Basel, Switzerland
( NHMB); National Museum of Natural Science, Taiwan
( NMNS); National Chung Hsing University, Taiwan ( NCHU).
Morphological methods
Morphological comparisons were conducted using both
optical and scanning electron microscopy. Colour variation
of live insects was recorded immediately after dissection from
the gall. Specimens were preserved in ethanol or dry at – 80 °C.
Specimens were dissected and examined in alcohol or
after slide mounting in Canada Balsam. Both adults and last
(fifth) instar nymphs were examined. Five to 30 specimens
were examined for each gall type and each stage. Terminology
follows Hodkinson & White (1979) and Burckhardt (1991)
for adults, and White & Hodkinson (1982, 1985) and
Muddiman et al. (1992) for nymphs.
Specimens for scanning electron microscopy were sonicated before dehydration. In a preliminary study, five variants
of the specimen preparation process were compared using
two individuals each. In the most elaborate protocol, specimens were pretreated with glutaraldehyde fixer (4%, 12 h)
and osmium tetroxide (2%, 4 h), dehydrated in a series of
increasing ethanol concentrations (30%, 40%, 50%, 60%,
70%, 80%, 90% and 95% each 10 min and 100% ethanol
1 h), then critical point dried in CO2. Three other treatments
consisted of different ethanol series (30–40–50–70–80–90–
100%; 70 – 80 – 90 – 95–100% and 70–100%), followed by
critical point drying, without pretreatment in glutaraldehyde
and osmium tetroxide. The final treatment was air drying
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
alone. Air-dried specimens shrank dramatically and this method
was abandoned. No significant differences were found between
specimens that were pretreated vs. not treated with glutaraldehyde and osmium tetroxide, nor between dehydration started
with a 30% vs. 70% ethanol concentration. The procedure
finally adopted was an ethanol series with concentrations
of 70–80–90–95–100%, followed by critical point drying.
Specimens were mounted on aluminium stubs with doublesided adhesive tape, sputter coated with gold–palladium
and examined and photographed using an Amray scanning
electron microscope. Two to four adults and nymphs of each
gall type were examined.
Electrophoretic methods
Allozyme electrophoresis was carried out using cellulose
acetate gels, following the methods of Hebert & Beaton (1989)
and Easteal & Boussy (1987). The Titan III cellulose acetate
gel apparatus from Helena Laboratories was used, with gels
measuring 94 × 76 mm. This technique allowed 12 samples
to be run side-by-side.
Prior to sample preparation, the body size of each individual
was measured using an electronic caliper. Frozen individual
psyllids were homogenized in 5 µL of distilled water in the
sample well, using a glass rod cut from a microscope slide.
Another 2.5 µL of distilled water was added to each well. Up to
13 runs, yielding 16 loci, could be obtained from each individual.
Buffer systems followed Richardson et al. (1986). Fifteen
loci were resolved in an initial survey of 27 enzyme stains
across 11 buffers. The optimal buffers and running conditions
for each locus are given in Table 1. Prior to loading, gels were
soaked in the same buffer as used for the runs. Gels were run
in a refrigerator (4 °C) at 180 V for 35 –90 min (Table 1).
Staining was carried out using an agar overlay, following
recipes from Hebert & Beaton (1989). The agar was washed
away after the gel was sufficiently stained. Gels were soaked
in water for at least half an hour and recorded by drawings,
photographs or xeroxing. All gels were subsequently dried
and filed in transparent pocket sheets for later reference.
Electrophoretic data analysis
Alleles were designated alphabetically from fast to slow. The
fit to Hardy–Weinberg equilibrium was analysed at each
variable locus using the Biosys-1 package of Swofford & Selander
(1981). Chi-square contingency table analyses, carried out
in Systat 5.2, were used for testing the heterogeneity of allele
frequencies for all loci among samples. Alleles for which the
expected value was less than five in more than one-fifth of the
cells were pooled (Sokal & Rohlf 1981). Allozyme frequency
data for samples from the same locality, gall type and cell position
were combined if they did not differ significantly. Phylogeny
estimation employed the distance Wagner and UPGMA routines
in Biosys-1 using Rogers’ distance (Rogers 1972).
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Psyllid gall inquiline • M.-M. Yang et al.
Table 1 Enzyme loci scored for analysis of Pachypsylla leaf gall-makers in different cell positions. The running condition for each locus,
including buffer and time, is provided. All the gels were run at a voltage of 180 mV.
Enzyme
E. C. #
Locus
Buffer
Run time
Fructose-1,6-diphosphatase
Fumarate hydratase
Glyceraldehyde-3-phosphate dehydrogenase
Glycerol-3-phosphate dehydrogenase
Isocitrate dehydrogenase
3.1.3.11
4.2.1.2
1.2.1.12
1.1.99.5
1.1.1.42
Tris-citrate 0.1 M pH 8.2
Tris-glycine 0.025 M pH 8.5
Tris-citrate 0.1 M pH 8.2
Phosphate 0.02 M pH 7.0
Tris-citrate 0.1 M pH 8.2
1h
1h
1.5 h
1h
1.5 h
Lactate dehydrogenase
Malate dehydrogenase
1.1.1.27
1.1.1.37
Malic enzyme
Peptidase
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Phosphoglucose isomerase
Triose phosphate isomerase
1.1.1.40
3.4.11
2.7.5.1
1.1.1.44
5.3.1.9
5.3.1.1
FDP
FUM
G3PDH
GPDH
IDH-1
IDH-2
LDH
MDH-1
MDH-2
ME
PEP-1
PGM
6PGDH
PGI
TPI
Tris-maleate-EDTA-MgCl2 0.05 M pH 7.8
Tris-maleate 0.015 M pH 7.2
Tris-maleate-EDTA-MgCl2 0.05 M pH 7.8
Tris-maleate 0.05 M pH 7.8
Tris-glycine 0.025 M pH 8.5
Citrate phosphate 0.01 M pH 6.4
Tris-maleate-EDTA-MgCl2 0.05 M pH 7.8
Citrate phosphate 0.01 M pH 6.4
Tris-citrate 0.1 M pH 8.2
35 min
1.5 h
35 min
1.5 h
45 min
1.5 h
1.5 h
1 h 10 min
1h
Isofemale progeny rearing experiment
A rearing experiment was conducted in spring 1993, using
wild-caught adult females and their laboratory-reared progenies, to test the hypothesis that side-cell nymphs cannot
form galls. This experiment also provided data on colour
differences between centre- and side-cell individuals.
Adult females were collected from the NAL and BB sites
where multiple-cell glabrous nipple galls were commonly found.
