ore

JHR 72: 27-43 (20 I 9) JOURNAL OF A peer-reviewed open-access journal
doi: 10.3897/jhr.72.36356 RESEARCH ARTICLE ME Hymenoptera

http://jhr.pensoft.net The Inerational Society of ymenoptersts, RESEARCH

Evolution of glandular structures on
the scape of males in the genus Aphelinus Dalman
(Hymenoptera, Aphelinidae)

Xanthe A. Shirley', James B. Woolley’, Keith R. Hopper’,

Nunzio Isidoro*, Roberto Romani?

| USDA-APHIS-PPQ, 2771 FeB Road, College Station, TX 77845, USA 2 Department of Entomology,
Texas A&M University, College Station, TX 77843, USA 3 USDA-ARS-BIIRL, Newark, DE, 19713, USA
4 Dipartimento Scienze Agrarie, Alimentari e Ambientali, Universita Politecnica Delle Marche, Via Brecce
Bianche, Ancona, 60131, Italy § Dipartimento Scienze Agrarie, Alimentari e Ambientali, Universita degli
Studi di Perugia, Borgo XX Giugno 74, Perugia, 06121, Italy

Corresponding author: James Woolley (jimwoolley@tamu.edu)

Academic editor: Petr Jansta | Received 20 May 2019 | Accepted 6 September 2019 | Published 31 October 2019
http://zoobank. org/BCB88C26-5 1 F8-45F6-B88C-5 D2BCDB48578

Citation: Shirley XA, Woolley JB, Hopper KR, Isidoro N, Romani R (2019) Evolution of glandular structures on the
scape of males in the genus Ap/elinus Dalman (Hymenoptera, Aphelinidae). Journal of Hymenoptera Research 72:
27-43. https://doi.org/10.3897/jhr.72.36356

Abstract

The pores and associated glands on male antennae in species of Hymenoptera are involved in mate recognition
and are diverse and widespread among taxa. However, nothing has been published about these structures in
species of Aphelinus (Chalcidoidea: Aphelinidae), a genus of parasitoid wasps with a long history in biological
control. Images from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of
Aphelinus varipes revealed pores on the ventral side of the male scape that were connected to glands. A survey
of the scapes of male antennae in 16 species in six species complexes of Ap/elinus, as well as two outgroup spe-
cies, Aphytis melinus and Centrodora sp., showed that pores were present in all except Centrodora sp. The pores
varied in several characters: the shape of the structures that carried them, pore size, elevation of the cuticle sur-
rounding the structures, the extent of a carina delimiting the area around the structures, and the number and
position of pores. The shape of the pore-bearing structures, the elevation of cuticle around these structures,
and the extent of the carina around them map well onto a molecular phylogeny of these Aphelinus species.

Combinations of pore characters are diagnostic of species complexes, and in some cases, species of Aphelinus.

Keywords
Aphelinus, antennal morphology, scanning electron microscopy, scape, sexual dimorphism, mate

recognition, glands

Copyright Xanthe A. Shirley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

28 Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

Introduction

Successful mating depends on discriminating between individuals of the same versus dif-
ferent species, as well as between potential mates that will yield progeny with high versus
low fitness. Communication with pheromones that attract conspecifics and provide cues
for their recognition is often a key component in the quest for mates by insects in general
and Hymenoptera in particular (for review, see Ayasse et al. 2001). However, more or less
elaborate courtship behavior may also be needed to secure mates (for reviews, see Brown
1999, Spieth 1974, Yuval 2006). In parasitic Hymenoptera, courting males often move
their antennae in specific patterns and touch females directly with them (for review,
see Gordh and DeBach 1978). Such behavior, usually called antennation, often occurs
after a male has approached a female, she has stopped walking, and he has climbed on
her back so that their bodies are parallel with his head above hers. Starting at this point,
males perform various behaviors that may include head-bobbing, antennal waving and
stroking, as well as other behaviors, like leg sweeps. Although male antennation during
courtship was described at least as early as 1910 (Girault and Sanders 1910), Barrass
(1960) in research on Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) pro-
vided perhaps the first quantitative analysis of courtship behavior, which included quan-
titation of male antennation. In one of a series of papers on reproductive isolation among
closely-related species of Aphytis Howard (Hymenoptera: Aphelinidae), Rao and Debach
(1969) described antennation behavior in Aphelinidae. Gordh and DeBach (1978) later
used a quantitative analysis of differences in courtship behavior, including antennation,
to distinguish among cryptic species of Aphytis in the lingnanensis species complex.

