Content uploaded by Netta Dorchin
Author content
All content in this area was uploaded by Netta Dorchin on Aug 11, 2015
Content may be subject to copyright.
RESEARCH ARTICLE
Morphological and Molecular Revision of the
Genus Ozirhincus (Diptera: Cecidomyiidae)—
Long-Snouted Seed-Feeding Gall Midges on
Asteraceae
Netta Dorchin
1
*, Jonas J. Astrin
2
, Levona Bodner
1
, Keith M. Harris
3
1Department of Zoology, Tel Aviv University, Tel Aviv, Israel, 2Alexander Koenig Zoological Research
Museum, Bonn, Germany, 3Independent researcher, Ripley, United Kingdom
*ndorchin@post.tau.ac.il
Abstract
The Palaearctic gall-midge genus Ozirhincus is unique among the Cecidomyiidae for its
morphology and biology. Unlike most other phytophagous gall midges, species in this
genus do not induce galls but develop inside achenes of Asteraceae plants. The heads of
adults are characterized by an unusually elongate proboscis, the function of which is
unclear. Despite a lot of attention from taxonomists in the 19th and early 20th century, a
proper revision of the genus has been hindered by complex host associations, the loss of
most relevant type material, and the lack of a thorough comparative study of all life stages.
The present revision integrated morphological, molecular, and life-history data to clearly
define species boundaries within Ozirhincus, and delimit host-plant ranges for each of them.
A phylogenetic analysis based on the mitochondrial COI and 16S genes confirmed the
validity of four distinct species but did not resolve the relationships among them. All species
are oligophages, and some may occur together on the same host plant. Species with wider
host-plant ranges have wider European and circum-Mediterranean distribution ranges,
whereas species with narrower host ranges are limited to Europe and the Russian Far East.
As part of the present work, O.hungaricus is reinstated from synonymy, O.tanaceti is syn-
onymized under O.longicollis, neotypes are designated for O.longicollis and O.millefolii,
and a lectotype is designated for O.anthemidis.
Introduction
Gall midges (Cecidomyiidae) constitute one of the largest families of Diptera, with more than
6200 described species and many that are undescribed or unknown [1]. Most of these species
are plant feeders in the larval stage, and induce galls, the complexity and morphological diver-
sity of which is rivalled only by gall wasps (Hymenoptera: Cynipidae) [2], [3]. Despite the ubiq-
uity of gall midges and their fascinating biology, the family is considered to be taxonomically
difficult because of the relative scarcity of useful characters, the small size of the midges, and
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 1/29
OPEN ACCESS
Citation: Dorchin N, Astrin JJ, Bodner L, Harris KM
(2015) Morphological and Molecular Revision of the
Genus Ozirhincus (Diptera: Cecidomyiidae)—Long-
Snouted Seed-Feeding Gall Midges on Asteraceae.
PLoS ONE 10(7): e0130981. doi:10.1371/journal.
pone.0130981
Editor: Sean Brady, Smithsonian National Museum
of Natural History, UNITED STATES
Received: January 24, 2015
Accepted: May 26, 2015
Published: July 2, 2015
Copyright: © 2015 Dorchin et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Sequences are available from the GenBank database
as detailed in the manuscript.
Funding: ND received a grant from the Israeli
Taxonomy Initiative (ITI) (http://taxonomy.tau.ac.il/en/).
Museum Alexander Koenig provided funds to JJA for
molecular work (www.zfmk.de). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
the fact that their study requires special techniques. Knowledge about gall midge systematics is
lacking, and many genera are in need of taxonomic revision or even placement to tribe [1].
Such revisions ideally combine morphological, molecular, and life-history data, as this
approach produces the most informative and reliable taxonomy, and may offer insight into the
evolution of the studied groups.