Star galls also occurred at both sites, but contained multiple
cells much less often than nipple galls. At the NAL site, the
glabrous nipple gall was more abundant than the star gall,
whereas at the BB site, the reverse was true. To ensure that
both kinds of gallers, as well as the inquiline, were included,
females were sorted in two ways. (1) First, females were separated into green vs. brown abdomen groups, because dissections
of galls had previously shown these colours to distinguish
side- vs. centre-cell individuals, respectively. (2) No discrete
morphological feature separates the glabrous nipple and
star gall-formers, but adults of the former are on average
markedly longer (Yang 1995). Therefore, to ensure that
centre-cell individuals of the glabrous nipple gall-maker as
well as star gall-maker were represented, the brown abdomen
adults were sorted into ‘large’ individuals (≥ 3 mm in length
including the wings), presumptive nipple gall-formers, and
‘small’ individuals (< 3 mm in length including the wings),
presumptive star gall-formers.
Individual wild-caught females were caged on hackberry
seedlings in the laboratory. There were four treatments:
(1) cages containing a single brown abdomen female, presumably a centre-cell individual; (2) cages containing a single green
abdomen female, presumably a side-cell individual; (3) cages
containing one brown plus one green abdomen female; and
100
(4) control cages containing no insects. In treatments (1) and
(3), both ‘large’ (≥ 3 mm) and ‘small’ (< 3 mm) brown abdomen
females were used (in separate replicates) to ensure inclusion
of centre-cell individuals of nipple galls.
To account for possible variation among seedlings, each
seedling hosted a replicate of each treatment. Thus, each
seedling bore six cages, each cage containing either a single
large brown abdomen female, a single small brown abdomen
female, a single green abdomen female, one large brown
abdomen plus one green abdomen female, one small brown
abdomen plus one green abdomen female, or no psyllids
at all.
The cages were 30 cm × 50 cm cylinders sewn from white
polyester fine-mesh organza, incorporating a median belt of
transparent mylar (4 cm wide, 0.012 mm thick) to permit
convenient inspection of the cage contents. These cages were
slipped onto branches and tied on with ‘Stretchrite’ round
cord elastic at both ends.
The number and timing of the replicates are shown in
Table 2. All experiments used seedlings of C. tenuifolia, except
for one batch of insects that was reared on C. occidentalis. The
seedlings were kept in a cold room (2–5 °C) starting in late
winter, and were placed in the laboratory at different times
the following spring (1993). The seedlings were kept near a
south-facing window in ambient light. Seedlings were used as
their foliage reached the appropriate stage of development
and when field-collected adults were available.
Females were removed from the cages after 10 days and
frozen at – 80 °C, and the colour of the nymphs was recorded.
Gall formation, or lack thereof, was recorded 1 month after
inoculation. Galls were dissected in September to examine
cell numbers within each gall.
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M.-M. Yang et al. • Psyllid gall inquiline
Table 2 Number and kinds of galls formed by progenies of single females in rearing experiments. This experiment tested for inquilinism of
side-cell individuals in glabrous nipple galls and star galls from two Maryland populations.
Population*
NAL
Host
(date of release)
Celtis
tenuifolia
(May 13)
Celtis
tenuifolia
(May 24)
BB
Celtis
tenuifolia
(May 17)
Celtis
occidentalis
(May 23)
Brown abdomen female
(centre-cell)
Green abdomen female
(side-cell)
Green + brown abdomen
females (centre + side)
Seedling #
Single-cell
Multiple-cell
Single-cell
Multiple-cell
Single-cell
Multiple-cell
1
2
3
4
5
31
32
33
34
11
12
13
14
15
21
22
23
24
3
54
22
18
14
8
6
7
—
—
—
6
6
8
5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10
1
19†
—
8
—
—
—
—
—
7
20
12†
9
—
—
—
—
6
14
—
—
5
—
—
—
—
—
5
18
—
6
—
—
—
*NAL, National Agricultural Library, Beltsville, Maryland; BB, Branchville Rd., Berwyn, Maryland.
†Green abdomen female died early in experiment.
The hypothesis of obligate inquilinism predicted that:
treatment (1) (centre-cell individuals only) would have
mono-cell galls only; treatment (2) (side-cell individuals
only) would have no galls; treatment (3) (both centre- and
side-cell individuals) would have both mono-cell and
multiple-cell galls; and treatment (4) (without psyllids) would
have no galls. The significance of the differences in frequency
of these outcomes among treatments was assessed by a χ2
contingency test.
Center-cell nymph removal experiment
A second experiment tested the dependency of side-cell on
centre-cell nymphs. Aggregates of newly hatched nymphs
were located in the field and the centre-cell nymph was
experimentally removed at various stages during gall formation, which occurs through gradual envelopment of the
feeding nymph by leaf tissue.
Manipulations for this experiment were conducted in the
field using a dissecting microscope fixed to a plastic manipulation and recording platform; this set-up could either be
fastened to a movable tripod or suspended from a shoulder
harness, so that dissections and data recording could be
performed with unencumbered hands. Insect pins were used
to pick the centre-cell nymph out of the leaf.
The treatments were: (1) centre-cell nymphs removed
early, i.e. before any nymphs were enclosed in the gall;
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
(2) centre-cell nymphs removed after the centre-cell individual was enclosed in the gall but the side-cell nymphs were
still exposed; (3) centre-cell nymphs removed when both the
centre- and side-cell nymphs were fully enclosed; and (4) control, in which galls in developmental stages corresponding to
the first three treatments were left undisturbed. Predictions
were that galls of treatment (4) would develop normally,
galls of treatment (1) would not develop and side-cell
nymphs would die, while galls in treatments (2) and (3) might
continue their development and side-cell nymphs might
survive.
Experiments were initiated at four time intervals in late
May 1993 at the NAL site. Leaf-gallers were abundant, and
several nymphal aggregations in different gall developmental
stages were often found on the same leaf. Each replicate, consisting of two or more treatments, was performed on a single
leaf when possible, or on different leaves of the same twig.
The experimental leaves were numbered and marked with
plastic tape on the twig just below the petiole. The position
of each gall on the leaf was sketched on paper at the time the
centre-cell nymph was removed, and the development (size)
of the gall was measured immediately and again 2 weeks,
2 months and 4 months later. The total number of replicates
for each treatment, which was determined in part by the
availability of galls in all the developmental stages, ranged
from 16 to 33.
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Psyllid gall inquiline • M.-M. Yang et al.
Table 3 χ2 tests for allele frequency differences between nymphal populations of leaf gall inhabitants in the same cell positions, all loci.