The well-documented role of antennation in courtship triggered investigations into
the morphology of antennae. Based on scanning electron microscopy (SEM) of anten-
nae, Goodpasture (1975) suggested that the numerous pores on the scapes of males of
some species of Monodontomerus Westwood (Hymenoptera: Torymidae) might secrete a
pheromone. Dahms (1984a) found pores on the ventral surface of male scapes in Melitto-
bia australica Girault (Hymenoptera: Eulophidae), for which he had described antenna-
tion a decade earlier (Dahms 1973). He proposed that these pores released a pheromone
involved in mate recognition, rather than being sensory organs, as had been previously
conjectured. This discovery prompted more studies on morphology of male antennae in
parasitic Hymenoptera, using SEM and transmission electron microscopy (TEM). Bin et
al. (1989) found three types of glands in antennae of Trissolcus basalis (Wollaston) (Hy-
menoptera: Platygastridae), and they suggested roles in courtship and mate recognition.
On the ventral side of the fourth antennomere of Amitus spiniferus (Bréthes) (Hymenop-
tera: Platygastridae), Isidoro and Bin (1995) found numerous pores on an elevated plate,
and these pores were attached internally to a complex of glandular cells. They suggested
that this structure released and spread a mate-recognition pheromone during courtship,
and Isidoro et al. (1996) suggested that such structures be called release-and-spread struc-
tures (RSS). Research has continued to delineate the taxonomic distribution and mor-
phological diversity of these structures (Bin et al. 1999, Romani et al. 2010).

RSS and their associated glands have been found on male antennae in other chalci-
doids: Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) (Amorn-

Glandular structures on scapes of male Aphelinus 29

sak et al. 1998), Leptomastix dactylopii Howard, Rhopus meridionalis (Ferriére), and Asitus
phragmitis (Ferriére) (Hymenoptera: Encyrtidae) (Guerrieri et al. 2001). They have also
been found in parasitoids in other superfamilies: Trissolcus basalis (Wollaston) (Hymenop-
tera: Platygastridae) (Bin and Vinson 1986), a variety of species of Cynipidae and Figiti-
dae (Cynipoidea) (Isidoro et al. 1999), Trichopria drosophilae (Perkins) (Hymenoptera:
Diapriidae) (Sacchetti et al. 1999), and Pimpla turionellae (Linnaeus) (Hymenoptera: Ich-
neumonidae) (Bin et al. 1999). In the last two species, the RSS and glands were shown to
be necessary for successful courtship and copulation (Bin et al. 1999, Romani et al. 2008).

Observations on courtship behavior in several species of Aphelinus Dalman (Hy-
menoptera: Aphelinidae) showed that males antennate during courtship (Kazmer et
al. 1996, Rhoades 2015), but nothing has been published on pores and glands on the
antennae of Aphelinus. However, antennal pores and glands have been reported on
the antennal clubs of males of Aphytis melinus DeBach (Romani et al. 1999), which
is closely related to Aphelinus (Kim and Heraty 2012). Furthermore, observations of
slide-mounted Aphelinus specimens using differential interference contrast microscopy
(DICM) showed that RSS are widespread in the genus, and that the arrangement and
shape of RSS differed among species complexes. Here we report results of an investiga-
tion of the glandular ultrastructure underlying RSS in scapes of male Aphelinus varipes
(Forster) using TEM, and a survey of variation in the RSS in 16 species in six species
complexes of Aphelinus using SEM, DICM, and macrophotography that documents

their diversity and shows that they are taxonomically and phylogenetically informative.