Ozirhincus Rondani is a small cecidomyiid genus that is unique for its morphology and biol-
ogy. Adults in this genus differ from other cecidomyiids in having an unusually elongate pro-
boscis and flattened head, and for developing in achenes of Asteraceae rather than inducing
galls, as do most other members of the tribe Lasiopterini [4], [1]. Members of this genus are
known only from host plants of the tribe Anthemideae and are restricted to the Old World,
with the exception of O.millefolii, which was introduced into North America in colonial times
[5], [4]. Based on its morphology and biology, the genus belongs to the subtribe Lasiopterina
[6], and is apparently closely related to Lasioptera, one of the largest and most loosely defined
genera of Cecidomyiidae [4]. The unique morphological characters exhibited by Ozirhincus
species, and their specialized life history, strongly suggest that the genus constitutes a mono-
phyletic group.
The genus has drawn the attention of many researchers since it was first described by Ron-
dani in 1840 [7], as attested by unusual nomenclatorial vicissitudes (reviewed by Verrall, [8]),
but despite this attention, its taxonomy remained confused until now. A major difficulty
stemmed from the fact that Ozirhincus species appeared to have a wide range of host plants,
and that some of these hosts support more than one species of gall midge at the same time and
place [9–12]. An added complexity was introduced by Möhn’s revision of Ozirhincus [11],
[12], which was based solely on larvae that were dissected from dried herbarium material. In
that revision, Möhn synonymized 5 of the previously known species and described 7 new ones,
basing all taxonomic decisions on problematic morphological characters of the mostly uninfor-
mative larvae. Recognizing that larval characters show high intraspecific variability and low
interspecific differences, and based on collecting efforts throughout Europe, Skuhravá [13]
reversed most of Möhn’s decisions, leaving Ozirhincus with 5 valid species, though without
clear characters to distinguish between them. The fact that most historical types of species
described in the 19
th
century are considered lost, led to further complications for clarifying the
taxonomy of this genus.
The objective of the present study was to settle the taxonomy of Ozirhincus through exten-
sive sampling from known and potential host plants and the combination of morphological
and molecular data. Our specific goals were: 1) to identify reliable morphological characters for
distinguishing between the species in the genus based on a thorough study of adults, pupae,
and larvae, 2) to clarify the complex host associations in the genus, and 3) to produce a phylog-
eny for the genus based on genetic markers.
Materials and Methods
Collecting and rearing of insects
Gall midges in the genus Ozirhincus do not cause the formation of galls, therefore rearing them
from their host plants requires collection of normal-looking inflorescences of potential host
plants towards the end of flowering, and keeping them in rearing cages in the laboratory until
adult emergence. In this study we screened 27 potential host plants (Table 1) in Germany,
Israel, and the UK, based on host records from the literature and the fact that Ozirhincus is
known only from plants of the tribe Anthemideae [1], [11,12]. Ozirhincus millefolii was also
collected in the USA, where it is widespread on its main host plant, Achillea millefolium, which
had been introduced from Europe, probably in colonial times [4]. In the following species
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 2/29
Competing Interests: The authors have declared
that no competing interests exist.
descriptions, only those names of plants that were confirmed as hosts in the present study are
given. Many other plant species have been mentioned in the literature but were not confirmed
here. Some of them were sampled in the present work but did not yield gall midges; for those
that were not sampled in the present study, further sampling and rearing will be needed to con-
firm their host status because host records were made based on larvae alone [11], [12], and it is
impossible to say to which Ozirhincus species those larvae belonged.
Inflorescences were collected in the field and transferred to the laboratory in large plastic
bags. They were then either kept in sealed bags, or placed as bouquets in water in ventilated
rearing cages until adult gall midges emerged from them or until they wilted without producing
gall midges. Plant species that did not yield gall midges were usually re-sampled several times
during their flowering season, sometimes in two or three consecutive years and at several local-
ities, to confirm that they are not hosts. Some inflorescences were dissected under a stereomi-
croscope to obtain larvae and pupae for morphological study.
To establish the monophyly of Ozirhincus, we chose outgroups representing four genera
from the two subtribes of Lasiopterini, the tribe to which Ozirhincus belongs. The subtribe
Lasiopterina is represented by Lasioptera, a large, cosmopolitan genus that is closely related to
Ozirhincus based on morphological characters, whereas the subtribe Baldratiina, which is
Table 1. Potential Asteraceae host plants screened in this study for the presence of Ozirhincus gall midges.