Contrast number
Contrast*
A1
Hairy nipple gall:
mono- vs. centre-cell (CMt)
Glabrous nipple gall:
mono- vs. centre-cell (NAL)
Glabrous nipple gall:
mono- vs. centre-cell (BB)
Glabrous nipple gall (mono- + centre-cells):
NAL vs. BB
Glabrous nipple gall (side-cell):
NAL vs. BB
Star gall (mono-cell):
NAL vs. BB
A2
A3
B1
B2
B3
χ2
Degrees of freedom
Significance level†
6.69
10
NS
14.90
13
NS
5.60
5
NS
35.69
33
NS
10.28
9
NS
10.67
11
NS
*Population abbreviations: BB, Branchville Rd., Berwyn; CMt, Catoctin Mountain Park, Thurmont; NAL, National Agricultural Library, Beltsville. All localities are in Maryland.
†NS = non-significant, P > 0.1.
Timing of adult female appearance
To compare the time of appearance of centre- vs. side-cell
females, females were collected at the NAL site every 5 days
in spring 1993, from April 15 to May 15. Females found on
the plant were randomly collected from different branches.
About 50 – 70 females were collected each time, and the
numbers of brown abdomen and green abdomen individuals
were recorded to determine the change in proportion of the
two colour morphs, putatively corresponding to gall-formers
vs. inquilines, respectively, over time.
Results
Morphological comparison
Initial gall dissections showed consistent colour differences
between the fifth instar nymphs from centre-cells and sidecells, especially in wing pad colour. The centre-cell nymph
always has dark wing pads, which are usually brown, whereas
the side-cell nymph always has light-coloured wing pads,
which are usually yellow. The nymphs from mono-cell galls
and from centre-cells within multiple-cell galls are brown in
general colour with green or red colour in the intersegmental
membranes. Side-cell nymphs are greenish yellow in general
body colour without differentially coloured intersegmental
membranes.
Field observations and laboratory rearing results also
suggested that first instar nymph and adult coloration are
associated with cell position. Side-cell nymphs in the first instar
are white, sometimes with dark maculation, and adult females
have a green abdomen. Center-cell individuals have yellow first
instar nymphs and adult females have a dark brown abdomen.
Differences in morphology between centre- and side-cell
individuals, apart from coloration, are slight. However,
side-cell individuals consistently differ from all leaf gall-formers
in the shape of the forceps of the male genitalia (see ‘Species
description’ below).
102
Electrophoretic data
Of the 15 loci analysed, three were monomorphic, while the
other 12 showed differences among individuals from different
gall types or cell positions.
Allozyme frequencies in centre-cell nymphs from multiplecell galls vs. mono-cell galls of the same gall type at the same
site were not significantly different (Table 3, contrast A1–
A3), and so these data were pooled. We also pooled the two
nearby populations (NAL and BB) of combined mono- and
centre-cell nymphs from glabrous nipple galls (Table 3, contrast B1), the NAL and BB populations of mono-cell nymphs
from star galls (Table 3, contrast B3) and the NAL and BB
populations of side-cell nymphs from glabrous nipple galls
(Table 3, contrast B2) for the same reason. There were six
populations in all after pooling (Table 3).
Within both the glabrous and the hairy nipple gall samples,
there are strong, highly significant frequency differences
between side-cell nymphs and centre-cell plus mono-cell
nymphs (Table 5, contrast C1–C2). The most pronounced
differences are at the malic enzyme (ME) locus (Table 4); in
both types of nipple galls, centre-cell nymphs have frequencies
above 0.90 for allele D and below 0.05 for allele F, while in
side-cell nymphs, frequencies are 0.29–0.57 for allele D and
0.41–0.60 for allele F.
There are also marked frequency differences between the
side-cell samples from hairy nipple galls (site CMt) as compared to sympatric mono-cell blister galls (Table 5, contrast
D1), and between side-cell nymphs from the glabrous nipple
gall (sites NAL/BB) and sympatric mono-cell star galls
(Table 5, contrast D2). Thus, the side-cell individuals are
unlikely to be conspecific with either of these co-occurring
gall types.
Allele frequencies in side-cell individuals from the two nipple
gall types differ significantly from each other, but much less
dramatically than each differs from sympatric centre-cell
Zoologica Scripta, 30, 2, April 2001, pp97–113 • © The Norwegian Academy of Science and Letters
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M.-M. Yang et al. • Psyllid gall inquiline
Table 4 Allele frequencies (after pooling) of nymphs from different cell positions, within various gall types collected in 1992 and 1993. Major
allele frequency differences between side-cell and centre-cell nymphs are underlined.
Population (Gall type, locality*, cell position†)
Enzyme
Hairy nipple
(CMt) m + c
Hairy nipple
(CMt) side
Blister
(CMt) mono
Glabrous nipple
(NAL + BB) m + c
Glabrous nipple
(NAL + BB) side
Star
(NAL + BB) mono
PGM
(N )
C
D
E
F
G
H
I
30
0.017
0.267
0.017
0.583
0.000
0.117
0.000
21
0.000
0.024
0.000
0.786
0.000
0.119
0.071
18
0.000
0.222
0.028
0.639
0.028
0.083
0.000
33
0.000
0.152
0.061
0.697
0.015
0.076
0.000
19
0.000
0.000
0.000
0.895
0.000
0.105
0.000
22
0.000
0.091
0.023
0.864
0.023
0.000
0.000
PGI
(N)
C
D
E
F
G
30
0.950
0.000
0.050
0.000
0.000
21
1.000
0.000
0.000
0.000
0.000
18
0.972
0.000
0.000
0.000
0.000
33
0.864
0.045
0.091
0.000
0.000
19
1.000
0.000
0.000
0.000
0.000
24
0.896
0.000
0.042
0.021
0.042
GPDH
(N)
A
B
C
D
23
0.000
0.000
1.000
0.000
18
0.000
0.000
1.000
0.000
17
0.000
0.029
0.971
0.000
33
0.000
0.121
0.879
0.000
19
0.000
0.000
0.974
0.026
24
0.083
0.042
0.875
0.000
ME
(N)
A
D
E
F
30
0.000
0.983
0.000
0.017
21
0.000
0.286
0.119
0.595
18
0.056
0.917
0.000
0.028
39
0.051
0.910
0.000
0.038
22
0.000
0.568
0.023
0.409
27
0.000
0.889
0.037
0.074
FUM
(N)
B
D
F
30
0.000
0.983
0.017
21
0.000
0.905
0.095
17
0.000
0.912
0.088
33
0.015
0.970
0.015
19
0.000
1.000
0.000
24
0.000
0.938
0.063
PEP-1
(N)
A
B
C
D
E
F
12
0.042
0.333
0.583
0.000
0.042
0.000
12
0.000
0.250
0.625
0.042
0.083
0.000
14
0.000
0.000
1.000
0.000
0.000
0.000
18
0.000
0.083
0.806
0.000
0.083
0.028
12
0.000
0.083
0.917
0.000
0.000
0.000
22
0.000
0.114
0.886
0.000
0.000
0.000
FDP
(N)
A
B
23
0.000
1.000
18
0.000
1.000
8
0.000
1.000
27
0.000
1.000
16
0.000
1.000
20
0.000
1.000
TPI
(N)
A
B
E
23
0.000
1.000
0.000
18
0.000
1.000
0.000
12
0.000
1.000
0.000
27
0.000
1.000
0.000
16
0.000
0.969
0.031
16
0.031
0.969
0.000
G3PDH
(N)
A
B
23
0.000
1.000
18
0.000
1.000
10
0.000
1.000
27
0.000
1.000
16
0.000
1.000
22
0.000
1.000
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
103
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Psyllid gall inquiline • M.-M. Yang et al.