Materials and methods

We follow the species complex classification of Hayat (1998), with one modification: we
treat the daucicola species complex as distinct from the mali complex (Hopper et al. 2012).
With TEM, we studied the structure of the cells underlying the pores in the male
scape of A. varipes. With SEM, we studied male scapes of two to three specimens each
from nine species of Aphelinus in six species complexes, along with species in two other
genera of Aphelininae: Aphytis melinus Debach and an undetermined species of Centrodo-
ra Forster (Hymenoptera: Aphelinidae) (Table 1). With DICM, we studied male scapes of
specimens from an additional seven species of Aphelinus in two species complexes (Table
1). Prior to preparation for imaging, the material was stored in molecular-grade ethanol.
For TEM, specimens were immersed in a solution of glutaraldehyde (2.5 ml/
25 ml solution) and paraformaldehyde (1 g/25 ml solution) in 0.1M cacodylate buffer
+5% sucrose, pH 7.2—7.3. The scape was removed from the rest of the antenna and
cooled at 4 °C for 3 h. The specimens were placed in 0.1M cacodylate buffer +5% su-
crose, pH 7.2—7.3, overnight at 4 °C, then the specimens were post-fixed in 1% OsO4
(osmium tetroxide) for 1 h at 4 °C and rinsed in the previous buffer. Dehydration in a
graded ethanol series from 60% to 99% was followed by embedding in Epon-Araldite
with propylene oxide as bridging solvent. Thin sections were made with a diamond
knife DiATOME ultra 45° (DiATOME AG, Biel, Switzerland) on a LKB Bromma ul-

tramicrotome (LKB®, Sweden), and mounted on formvar-coated, 50-mesh grids. The

30 Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

sections were stained with uranyl acetate (20 min, room temperature) and lead citrate
(5 min, room temperature). Finally, the sections were investigated with a TEM Philips
EM 208 (Thermo Fischer Scientific, Hillsboro, Oregon, USA). Digital images with
1376x1032 pixels, 8 bit, uncompressed greyscale in TIFF files were obtained using a
high-resolution digital camera MegaViewllI (SIS) connected to the TEM.

For SEM, specimens were critical-point-dried (CPD) using a Tousimis Samdri-790
(Tousimis Research Corporation, Rockville, Maryland, USA), following the manufactur-
er’s protocol. After CPD, two to three males from each accession (Table 1) were mounted
onto standard 12.7mm Ted Pella pin stubs using black carbon tape. Specimens were then
gold sputter-coated using a Technics Hummer I (Anatech Ltd, Battle Creek, Michigan,
USA), following the manufacturer's protocol. Gold was sputtered on specimens for a
total of five minutes, in one-minute intervals separated by one minute. All antennomeres
on both the left and the right antennae were examined on each specimen. SEM images
were acquired using a Tescan Vega 3 microscope (Tescan USA, Warrendale, Pennsylvania,
USA) with secondary electron-emission in high vacuum and beam-acceleration voltages
ranging from 15 kV to 30 kV. The beam intensity was either 3 or 5 (range is 1-20), and
the scan speed was either 6 or 7 (corresponding to 32 or 100 msec/pixel). For DICM,
specimens were slide-mounted in Canada balsam using a protocol modified from Noyes
(1982) and observed with an Olympus BH2 compound microscope equipped with differ-
ential interference contrast and planapochromat objectives. DICM images were captured
with a Jenoptik ProGres CT5 digital camera using Image Pro Plus 7.0. Macrophoto-
graphic (MP) images were taken of card-mounted specimens using a Macropod Pro setup
(https://macroscopicsolutions.com/) consisting of Mitutoyo long-working distance 50x
and 100x planapochromat objectives, a Canon 200 mm prime lens, and a Canon EOS
5D Mark II camera. DICM and MP images were focus-stacked using Zerene Stacker
1.04 and final preparation of images from SEM, DICM, and MP was done in Adobe
Lightroom. Sample size for specimens examined by DICM and MP varied from a few
specimens to ten specimens, or more in cases in which abundant material was available.

Voucher specimens of material examined have been deposited in the Texas A&¢M
University Insect Collection with Voucher #733. Voucher numbers in Table 1 refer to
specimens deposited in the Beneficial Insect Introduction Research Laboratory (USDA/
ARS), as voucher specimens of the original collections from which colonies were founded.

Five morphological traits of the RSS were recorded for 14 species of Aphelinus, as
well as for Aphytis melinus (Table 2). Eight of these 14 species of Aphelinus were studied
using SEM, the remainder were studied using DICM. ‘The data were entered into mx,
an open-source, web-based database management system (code and documentation
available at http://mx.phenomix.org), and the results were also exported as a Nexus
file. Diagnostics (Consistency Index and Retention Index) were calculated for each
trait using PAUP* with the traits mapped onto a molecular phylogeny from Heraty et
al. (2007), modified with additional unpublished data. PAUP* (4.a build 159: Swof-
ford 2002) and the Trace Character History algorithm using the Parsimony Ancestral
States in Mesquite (Maddison and Maddison 2018) provided the most parsimonious
reconstructions of character state changes on the phylogeny.