Plant Asteraceae tribe Confirmed as a host?
Aaronsonia factorovskyi Anthemideae -
Achillea fragrantissima Anthemideae -
Achillea millefolium Anthemideae +
Achillea ptarmica Anthemideae +
Achillea santolina Anthemideae -
Anthemis bornmuelleri Anthemideae +
Anthemis cotula Anthemideae +
Anthemis palestina Anthemideae -
Anthemis pseudocotula Anthemideae +
Anthemis rascheyana Anthemideae +
Anthemis retusa Anthemideae +
Anthemis tinctoria Anthemideae +
Artemisia arborescence Anthemideae -
Artemisia judaica Anthemideae -
Artemisia monosperma Anthemideae -
Artemisia sieberi Anthemideae -
Bellis perrenis Astereae -
Chrysanthemum coronarium Anthemideae +
Chrysanthemum segetum Anthemideae +
Erigeron sp. Astereae -
Leucanthemum vulgare Anthemideae +
Matricaria aurea Anthemideae -
Matricaria recutita Anthemideae -
Tanacetum parthenium Anthemideae +
Tanacetum santolinoides Anthemideae -
Tanacetum vulgare Anthemideae +
Tripleurospermum inodorum Anthemideae +
doi:10.1371/journal.pone.0130981.t001
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 3/29
restricted to host plants of the Chenopodiaceae is represented by the genera Baldratia,Careo-
palpis, and Stefaniola. All methods described for Ozirhincus apply also to the outgroups.
Molecular methods
Genomic DNA was extracted from whole adult or immature midges using mostly individual
silica-membrane columns from the Blood and Tissue kit by Qiagen (Hilden, Germany). DNA
extracts are available from the ZFMK Biobank, Bonn (DNA voucher IDs are given in Table 2).
For PCR amplifications, we used the Qiagen Multiplex PCR kit, following the manufacturer's
specifications and based on 2–2.5μl undiluted DNA template in 20μl total reaction volumes.
The GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA) was used to
amplify two mitochondrial genes. A 658 bp fragment from the 5' part of the mitochondrial
cytochrome oxidase subunit I (COI) gene (the so called ‘DNA barcoding gene’) was amplified
using the primers LCO1490 (F) and HCO2198 (R) [14]. Failed reactions were repeated with
the primer combination LCO1490 (F) and C1-N-2191 (R, aka 'Nancy') [15]. A 532–533 bp
fragment from the 3' end of the 16S ribosomal RNA gene was amplified using the primers
16S-ar-JJ and 16S-1472-JJ [16]. For both genes, the touchdown PCR protocol started at an
annealing temperature of 55°C, decreasing to 40°C for the remaining 25 cycles. Following enzy-
matic clean-up (Exo/SAP), double-stranded sequencing was conducted on an automated ABI
3730XL sequencer (Applied Biosystems) at the Macrogen facility, Amsterdam, NL. Sequences
were assembled, inspected and aligned using Geneious vers. R7 (Biomatters, Auckland, New
Zealand). The COI + 16S datasets were concatenated in BioEdit [17], resulting in a combined
alignment of 1191 bp. Sequences are deposited in GenBank (http://www.ncbi.nlm.nih.gov/)
and accession numbers are provided in Table 2.
Phylogenetic analysis
MODELTEST ver. 3.7 [18] consistently identified the GTR+G model of nucleotide substitution
[19] as the best-fit model for the COI data. For 16S, heterogeneous models were suggested by
different algorithms (hierarchical likelihood ratio test, Akaike information criterion, Bayesian
information criterion) and conditions (Bayesian information criterion), but most converged
on assuming a number of substitution types of 6, so that the same model was applied as for
COI. Bayesian analyses were conducted using MrBayes vers. 3.2.0 [20]. Specific parameters for
the GTR+G model were equated by MrBayes. Parameters were unlinked between the 3rd ver-
sus 1st plus 2nd codon positions and for 16S. We ran two independent replicates (4 chains
each) for 70 million generations per analysis. Every 1000th tree was sampled. Negative log-like-
lihood score stabilization was determined in a separate visualization and the trees for the first
19.000 generations were discarded as burn-in accordingly. We thus retained 139,962 trees,
which were used for building a 50%-majority rule consensus tree with posterior probabilities.