Table 4 continued
Population (Gall type, locality*, cell position†)
Enzyme
Hairy nipple
(CMt) m + c
Hairy nipple
(CMt) side
Blister
(CMt) mono
Glabrous nipple
(NAL + BB) m + c
Glabrous nipple
(NAL + BB) side
Star
(NAL + BB) mono
IDH-1
(N )
C
D
E
F
G
30
0.000
0.233
0.767
0.000
0.000
18
0.000
0.389
0.611
0.000
0.000
18
0.000
0.972
0.028
0.000
0.000
39
0.000
0.103
0.872
0.026
0.000
22
0.000
0.023
0.932
0.000
0.045
27
0.204
0.204
0.500
0.093
0.000
IDH-2
(N )
C
D
E
29
0.000
1.000
0.000
21
0.000
1.000
0.000
15
0.000
1.000
0.000
39
0.026
0.949
0.026
22
0.000
1.000
0.000
27
0.000
1.000
0.000
MDH-1
(N )
A
B
C
D
E
F
G
28
0.000
0.071
0.036
0.875
0.000
0.018
0.000
21
0.000
0.024
0.048
0.405
0.119
0.381
0.024
13
0.000
0.346
0.000
0.654
0.000
0.000
0.000
33
0.061
0.152
0.000
0.742
0.000
0.030
0.015
19
0.000
0.158
0.000
0.632
0.079
0.079
0.053
16
0.063
0.219
0.000
0.719
0.000
0.000
0.000
MDH-2
(N )
A
B
C
26
0.000
0.000
1.000
18
0.000
0.000
1.000
14
0.000
0.000
1.000
39
0.000
0.000
1.000
22
0.000
0.000
1.000
27
0.000
0.000
1.000
6PGDH
(N )
A
B
C
D
E
F
G
28
0.000
0.018
0.018
0.839
0.000
0.071
0.054
21
0.024
0.024
0.000
0.952
0.000
0.000
0.000
16
0.000
0.000
0.000
0.813
0.031
0.156
0.000
39
0.000
0.000
0.000
0.987
0.000
0.013
0.000
22
0.000
0.000
0.000
0.841
0.068
0.091
0.000
27
0.000
0.000
0.000
0.870
0.019
0.111
0.000
LDH
(N )
C
D
E
F
30
0.033
0.833
0.133
0.000
21
0.095
0.881
0.024
0.000
17
0.118
0.824
0.000
0.059
38
0.000
0.868
0.092
0.039
22
0.068
0.932
0.000
0.000
27
0.000
0.833
0.019
0.148
*Abbreviations for locality: BB, Branchville Road, Berwyn, MD; NAL, National Agricultural Library, Beltsville, MD; CMt, Catoctin Mountain Park, Thurmont, MD.
†Cell position within an individual gall: m or mono, mono-cell; c, centre-cell; side, side-cell.
Contrast number
Contrast*
C1
Hairy nipple gall (CMt):
(mono- + centre-) vs. side-cell
Glabrous nipple gall (NAL + BB):
(mono- + centre-) vs. side-cell
CMt: Hairy nipple gall (side-cell)
vs. blister gall (mono-cell)
NAL + BB: Glabrous nipple (side-cell)
vs. star gall (mono-cell)
Side-cell: Hairy nipple gall
vs. glabrous nipple gall
C2
D1
D2
E1
χ2
Degrees of freedom
Significance level
87.95
13
†
63.87
15
†
106.87
16
†
58.36
14
†
53.78
13
†
Table 5 χ2 tests for allele frequency
differences between: nymphs in different cell
positions within the same gall type; side-cell
nymphs and other co-occurring mono-cell
gall types; and side-cell nymphs from
different gall types, all loci.
*Population abbreviations: BB, Branchville Rd., Berwyn; CMt, Catoctin Mountain Park, Thurmont; NAL, National
Agricultural Library, Beltsville.
†P < 0.005.
104
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M.-M. Yang et al. • Psyllid gall inquiline
Fig. 1 Distance Wagner (midpoint rooting) (A) and UPGMA (B) trees using Rogers’ distance ( Rogers 1972) on allele frequencies at 15
allozyme loci, for eight populations of Pachypsylla leaf gall nymphs from different gall types and cell positions. Populations are given in
parentheses. m, mono-cell gall; c, centre-cell of multiple-cell gall; side, side-cell of multiple-cell gall; BB, Branchville, Berwyn; CMt, Catoctin
Mountain Park, Thurmont; NAL, National Agricultural Library, Beltsville.
individuals ( Table 5, contrast E1). These samples are from
populations ~ 85 km apart, in very different habitats, and the
degree of differentiation between them is typical of differences
between samples of any one gall type from the same two localities. Both in the present study ( Fig. 1A) and in an extensive
survey of allozyme variation within and among the species of
Pachypsylla and related genera ( Yang 1995), the two side-cell
populations are grouped together whenever a phylogenetic
(additive tree) grouping method is applied, although they are
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
sometimes separated (by one additional node) on phenetic
( UPGMA) trees ( Fig. 1B). The side-cell samples never grouped
with the nipple gall types in which they developed.
Isofemale progeny rearing experiment
None of the offspring of the 18 green abdomen, presumably
side-cell, females formed galls, nor were any galls found in
the control bags. In contrast, offspring of 12 of the 18 brown
abdomen females caged alone did form galls (Table 2).
105
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Psyllid gall inquiline • M.-M. Yang et al.
The difference between green and brown abdomen females
in fraction of progenies forming galls is highly significant
( χ2 = 18.0, d.f. = 1, P < 0.005). The progenies of brown
abdomen females caged alone produced only single-cell galls.