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32 Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

Table 2. Characters of release and spread structure on male scapes of Aphelinus species and Aphytis me-

linus.
Species ’ Pore Pore region
Species SS ee a oe Ie oe oo ee ee
complex Number Size* Shape? Location‘ Carina’ _Elevation*
N.A. Aphytis melinus 2 small flat proximal complete none
subflavescens Aphelinus perpallidus 2 large crenulated ridge —_ proximal complete depressed
asychis A. asychis 4 small flat midpoint none elevated
A, sinensis 5 small flat midpoint none elevated
abdominalis A. abdominalis 3 small cone, round top distal complete depressed
daucicola _A. daucicola 5 large cone, flat top proximal complete depressed
mali A. coreae 2 large cone, flat top midpoint complete depressed
A. glycinis 2,3,5 large cone, flat top midpoint complete depressed
A. rhamni 2,3 large cone, flat top midpoint complete depressed
varipes A. atriplicis 3 large cone, flat top proximal half depressed
A. certus 3 large cone, flat top midpoint half depressed
A. near certus 3 large cone, flat top midpoint half depressed
A. hordei =) large cone, flat top midpoint half depressed
A. kurdjumovi s large cone, flat top midpoint half depressed
A. nigritus 3 large cone, flat top midpoint half depressed
A. varipes 3 large cone, flat top midpoint half depressed

* small = diameter of setae base (Fig. 2A—C, E); large, diameter >2x setae base (Figs 2D, 3A-—F)

> flat = not raised above surrounding cuticle (Fig. 2A, B, E); cone, flat top = on cones, truncated and flat on top (Figs
1A, 3A-F); cone, round top = on cones, rounded on top (Fig. 2C); crenulated ridge (Fig. 2D)

‘distal = most proximal pore distal to scape midpoint; proximal = most proximal pore proximal to scape midpoint;
midpoint = most proximal at scape midpoint

‘complete = carina completely surrounds pores (Figs 2C—E, 4A); half = at proximal end of pores only (Fig. 4B); no
carina around pores (Figs 2A, B, 4C)

none = neither depressed or elevated relative to surrounding cuticle (Fig. 2E); elevated = elevated relative to surround-
ing cuticle (Fig. 2A, B); depressed = depressed relative to surrounding cuticle (Figs 2C, D, 3A—F)

Results

The male scape in A. varipes is characterized by the presence of cuticular modifications
located on the ventral side, defining a specialised area (Fig. 1A). The ventral margin
presents three subconical elevated areas with truncated and slightly concave tops that
carry single pores and which are surrounded by a depressed region delimited by a ca-
rina (Fig 1B). In some specimens, a secretion oozing from the pore can be observed
accumulating on top of the flattened area (Fig. 1C). Internal observations revealed the
presence of a large cell that occupies about half of the whole internal scape volume
behind each pore (Fig. 1D). This cell has a large nucleus located in the basal region, a
cluster of electron-lucid vesicles located in the middle region, and a straight evacuat-
ing duct connected with the external pore (Figs 1D, E). The antennal muscles located
within the scape are not connected with the glands, and they are located in the remain-
ing half of the internal scape volume together with the antennal nerve (Fig. 1D, F).
Sections taken in a different plane show the presence of a well-developed end apparatus
located in the middle region of the cell and characterized by an aporous structure sur-
rounded by microvilli (Fig. 1E G). Large clumps of electron-lucid vesicles surround

the end apparatus (Fig. 1G). The cytological features of the cell are those typical of

Glandular structures on scapes of male Aphelinus bb)

intensively active cells, i.e. the presence of a large nucleus and cytoplasm showing the
presence of abundant mitochondria and ribosomes (Fig. 1H). The cuticular evacuating
duct is connected to the end apparatus and is produced by an associated duct cell with
very reduced cytoplasm and a small nucleus (Fig. 1H). The secretion gathered at the
level of the end apparatus flows through the evacuating duct and reaches the external
pore where it is released.