We reconstructed the evolution of proboscis length as an unordered multistate character
onto our phylogenetic tree using the maximum parsimony approach, as implemented in MES-
QUITE vers. 3.03 [21].
Taxonomy
Larvae, exuviae, and adults of the gall midges were preserved in 70% ethanol for morphological
study, and were later mounted on permanent microscope slides in euparal according to the
method outlined by Gagné [22]. Specimens are mounted on slides individually unless other-
wise noted. Relevant historical material from the Rübsaamen, and Möhn collections was exam-
ined, and its condition evaluated. Material in these collections had originally been mounted on
temporary microscope slides in glycerin, or stored in 70% ethanol, and many of the specimens
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 4/29
are either lost or have deteriorated to the point that they cannot be used for taxonomic study.
Some of that material that was found to be in reasonable condition was remounted on perma-
nent microscope slides in euparal for the purpose of the present study and to ensure its integ-
rity and long-term preservation.
Illustrations of morphological structures were made with the aid of a drawing tube or a
Leica DFC295 camera mounted on a Leica DM1000 LED compound microscope. Pupae were
studied under a scanning electron microscope. Some adults were also pinned to preserve the
color pattern created by the thick covering of scales particularly on the abdomen. Proboscis
length was measured from the suture at the base of the labrum to the tip of the labella, and
expressed in relation to the length of the antennal scape. Ovipositor length was measured from
the base of the eighth abdominal segment to the apex of the cercus, and expressed in relation to
the length of segment 8. Some adults and immature stages were preserved in 96% ethanol for
molecular study. We compared newly collected material to types and other relevant material
deposited in the Staatliches Museum für Naturkunde, Stuttgart, Germany (SMNS), Museum
für Naturkunde, Berlin, Germany (ZMHB), the Naturhistorisches Museum, Vienna, Austria
(NHMW), the Natural History Museum in London, UK (BMNH), and the private collections
of Marcela Skuhravá (Prague) and Eddy Dijktra (Delft). Terminology for adult morphology
follows McAlpine et al. [23], and terminology for immature morphology follows Gagné [4].
The specimens examined in this work are deposited in the National Collection of Insects, Zoo-
logical Museum, Tel Aviv University (TAUI) unless otherwise indicated.
Results
Phylogenetic analysis
The complete molecular dataset of COI and 16S consisted of 1191 bp. Unaligned fragment
lengths for 16S were 532–533 bp, i.e. included only a single-base indel. The COI fragment con-
sisted of 658 positions and contained no internal gaps. The phylogenetic analysis yielded four
strongly supported and genetically quite distant clades, clearly representing four valid species
in the genus Ozirhincus (Fig 1), which can be distinguished from each other based on unique
combinations of larval, pupal, and adult morphological characters. However, the analysis could
not resolve the phylogenetic relationships among the four species. The ancestral states analysis
did not yield conclusive results regarding the history of proboscis length in the genus (see char-
acter states in Fig 1). It is likely that the short proboscis in O.anthemidis represents the ances-
tral state, similar to the situation in Lasioptera, and that the typical elongate proboscis evolved
only once in the history of the genus, but other scenarios cannot be ruled out.
Mapping the identity of host-plant genera on the phylogenetic tree (colors in Fig 1) suggests
that O.millefolii is restricted to the genus Achillea, whereas each of the remaining species is
capable of developing in host plants of various genera. It also indicates that certain host plants
support more than one Ozirhincus species at the same time and place: Tripleurospermum ino-
dorum is used by O.longicollis and O.hungaricus, while some Anthemis spp. are hosts to both
O.longicollis and O.anthemidis. In all of these scenarios it is possible to differentiate among
the gall-midge species based at least on their adult and pupal morphology.