In contrast, when brown and green abdomen females were
caged together, both single- and multiple-cell galls were
formed in all eight of the replicates that produced any galls at
all, except that in the two cages in which the green abdomen
female died early in the experiment, only single-cell galls
were formed. The difference in proportion of gall-producing
progenies that included multiple-cell galls, between trials
with brown abdomen females only and those with both green
and brown abdomen females, is highly significant (χ2 = 12.9,
d.f. = 1, P < 0.005).
In the trials containing only the progeny of a green abdomen
female, galls were never formed. Hatchlings were observed
crawling on the leaves, but later instars were never seen.
Likewise, in the field, Pachypsylla nymphs past the hatchling
stage were never seen outside of galls. We infer that neither
the inquiline nor the leaf-gallers can develop as free-living
nymphs.
Center-cell nymph removal experiment
Galls in which the centre-cell nymph was left intact until the
third stage (all nymphs fully enclosed by the gall) mostly
completed normal development (> 91%, n = 23). However,
removal of the centre-cell nymph at any of the three stages
invariably resulted in significantly smaller galls than the
corresponding control, as judged by the non-overlap of 95%
confidence limits seen in the plot of gall diameter vs. observation date in Fig. 2. While one could argue that differences
in gall size were due to gall damage when the centre-cell
individual was removed, this explanation is unsatisfactory in
cases where the centre-cell individual was removed at the first
stage, before it was enclosed by the gall.
None of the inhabitants of galls in which the centre-cell
nymph was removed at the first stage survived, whereas the
corresponding undisturbed controls developed until emergence (Fig. 2A). Some of the treated leaves fell off the tree
before the last examination, reducing the number of observations in each category at the last stage from 16–33 to 3–15.
Leaves bearing the controls for trials in which the centre-cell
nymphs were removed at the first stage dropped from the tree
before the last examination was performed; in one case a
dropped control leaf was found on the ground. Therefore, no
measurements of this first control group were taken in the
fourth time period (Fig. 2A). However, the leaf found on
the ground had three galls and all had 1–3 obvious psyllid
emergence holes, suggesting that the insects survived until
adulthood. Those galls with centre-cell individuals removed
at the second and third stages kept developing until the adult
stage, although survivorship was lower (42.8% and 55.6%,
106
Fig. 2 Change in gall diameter through time for treatment
(centre-cell individual destroyed) vs. control (centre-cell individual
undisturbed). Destruction was conducted at three different times
during gall initiation. —A. Both centre- and side-cell nymphs
exposed. —B. Centre-cell nymph enclosed but side-cell nymphs
exposed. —C. Both centre- and side-cell nymphs enclosed in galls.
Observation time 1, time when destruction implemented; time 2,
2 weeks after time 1; time 3, 2 months after time 1; time 4, 4 months
after time 1. Number of individuals surviving until emergence/total
observed individuals is given at time 4 in parentheses.
respectively) than in the corresponding controls (86.7% and
100%, respectively; Fig. 2B,C). Summing across treatments,
the overall survivorship within intact gall controls was
91%, whereas survivorship within galls with the centre-cell
Zoologica Scripta, 30, 2, April 2001, pp97–113 • © The Norwegian Academy of Science and Letters
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M.-M. Yang et al. • Psyllid gall inquiline
Fig. 3 Change in proportion of centre- to side-cell females of
hackberry leaf gall-makers at Beltsville, Maryland, in the spring of
1993. Numbers in parentheses below each date indicate total
number of individuals collected.
individual removed was 40%, a highly significant difference
( χ2 = 12.8, d.f. = 1, P < 0.005).
Timing of female appearance
Leaf gall-maker females were occasionally found in early
April, but did not become abundant until mid-April. On
April 15, 1993, none of the females caught in the field had
green abdomens ( Fig. 3). Ten days later (April 25), the proportion of green abdomen females, presumably side-cell
individuals, increased to 10%, and in the first 2 weeks of May,
it rose to around 70%, where it remained for the rest of the
sampling period.
Discussion and conclusions
The results strongly support the hypothesis that, at least
within nipple galls, side-cell individuals are a different species
than centre-cell and mono-cell species. The evidence is as
follows: (1) the adult abdomen of side-cell individuals is
green while that of centre- and mono-cell individuals is
brown; (2) first instars of side-cell individuals are white, often
with dark maculation, while those of centre- and mono-cell
individuals are yellow; (3) fifth instars of side-cell individuals
have light-coloured wing pads, and a greenish yellow body
without intersegmental markings; fifth instars of centreand mono-cell individuals have dark brown wing pads, and
a brown body with green or red intersegmental markings;
(4) sympatric samples of side-cell and centre- and mono-cell
individuals, often from the same gall, show dramatic differences
in allozyme frequencies, particularly at the ME locus; (5) green
abdomen (side-cell) adults appear later in the season than
brown abdomen (centre- and mono-cell) adults.
Strong allozyme frequency and coloration differences
between side-cell samples and sympatric blister or star gallers
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
permit rejection of Moser’s (1965) hypothesis that side-cells
are ‘marginal galls’ overgrown by a nipple gall. The fact that
multiple adults emerging from a single gall are not necessarily conspecific may help explain some of the confusion about
species recognition in Pachypsylla leaf-gallers.
The close morphological and allozyme similarity between
the two geographical populations of inquilines examined in
detail is consistent with the hypothesis that these entities
represent a single inquiline species. Phylogenetic analyses of
allozyme frequencies and of discrete allozyme characters
combined with morphological characters, for a broad sample
across Pachypsylla (Yang 1995), invariably group the side-cell
populations together on the basis of two unique apomorphies:
the abrupt apical tapering of the forceps of the male genitalia
(see ‘Species description’ below) and the high frequency of
the ‘F’ allele for malic enzyme. Failure of the two inquiline
samples to group together in the UPGMA tree in this study,
based on overall similarity of allozyme frequencies, probably
reflects the small divergence and lack of absolute separation
in allozymes among species in the leaf gall-forming complex
more generally. From a broad survey of the distribution of
multiple-cell leaf galls (see ‘Species description’), it appears
that the inquiline is associated with most of the Pachypsylla
leaf-galler species, and seems to occur broadly across their
collective range.
Results of rearing and centre-cell removal experiments
fully support Riemann’s (1961) hypothesis that side-cell individuals are true, obligate inquilines. Galls were never formed
independently by progeny of side-cell females, and side-cell
nymphs never survived in the field if the centre-cell nymph
was removed before the gall was well established. Conversely,
galls with side-cells were never formed by the progeny of
centre-cell females alone; the nymphal habit of feeding next
to a gall-inducer is presumably unique to the side-cell form.