The morphology of these release-and-spread structures varies among Aphelinus spe-
cies and Aphytis melinus (Figs 2, 3; Table 2). The pores in the asychis complex are sessile,
ie. not raised above surface of surrounding cuticle, and are on a convex region not sur-
rounded by a carina (Fig. 2A, B). In the other species complexes of Aphelinus, the pores
are in a depressed region of the cuticle, but individual pores are on elevated structures
(Figs 2C, D, 3A—E), which vary from smoothly rounded (Fig. 2C), to crenulated ridges
(Fig. 2D), or truncated and volcano-like (Fig. 3A—F). In the two outgroup species, pores
are present and sessile in Aphytis melinus (Fig. 2E), and absent in Centrodora sp. (Fig. 2F).

Mapping six characters of the release-and-spread structures (Table 2) onto a mo-
lecular phylogeny of these Aphelinus species and Aphytis melinus revealed two broad
phylogenetic patterns for different sets of characters; one set had shared states for the
mali and varipes complexes and different states in the other species complexes, and the
other set had different states in all the species complexes (Fig. 5). For the shape of the
structure bearing each pore (Fig. 5A) and the elevation of the region around the pores
(Fig. 5C), consistency and retention indexes are 1.0, indicating no homoplasy and
potentially useful phylogenetic signal. States for these characters are the same in the
mali and varipes complexes, as well as Aphelinus daucicola Kurdjumoy, the shape being
raised, conical, truncated, and flat or slightly concave on the top with the region around
these structures being depressed. The shape varies among the other species, with Apheli-
nus abdominalis (Dalman) having raised structures that are rounded on top, and Aphe-
linus perpallidus Gahan having pores on a crenulate ridge, in both cases surrounded by
a depressed region. Species in the asychis complex, as well as Aphytis melinus have sessile
pores, suggesting that sessile pores might be ancestral. However, the surrounding region
is elevated in the asychis complex, but neither depressed nor elevated in Aphytis melinus.
Pore size (Fig. 5B) is also the same in the mali and varipes complexes, as well as A. dauci-
cola, and the same in the asychis complex, A. abdominalis and Aphytis melinus, but the
consistency index is 0.5 and the retention index is 0.67 because of parallel increase in
pore size in A. perpallidus and in the lineage consisting of A. daucicola through Aphelinus
certus Yasnosh. For the carina around the pores (Fig. 5D), the consistency and retention
indexes are 1.0, indicating no homoplasy and strong phylogenetic signal. However, the
state of the carina differs between the varipes complex and mali complex. Species in the
varipes complex have a carina around the proximal end of the pores, whereas species
in the mali complex, as well as A. daucicola, A. perpallidus, and Aphytis melinus have a
carina completely surrounding the pores, suggesting that a complete carina is ancestral
in Aphelinus, although it is absent in the asychis complex. Both the number (Fig. 5E)
and location of pores (Fig. 5F) have consistency and retention indexes less than 1 (0.6
and 0.5 for pore number; 0.4 and 0.25 for pore location), indicating a fair amount of

34 Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

WY 4

mF

Figure 1. Structure of the male scape in Aphelinus varipes. A—C Scanning electron microscope images show-
ing in A the modified male scape (Sc) with the presence of a ventral carina on which three specialized struc-
tures (arrowheads) are observed B detail of the carina revealing three elevated areas (Ea) C close up of the pre-
vious in which a single apical pore (Po) per Ea is clearly visible, as well as secretion (Scr) oozing from the pore
itself. D-H Transmission electron microscope images showing internal ultrastructural features of the male
scape D cross section of the scape taken through one of the elevated areas, showing the secretory cell (Scl) that
occupies about half of the scape internal volume; Scl has a large nucleus (Nu) located basally, a central cluster
of electron-lucid secretory vesicles (Sv) and a straight evacuating duct (Ed) connected with the external pore
(Po); the rest of the scape volume is occupied by muscles (Mu) and the antennal nerve (An) E close-up view
of the previous image, showing the cuticular evacuating duct (Ed) running straight towards the external pore
F cross section of the scape taken in a different view, showing the large secretory cell (Scl) with a centrally posi-
tioned end apparatus (Ea) surrounded by numerous secretory vesicles (Sv); the evacuating duct (Ed), muscles
(Mu), and antennal nerve (An) can be seen G detail of the end apparatus (Ea), which appears perforated and
surrounded by microvilli (Mv); secretory vesicles (Sv) are above H detail of the duct cell (Dc) characterised
by a very reduced cytoplasm and a small nucleus (Nu); the duct cell is surrounded by the cytoplasm of the
secretory cell, that reveals the presence of ribosomes (Ri) and mitochondria (Mt); the evacuating duct (Ed) is
visible in cross section. Scale bars: 50 um (A); 10 um (B, D, F); 5 um (C); 2.5 um (E); 1 um (G); 2 pm (H).