Taxonomy
Ozirhincus Rondani 1840
Type species: Ozirhincus longicollis Rondani 1840: 16; by monotypy
Ozirhincus is a small genus in the tribe Lasiopterini, apparently closely related to Lasioptera.
Like other Lasiopterini, it is characterized by a thick covering of scales (Fig 2A–2C), an irregu-
lar number of antennal flagellomeres that are gynecoid in the male, a very short R
4+5
that
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 5/29
Fig 1. Phylogenetic tree of Ozirhincus Rondani based on Bayesian analysis of partial sequence of the cytochrome oxidase subunit I (COI) and
ribosomal RNA16S mitochondrial genes. Support values are shown next to nodes, above branches. Character states representing proboscis length as
suggested by the ancestral states analysis are shown below branches (in square brackets). Letters and numbers following species name refer to collecting
localities and dates (details in Table 2). Colors correspond to host-plant genera.
doi:10.1371/journal.pone.0130981.g001
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 6/29
reaches C proximal to mid-length of wing, longitudinally divided mediobasal lobes in the male
that sheath the aedeagus almost to its apex, a protractible ovipositor with a lateral group of
hooked setae on segment 8 of the female abdomen, and variously modified setae on the cercal
segment. In the closely related Lasioptera, it was argued that these modified setae in the female
function in collecting and carrying conidia [24], [25], but their function in Ozirhincus is
unclear because species in this genus do not appear to be associated with fungal symbionts.
Adults in this genus have unusually elongate mouthparts that form a short to very long probos-
cis, composed mostly of the strongly setose labrum and labella, and a considerable elongation
of the frontoclypeal membrane (Fig 3A–3E). The head in most species is flattened, with the
occiput encroaching the eye area. Palps are four-segmented. Tarsal claws are toothed on all
legs. The body is covered by black and white scales that form dorsal transverse stripes on the
abdomen. Larvae have a bifid spatula in the third instar, with a reduced number of 3–4 lateral
asetose papillae on each side, and usually 2–3 setose papillae on each side of the terminal seg-
ment. Pupae have pointed antennal bases (‘antennal horns’) that terminate in one or two tips.
All species develop in achenes of Asteraceae belonging to the tribe Anthemideae (Fig 2D).
Pupation takes place inside the achene and the species complete at least two generations a year.
Fig 2. Ozirhincus hungaricus.a-c. Female on Tanacetum vulgare inflorescence (photos: Hedy Jansen); d. Tanacetum vulgare flowers containing O.
hungaricus larvae (upper row), and normal flowers (lower row).
doi:10.1371/journal.pone.0130981.g002
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 7/29
Key to the species of Ozirhincus Rondani
1. Proboscis short (Fig 3A): length, from base of labrum to tip of labella, shorter than height of
eye. Occiput not encroaching eye area. Fourth palp segment about as long as third. On
Chrysanthemum and Anthemis spp....................................Ozirhincus anthemidis (Rübsaamen)
-. Proboscis long (Fig 3B–3E): length, from base of labrum to tip of labella, longer than
Fig 3. Adult heads. a. Ozirhincus anthemidis, male; b. O.hungaricus, male; c. O.millefolii, female; d. O.longicollis, female; e. O.longicollis, male, showing
typical shape of flagellomeres.
doi:10.1371/journal.pone.0130981.g003
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 8/29
height of eye. Occiput encroaching eye area. Fourth palp segment usually notably longer
than third. On various Anthemideae genera.....................................................................................2
2. Antennae with 11–12 flagellomeres. Pupal antennal horns with single pointed tip (Fig 4C
and 4D). 3
rd
instar larva with 4 lateral and 3 terminal papillae on each side. On Tanacetum
and Tripleurospermum spp............................................................... Ozirhincus hungaricus Möhn
Fig 4. Pupae heads. a. Ozirhincus anthemidis, frontal; b. O.anthemidis, lateral; c. O.hungaricus, frontal; c. O.hungaricus, lateral; e. O.longicollis, frontal; f.