As would be expected, if the inquiline nymphs need to feed
close to already initiated galls, the phenological observations
also show that, on average, the side-cell females return to the
host to mate and lay eggs later than centre-cell or mono-cell
females (Fig. 3).
While side-cell individuals cannot initiate a gall, it is possible
that they could influence subsequent gall development. In cynipids, the species-specific gall morphology is sometimes altered
by the presence of inquilines (Evans 1965; Shorthouse
1980; Askew 1984; Meyer 1987). Although one cannot be
sure whether there are side-cell individuals in a Pachypsylla
leaf gall without dissection, it is sometimes possible to distinguish mono-cell galls from multiple-cell galls by appearance.
Especially in the glabrous nipple gall, the mono-cell gall is
usually perfectly rounded, while protrusions on the gall
margin are sometimes evident when side-cells are present.
This observation, and the fact that the inquilines are enclosed
in separate cells and sometimes survive even after removal of
107
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Psyllid gall inquiline • M.-M. Yang et al.
the gall initiator once the gall is well established, suggest that
side-cell individuals can modify gall development to some
degree.
It seems clear that the inquiline is derived from an ancestor
that made an enclosed gall. In the phylogenetic analysis of
Pachpsylla ( Yang 1995), the inquiline was placed well within a
monophyletic group that includes leaf gall-makers. All other
members of this and the other lineages within the genus form
enclosed galls, which is thus very likely to have been the
ancestral habit. Judging from its close similarity in morphology
and allozymes to the gall-formers, the Pachypsylla inquiline
appears to have diverged quite recently from its gall-forming
congeners.
Among gall-forming Sternorrhyncha generally, inquilines
appear to be rare, although if they are typically as similar to
their hosts as in Pachypsylla, there are undoubtedly more
undiscovered cases. Apart from Pachypsylla, the only obligate
hemipteran inquiline that we are aware of is the aphid Eriosoma
yangi, although a number of aphids seem to be facultatively
inquiline parasites (Akimoto 1988a,b). In these cases, the fundatrices invade and take over each others’ galls, particularly
in species where the gall is formed at some distance from the
site of induction. Inquiline species may be substantially more
numerous in other gall-forming groups. In Cynipidae, for
example, inquilinism characterizes about 175 species in six or
more genera; however, these genera probably form a monophyletic group, and may represent a single origin of the
inquiline habit, with subsequent radiation (Ronquist 1994).
Among Palaearctic gall midges (Cecidomyiidae), inquilines
appear to have arisen independently in a number of genera
and comprise about 90 species, about 6% of the total (Skuhravá
et al. 1984). On the other hand, obligate inquilinism is apparently unknown in several other groups with substantial
numbers of gallers, such as tephritid flies (Freidberg 1984)
and scale insects (Beardsley 1984).
All instances of obligate inquilinism in the aforementioned
groups seem to have arisen from gall-forming ancestry. The
selective factors that favour this evolutionary step are unclear.
Akimoto (1981, 1988a) hypothesized that facultative inquilinism
was a preadaptation for obligate inquilinism in Eriosoma aphids,
which he postulated arose by dispersal beyond the range of
the primary host. This could result in strong selection for
invasion of galls formed by related species, if the dispersants
could not form their own galls on the available hosts. Subsequent
specialization for this habit might preclude the re-acquisition
of gall induction, even upon re-colonization of the ancestral
host. This scenario seems doubtful for Pachypsylla, however,
given that the inquiline seems to have a broader host plant
range than the individual gall-forming species.
An alternative explanation, not invoking unique historical
circumstances, is that inquilinism represents a strategy for
avoiding high mortality risk associated with gall initiation.
108
Sources of such risk might include gall failure due to phenological mismatch with host development, and predation or
desiccation during a longer period of exposed feeding. Under
this hypothesis, which seems plausible for Pachypsylla, the
potential benefits of obligate inquilinism might be outweighed
by the risk of mortality from failure to find a host. Thus, we
might expect obligate inquilines to be most likely to arise in
groups in which gallers are very abundant, and in which
mortality risk during gall formation is high. Pachypsylla
leaf-gallers are often extremely abundant; their mortality
rates during gall initiation have yet to be measured.
Finally, what is the effect of inquilines on the gall-forming
host, and how might it influence evolution of the inquiline–
host interaction? Although inquilines can be viewed as
parasites of gall tissue, they cannot simply be assumed to be
detrimental to the host. Meyer (1987: 177) defined inquilines
as ‘insects living commensally in the gall cortex’, implying
that they do little harm to the true gall-inducer. The inquiline
Synergus pallicornis in galls of the cynipid Cynips quercusfolii is
an example (Askew 1961). However, other studies on cynipid
galls have demonstrated that inquilines have negative effects
on gall-inducers by food deprivation, or even by directly
killing the host at an early stage of development (Evans 1965,
1967; Shorthouse 1973, 1980; Washburn & Cornell 1981).
For example, the inquiline Periclistus kills the gall-former
Diplolepis at oviposition (Shorthouse 1980).
The fitness effects of the Pachypsylla inquiline have not
been measured, but anecdotal evidence suggests a detrimental
effect. Riemann (1961) reported that the centre-cell nymph
can be killed by expansion of the side-cells. Heard & Buchanan
(1998) studied the larval performance of the hackberry nipple
gall-maker, P. celtidismamma, when a leaf contained: a single
gall with only P. celtidismamma; multiple galls with only
P. celtidismamma; and galls containing P. celtidismamma and its
inquiline. Their results showed that larval performance was
best on leaves that contained multiple P. celtidismamma and
no inquiline and was worst when a gall contained inquilines.
Their explanation was that there is positive intraspecific facilitation with multiple single-species infestations, but that
there is negative impact in inquiline-infested galls because of
reduced resources. We speculate that the selective advantage
of avoiding the inquiline could have promoted the divergence
of phenology and gall morphology in the leaf gall-forming
complex of Pachypsylla.
Genus Pachypsylla Riley, 1983
Pachypsylla cohabitans Yang and Riemann sp. n.
(Figs 4 and 5)
Holotype. m (pinned) UNITED STATES, Maryland, Beltsville,
National Agricultural Library ( NAL), 22 September 1992, ex
glabrous nipple gall on Celtis tenuifolia (USNM).