Glandular structures on scapes of male Aphelinus

2E

Figure 2. Scanning electron microscope images of the ventral surfaces of male scapes in four Aphelinus
species and two outgroup species. AA. asychis B A. sinensis C A. abdominalis D A. perpallidus E Aphytis
melinus F Centrodora sp.

Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

Figure 3. Scanning electron microscope (A, B, C, D) and macrophotographic (E,F) images of the ventral sur-

faces of male scapes in five Aphelinus species. A.A. albipodus BA. varipes C A. coreae D A. daucicola E, F A. mali.

Glandular structures on scapes of male Aphelinus 37

Figure 4. Three states of character representing form of carina on Aphelinus male scapes, lateroventral view.

A Pores completely surrounded by carina B carina at proximal end of pores only C no carina around pores.

38

(a) Supporting structure shape

Hl not raised above cuticle

i raised, conical, flat on top
[41 raised, conical, rounded on top
MM raised on crenellated ridge

certus

near certus
nigritus
varipes
atriplicis
kurdjumovi
hordei
coreae
rhamni
glycinis
mali
daucicola
sinensis
asychis
abdominalis
perpallidus
Aphytis melinus

(c) Elevation of region
around pore structures
[i neither depressed or elevated

GS elevated
HI depressed

certus

near certus
nigritus
varipes
atriplicis
kurdjumovi
hordei
coreae
rhamni
glycinis
mali
daucicola
sinensis
asychis
abdominalis
perpallidus
Aphytis melinus

(e) Number of pores

Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

(b) Pore size

certus

MM large, diameter at least 2x setae base

: near certus
Hi small, diameter of base of setae

nigritus
varipes
atriplicis
kurdjumovi
hordei
coreae
rhamni
glycinis
mali
daucicola
sinensis
asychis
abdominalis
perpallidus
Aphytis melinus

(d) Carina status

[ij completely surrounds pores
1 at proximal end of pores only
MH nocarina

certus

near certus
nigritus
varipes
atriplicis
kurdjumovi
hordei
coreae
rhamni
glycinis
mali
daucicola
sinensis
asychis
abdominalis
perpallidus
Aphytis melinus

(f) Pore location

age) GS distal to scape midpoint proximal pore certus

= ee near certus HH proximal to scape midpoint near certus

— pipes MM at scape midpoint rite

Wi five varipes varipes
atriplicis atriplicis
kurdjumovi kurdjumovi
hordei hordei
coreae coreae
rhamni rhamni
glycinis glycinis
mali mali
daucicola daucicola
sinensis sinensis
asychis asychis
abdominalis abdominalis
perpallidus perpallidus
Aphytis melinus

Aphytis melinus

Figure 5. Characters of release-and-spread structures mapped onto molecular phylogeny of Aphelinus
plus Aphytis melinus. A Supporting structure shape B pore size C elevation of region around pore struc-
tures D carina status E number of pores F pore location.

Glandular structures on scapes of male Aphelinus a9

homoplasy for these characters. The varipes complex is consistent in pore number with
all species having three pores. Nine species in the varipes and mali complexes have the
most proximal pore at the scape midpoint. However, Aphelinus atriplicis Kurdjumoy,
Aphelinus mali (Haldeman), and A. daucicola, have the most proximal pore proximal to
the scape midpoint. For A. mali and A. daucicola this may have arisen because they both
have five pores, which might force the most proximal pore further towards the head.
Species in the asychis complex have four or five pores, but like most species in the varipes
and mali complexes, the most proximal pore is at the scape midpoint. However, species
in the asychis complex have small, sessile pores that are close together, which may allow
them to fit more distally than found in A. mali and A. daucicola. On the other hand,
both A. perpallidus and Aphytis melinus have only two pores but have the most proximal
pore proximal to the scape midpoint, suggesting that fit to the available length may be
not be important for pore placement.