O.millefolii, frontal.
doi:10.1371/journal.pone.0130981.g004
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 9/29
- Antennae with 8–10 flagellomeres. Pupal antennal horns bifid (Fig 4E and 4F). 3
rd
instar
larva with 3 lateral and 2 terminal papillae on each side. On various Anthemideae genera. 3
3. Antennae with 8, occasionally 9 flagellomeres. Pupal antennal horns widely separated (Fig
4F). On Achillea spp.......................................................................... Ozirhincus millefolii (Wachtl)
- Antennae with 10, very rarely 9 flagellomeres. Pupal antennal horns not widely separated
(Fig 4E). On Tripleurospermum,Tanacetum,Leucanthemum and Anthemis
spp....................................................................................................... Ozirhincus longicollis Rondani
Descriptions
In the following descriptions, the names of host plants are given first because the identity of the
host plant is one of the most important characters that may aid in species identification. Only
those plant species that were confirmed as hosts in the present study are listed.
Ozirhincus anthemidis (Rübsaamen 1916)
Clinoryncha anthemidis Rübsaamen 1916: 561
Ozirhincus dalmaticus Möhn 1966
Ozirhincus kabylensis Möhn 1966
Host plants. Chrysanthemum coronarium,C.segetum,Anthemis arvensis,A.bornmuelleri,
A.rascheyana,A.retusa,A.tinctoria.
Adult. Head (Fig 3A): Eye facets round; more spaciously arranged on vertex than laterally;
eye bridge 3-4-facets long. Antenna (Fig 5A–5C): scape wide trapezoidal; pedicel globose; num-
ber of flagellomeres 11–12 in both sexes, rarely 10, number occasionally differs between anten-
nae of same individual (n = 102♀, 104♂); flagellomeres globular to almost quadrate in female,
more cylindrical in male (Fig 5B and 5C); first two flagellomeres usually partially to entirely
fused, apical flagellomere often longer, evidently composed of 2–3 entirely or partially fused
units; adjacent flagellomeres sometimes fused in mid antenna (Fig 5C); each flagellomere with
two whorls of appressed circumfila and two rows of strong setae originating from prominent
sockets, one row proximal to and one row between circumfila (Fig 5A); entire flagellomere sur-
face other than neck covered by microtrichia. Palpus 4-segmented; segment 1 only slightly lon-
ger than wide, segments 3–4 about same length, 3 times longer than wide, with several strong
setae and otherwise setulose. Frontoclypeal membrane with several strong setae on each side.
Labrum about 4 times as long as width at base, parallel sided on basal two thirds, tapering from
apical third towards setulose apex, with a few strong setae dorsally. Labella (Fig 6A) about 2.5
times as long as wide, somewhat concave medially, tapered apically, with several strong setae
and densely setose along medio-apical surface.
Thorax: Dark grey, covered by white scales and setae. Anepimeron with group of 15–20
setae; other pleura without setae. Legs: dorsal part densely covered by black scales other than a
patch of white scales on basal half of first tarsomere (Fig 2A–2C); ventral part densely covered
by white scales. Tarsal claws (Fig 6C–6E) evenly curved, with thin tooth, strongly curved close
to base; empodia longer than bend in claw; pulvilli about 0.3 times as long as claw. Wing: hya-
line, with sparse delicate hairs on entire surface and long hairs along posterior margin; length
1.09–1.76 mm in males (n = 65), 1.09–1.66 mm in females (n = 58); R
4+5
joins C around mid-
length of wing, densely covered by mixed black and white scales; C with break after meeting
point with R
4+5
, densely covered by black scales almost to wing apex, except for patch of white
scales at meeting point with R
4+5
(Fig 2A–2C); M straight, CuA unforked. Stem of halter light
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 10 / 29
orange, without scales; knob densely covered by black scales on basal third, white scales cover-
ing remainder.