Paratypes. 1m, 2ff (slide mounted), NAL, 16 October 1993,
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M.-M. Yang et al. • Psyllid gall inquiline
Fig. 4 Scanning electron micrographs of Pachypsylla cohabitans adult. —A. Head, frontal view. —B. Enlargement of area around frons, showing
surface texture of vertex and genae. —C. Antenna. —D. Forewing (arrow indicates enlargement area in E ). —E. Surface texture and setae along
veins of forewing. —F. Tarsus, ventral view. —G. Male genitalia, lateral view. —H. Female genitalia, lateral view (arrow indicates the lateral
process beside female circum anus). a, anus; ae, aedeagus; c, claws; cr, circumanal ring; cu1, cell cu1; e, empodium; f, frontal ocellus; fp,
parameres; g, genal cone; m2, cell m2; p, protiger; sp, subgenital plate; v, vertex.
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
109
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Psyllid gall inquiline • M.-M. Yang et al.
ex glabrous nipple gall on Celtis tenuifolia (USNM); 2ff,
Maryland, Berwyn, Branchville Road (BB), 16 October 1993, ex
glabrous nipple gall on Celtis tenuifolia (USNM); 3mm, 5ff
(slide mounted), 9 November 1993, Maryland, Catoctin
Mountain Park, Thurmont (CMt), ex hairy nipple gall on
Celtis occidentalis (USNM); 6mm, 9ff (pinned), NAL, 22 November 1992, ex glabrous nipple gall on Celtis tenuifolia (USNM);
13mm, 12ff (pinned), NAL, 21 November 1993, ex glabrous
nipple gall on Celtis tenuifolia (USNM; BMNH; NHMB;
NCHU); 2mm, 3ff (pinned), BB, 19 September 1992, ex
glabrous nipple gall on Celtis tenuifolia (USNM); 2mm, 3ff
(pinned), BB, 24 September 1992, ex glabrous nipple gall on
Celtis tenuifolia (USNM); 1m, 1f (pinned), BB, 25 September
1992, ex glabrous nipple gall on Celtis tenuifolia (USNM);
2mm (pinned), BB, 26 September 1992, ex glabrous nipple
gall on Celtis tenuifolia (USNM); 2mm (pinned), NAL,
18 September 1993, ex star gall on Celtis tenuifolia (USNM);
1m (pinned), BB, 19 September 1992, ex star gall on
Celtis tenuifolia (USNM); 3mm, 2ff (pinned), BB, 28 September 1992, ex hairy nipple gall on Celtis occidentalis (USNM);
3mm, 2ff (pinned), CMt, 5 September 1992, ex hairy nipple
gall on Celtis occidentalis (USNM); 1f (pinned), CMt, 26 September 1992, ex hairy nipple gall on Celtis occidentalis
( USNM); 1m (pinned), North Dakota, Fargo, 7 October 1993,
ex hairy nipple gall on Celtis occidentalis (USNM); 2 fifth
instar nymphs (slide mounted), CMt, 13 September 1992, ex
hairy nipple gall on Celtis occidentalis (USNM); 9 fifth
instar nymphs (slide mounted), CMt, 29 September 1992
(USNM; BMNH; NHMB; NCHU); 10 fifth instar nymphs
(slide mounted), NAL, 9 October 1992, ex glabrous nipple
gall on Celtis tenuifolia (USNM; BMNH; NHMB; NCHU);
6 fifth instar nymphs (slide mounted), BB, 9 October 1992, ex
glabrous nipple gall on Celtis tenuifolia (USNM); 6 fifth instar
nymphs (spirits), NAL, 16 October 1993, ex glabrous nipple
gall on Celtis tenuifolia ( USNM); 3 fifth instar nymphs (spirits),
BB, 19 September 1992, ex glabrous nipple gall on Celtis
tenuifolia ( USNM); 3 fifth instar nymphs (spirits), BB,
9 October 1992, ex glabrous nipple gall on Celtis tenuifolia
(USNM); 1 fifth instar nymph (spirits), NAL, 18 October
1992, ex star gall on Celtis tenuifolia (USNM); 2 fifth instar
nymphs (spirits), NAL, 16 October 1993, ex star gall on
Celtis tenuifolia (USNM); 6 fifth instar nymphs (spirits), CMt,
28 September 1993, ex hairy nipple gall on Celtis occidentalis
( USNM ); 2 fifth instar nymphs (spirits), North Dakota,
Fargo, 5 October 1993, ex hairy nipple gall on Celtis occidentalis (USNM).
Etymology. The species epithet is a Latin noun in apposition
meaning ‘the one who is living with’ and refers to the obligate
inquiline characteristics of this species.
Fig. 5 Nymphal characters of Pachypsylla cohabitans. —A. Fifth instar
nymph. —B. First instar.
110
Diagnosis. The inquiline and the true leaf gall-formers among
hackberry psyllids are extremely similar. The inquiline species
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M.-M. Yang et al. • Psyllid gall inquiline
is most easily distinguished from the true gall-formers by
colour. The abdomen of the adult female inquiline is green,
whereas in the females of the star as well as glabrous and hairy
nipple gall-formers it is brown. In addition, the general body
colour of the first instar nymph is white, usually with dark
maculation, in inquilines, while in the two nipple gallers and
star gallers the first instar is yellow. In the fifth instar nymph,
in the inquiline the wing pads are yellow, while in both the
glabrous and hairy nipple gallers and in disc gall-formers they
are brown. In the star galler, the wing pads are somewhat
lighter than in the nipple gallers, and are not always distinct
from the condition in the inquiline. In the inquiline, the apex
of the male parameres is angled, while in gall-formers of
the two nipple gall types, it is smoothly rounded. In the star
gall-maker it forms a sharper angle, nearly 90°, more than in
the inquiline species.
Description
Adult male ( holotype). General body colour yellowish brown
with dark brown maculation. Eyes red. Antennae yellowish
brown with dark brown annulus apically on segments III to
VIII, on basal half of segment I to II; completely dark on
segments IX–X. Forewing transparent, mottled and spotted
with round brown dots throughout except in a clear subapical
oblique band. Hindwing clear with some brown dots on anal
region. Entire body punctate and shortly pubescent, most
prominently on head and thoracic dorsum. Dorsum strongly
arched; body robust.
Head. Vertex nearly or quite perpendicularly inclined to
longitudinal body axis. Genal cones ( Fig. 4A) subconical,
shorter than half length of vertex. Both vertex and genal
processes covered with wrinkles and short hairs (Fig. 4B),
hairs longer at apex of genal cones. Antenna (Fig. 4C) length
nearly equal to width of head; width of flagellum increasing
distally, especially from segments VI to VIII.
Thorax. Strongly arched and broad, widest part of thorax
slightly wider than head width. Forewing (Fig. 4D) obovate,
broadest at apical one-third; cu1 and m2 cells long and
narrow, 5 – 6 times as long as wide. Fore- and hindwings
with spinules over membranous area (Fig. 4E). Pterostigma
distinct. Hind coxa with ventral crescentic denticulate patch
on outer side above meracanthus, covered with sharply
pointed structure. Inner side of crescentic denticulate patch
with obvious grooves and ridges in fan-like arrangement.