Discussion

There is considerable variation among Aphelinus species in the characters of the release-
and-spread structures on male scapes. Although three characters show some homoplasy
with CI values of 0.50 to 0.75 (pore number, size, and location), three characters
have CI = 1.0 (supporting structure, elevation of cuticle, and extent of carina). Com-
binations of characters are diagnostic for species complexes of Aphelinus (Table 2),
and some combinations are diagnostic for species within complexes. Furthermore, the
morphologically distinctive aspects of the male scape in these Aphelinus species mapped
reasonably well onto the molecular phylogeny. It is intriguing that pores were found
on the male scapes of one outgroup taxon, Ap/ytis melinus, but were absent from the
male scapes of Centrodora sp. Along with Aphelinus, both outgroups are members of
Aphelininae, but Ap/yztis and Centrodora are in the subtribe Aphytini (Hayat 1998) and
are closely related members of a clade separate from the lineage containing Aphelinus
in the morphological phylogeny of Kim and Heraty (2012). A different RSS structure
on the antennal club (terminal antennomere) of Ap/ytis males may also play a role in
male antennation during courtship (Romani et al. 1999). A broader survey of RSS
structures on male antennae in Aphelinidae would be worthwhile.

Aphelinus varipes males have specialized secretory structures on the scape, which are
connected with the external pores. The glandular units have secretory cells releasing secre-
tions in electron-lucid vesicles. These vesicles surround an end apparatus connected with
the cuticular evacuating duct (produced by a duct cell) that allows the external release of
the secretion. These features are typical of secretory cells belonging to class III, according
to the classification of insect epidermal glands proposed by Noirot and Quennedey (1974,
1991) and Quennedey (1998). Male antennal glands in this class have been reported for
other groups of Hymenoptera, including Symphyta and Aculeata (Bin and Vinson 1986,
Bin et al. 1999, Isidoro et al. 1999, Romani et al. 2005, Romani et al. 2008), and func-
tionally, these structures have been related in most cases with courtship behavior by males.

40 Xanthe A. Shirley et al. / Journal of Hymenoptera Research 72: 27-43 (2019)

However, despite the numerous cases reported, occurrence of male antennal glands on the
scape is relatively uncommon. Dahms (1984a) described the presence of antennal glands
on the profoundly modified scape of males of Melittobia australica, which are related to
stereotyped antennal movements during courtship involving the scape itself.

Dahms (1984b) used differences among structures on the ventral area of male
scapes in a revision of the genus Melittobia Westwood (Hymenoptera: Eulophidae).
Dahms compared his work on Melittobia with that of van den Assem et al. (1982) on
Melittobia and noted that both antennation and morphology of the structures on male
scapes varied among Melittobia species groups. The species group that did not have
extensively modified structures did not antennate as strongly in its courtship as the
species groups that had more modified male scapes.

In Aphelinidae, male antennal glands have been reported in Encarsia asterobemisiae
Viggiani and Mazzone (Hymenoptera: Aphelinidae), Encarsia aurantii (Howard) and
Encarsia opulenta (Silvestri) (Pedata and Isidoro 1993), however, in these species RSS
are located on the flagellar segments, not the scape. The functional significance of RSS
on the scapes of Aphelinus and Aphytis (and perhaps other Aphelinidae) presents an
intriguing problem in functional morphology associated with mate recognition. By
demonstrating the diversity in forms of RSS and by placing this diversity in a phyloge-
netic context, our results are a first step in unraveling this puzzle. Knowledge of these
structures may help in understanding mate selection in this genus, as well as adding to
the knowledge about sex glands in parasitic Hymenoptera in general.

Acknowledgements

We thank Andreas Holzenburg and Mike Pendleton of Texas A&M Microscopy Cent-
er for assistance with SEM imaging. Thanks to Kim Carr, Texas A&M, for the pre-
liminary male scape survey. Thanks also to Jewel Coffey, Bryant McDowell, and Itzel
Cetina, Texas A&M, for help with specimen preparation. We thank Petr Jansta and
Ovidiu Popovici for their excellent comments and suggestions for the manuscript.
TEM and SEM images of A. varipes and SEM images of A. varipes were done at the
Centro Universitario di Microscopia Elettronica (CUME; Universita degli Studi di
Perugia, Italy). This research was supported by the following grants from the National
Science Foundation, USA: DEB 1257601 and DEB 1555790, and it was part of the
M.S. thesis of XAS at Texas A&M University.

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