Meracanthus about 1.4 times as long as wide. Hind tibia
without basal spine, with nine dark spurs apically. Two black
spurs on basal segment of hind tarsus; two sharp claws on
apical segment, with two setae between lobes of empodium
(Fig. 4F).
Male genitalia (Fig. 4G). Proctiger bipartite; terminal segment
covered with dense setae; distal half of basal segment covered
with hairs, less dense than terminal segment. Parameres, in
© The Norwegian Academy of Science and Letters • Zoologica Scripta, 30, 2, April 2001, pp97–113
lateral view, abruptly narrowed subapically, lateral margins
parallel. Aedeagus hooked at apex of head of distal segment;
base of head at junction of basal rod forming sharp angle.
Adult male (paratypes). Differ from holotype as follows. General
body colour yellowish brown or green. Antenna (Fig. 4C)
length sometimes slightly shorter than width of head. Hind
tibia with variable number (8–10) of dark spurs apically.
Adult female (paratypes). Differ from holotype as follows.
Abdomen yellowish green. Genitalia (Fig. 4H) triangular in
lateral view, wide at base, pointed towards end. Proctiger with
paired, thumb-like processes at lateral proximal side; setae of
homogeneous size, evenly distributed. Circumanal ring with
single row of pores.
Fifth instar nymphs (Fig. 5A). General body colour yellowish
green with yellow wing pads. Eyes red. Antenna 10-segmented.
Dorsal surface of head lacking conspicuous sclerotized areas.
Clusters of simple setae over head and thorax. Trochanter
present. Abdomen densely covered with transverse rows of
simple setae. One pair of ventral plates anterior of caudal
plate. Paired, small, semicircular plates in mediodorsal area
near caudal plate. Circumanal ring absent. Caudal plate in
ventral view entire, with pointed sclerotized caudal spurs,
central apical spur acute; apical caudal spurs present in apical
two segments only; abdominal caudal spurs absent.
First instar nymphs (Fig. 5B). General body colour white,
usually with dark maculation on each segment. Eyes red.
Body oval, no indentation on margins between thorax and
abdomen. Antenna three-segmented. Base of antenna very
close to margin of head, antenna beyond first segment
outside margin of head when extended. Wing buds absent.
Legs with two long setae on outer apex of tibia, nearly as long
as tibia. Ventral side of all coxae with one stout seta pointing
medially. Simple setae along margin of head and abdomen.
Anus membranous, forming tube at end of body.
Distribution. The inquiline species is closely associated with
the distribution of Pachypsylla leaf-gallers, and seems to
occur broadly across their collective range. Direct observations of multiple-cell leaf galls, which we assume indicates
the presence of the inquiline, were made for: hairy nipple
galls (P. celtidismamma) in MD, VA, NY, OH, IN, AR, ND on
Celtis occidentalis; glabrous nipple galls (P. celtidisglobulus) in
MD, VA, LA, TX, AR on C. tenuifolia; star galls (P. celtidisasterisca)
in MD, VA, LA, TX on C. tenuifolia and C. lavigata; disc galls
(P. celtidisumbilicus) in MD, VA, AR (multiple cells are rare in
this gall type) on C. occidentalis; and blister galls in TX, AZ,
LA on C. reticulata and C. lavigata. In the above blister gall
samples, multiple-cell galls show a less obvious distinction
111
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Psyllid gall inquiline • M.-M. Yang et al.
between centre- and side-cells than is apparent in other gall
types. The only leaf gall samples we examined which never
had inquilines were those of blister galls on Celtis occidentalis
in MD, VA, NY, OH, IN, AR, ND.
Indirect evidence of the occurrence of inquiline species
was also obtained by examination of the abdomen colour of
pinned female specimens in the USNM psyllid collection.
The problem with these data is that teneral females of the
leaf-gallers can also have light abdomens. Light abdomen
females were taken from hairy nipple galls in Ottawa, Canada;
from nipple galls of an uncertain type in AZ, UT, SD, MD,
DC; and from blister galls in AZ, TX, OK, GA, WV, NY.
Host plants and biology. This species is an obligatory inquiline
which is unable to induce a gall by itself. The newly hatched
nymph settles near the leaf gall-former nymph during
gall initiation and is incorporated into the gall of a different
Pachypsylla species. The true gall-former is located in the
centre-cell of the gall, whereas the inquilines are confined
in separate lateral cells surrounding the centre-cell. Although
the centre-cell lumen may sometimes be displaced sideways,
it can be distinguished by its connection to the centre nodule
on the underside of the gall.
Note. J. Riemann is considered an author of the new species,
although not an author of this publication. This act is not in
conflict with the International Code of Zoological Nomenclature. Riemann was the first to hypothesize that it was a
separate inquiline species, he coined the name, and he wrote
the first description of the species (Riemann 1961). Unfortunately, this information was never published.
Acknowledgements
We thank R. Denno, G. Roderick, C. Fenster, W. Lamp,
J. Riemann, J. Moser and T. Friedlander for much valuable
help and advice. We are most grateful to D. Burckhardt and
N. Vandenberg for their detailed review of the manuscript
and for their many helpful suggestions for improving the
final copy. Special appreciation is due to G. Roderick for help
in data analyses and computer consulting; he and L. Garcia
de Mendoza also greatly facilitated the electrophoretic work.
Helpful guidance on the SEM technique was provided by
J. Plaskowitz, and financial support for SEM work was
generously provided by M. Stoetzel, Systematic Entomology
Laboratory, USDA. We are grateful to M. Hardin, T. Jacobs
and staff at Catoctin Mountain National Park and Beltsville
Agricultural Research Center, USDA, for permission to
collect. We thank A. Mitchell for his help in dissecting galls;
S. Cho, G. Hormiga, A. Wijesekara and G. Miller for helpful
discussions and technical support; K. T. Park for his help
in electrophoresis; D. Creel for her help in slide mounting;
L. Garcia de Mendoza and laboratory colleagues for help
112
with field work; and many laboratories of the Department of
Entomology, University of Maryland, for the generous loan
of vehicles. We also thank Yimay Hsu and Linshue Liao of
the National Museum of Natural Science, Taiwan, for their
kind help in preparation of scanning photographs. Financial
support was provided by the Theodore Roosevelt Fund of
the American Museum of Natural History, the Maryland
Agricultural Experiment Station, the Smithsonian Institution,
the Graduate School of the University of Maryland and the
USDA NRI-CGP.
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