Fig 5. Antennae. a. Ozirhincus anthemidis, male apical flagellomeres, setae not shown; b. O.anthemidis, male flagellomeres, some setae shown; c. O.
anthemidis, female flagellomeres; d. O.longicollis, male apical flagellomeres; e. O.longicollis, male (left), female (right); f. O.millefolii, male (left), female
(center and right). Scale bars = 0.1 mm.
doi:10.1371/journal.pone.0130981.g005
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 11 / 29
Female abdomen (Fig 7A and 7B): Dorsum with dense covering of black and white scales:
each tergite with wide transverse stripe of black scales on most of surface, and a narrow strip of
white scales along posterior margin. Pleuron and venter with white scales. Tergites 1–6 rectan-
gular, with anterior pair of trichoid sensilla, posterior row of strong setae, and otherwise evenly
covered by scales; tergite 7 much smaller than preceding, pigmentation evanescent laterally at
midlength, with anterior pair of trichoid sensilla and posterior row of setae; tergite 8 divided
longitudinally into two elongate sclerites (Fig 7C), each with wide anterior area with pointed
dorsal extension and wide posterior area with pointed ventral extension connected by long,
narrow band; each sclerite with trichoid sensillum on anterior part of narrow band, and a
Fig 6. Proboscis. a. Ozirhincus anthemidis;b.O.hungaricus. c-e. O.anthemidis, fifth tarsomere, claw and acropod. c. Lateral; d. Lateral, showing both
claws; e. Ventral. Lb–Labella, Lr–Labrum, Pl–Palp. Scale bars = 0.1 mm.
doi:10.1371/journal.pone.0130981.g006
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 12 / 29
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 13 / 29
group of strong, posterior setae on widened posterior area. Eighth tergite 1.65–2.77 times as
long as seventh tergite (n = 56). Sternites 2–7 rectangular, with pair of closely approximated tri-
choid sensilla, posterior row of setae, and several setae laterally and medially, more numerous
on more posterior segments; sternite 8 not apparent. Ovipositor long, protrosible, 3.57–7.47
times as long as eighth tergite (n = 57), with pigmented lateral sclerite along segment 9 and lat-
eral group of strong, arched setae originating from prominent sockets, pointed mostly ven-
trally. Cercal segment (Fig 7B) with dorsolateral sclerotized plate more strongly pigmented
along posterior area than elsewhere, laterally with 10–20 short, strong and straight setae; poste-
rior pigmented area with small dorsal projection and bearing 3–4 very long, hook-like, blunt
setae. Apical lamella cylindrical, evenly setulose, with numerous strong setae mostly concen-
trated on dorsal and apical areas. Hypoproct setulose.
Male abdomen (Fig 8A): Color pattern and scale covering as in female. Tergite 1 rectangu-
lar, with posterior row of strong setae, and evenly scattered scales; tergites 2–6 similar but
larger and with anterior pair of trichoid sensilla; tergite 7 weakly sclerotized posteriorly, with
fewer posterior setae, not forming a row; tergite 8 narrow, band-like, without setae other than
anterior trichoid sensilla. Sternites 2–7 rectangular, with pair of closely approximated trichoid
sensilla and 1–2 posterior rows of strong setae; posterior sternites with several strong setae
medially, and otherwise evenly covered by scales; sternite 8 less pigmented and more setose
than preceding but without trichoid sensilla. Terminalia (Fig 8B–8E): Gonocoxite cylindrical,
about same width throughout length, with mediobasal lobe divided into prominent, globose,
densely setose dorsal lobe, and elongate, ventral lobe tapering towards apex, and further subdi-
vided into two longitudinal ridges on apical two thirds, sheathing aedeagus almost to apex (Fig
8D). Gonostylus widest at about mid length, tapering to wide comb-like tooth, with numerous
setae, setulose on basal half dorsally and basal two thirds ventrally, remaining part with shallow
ridges (Fig 8E). Aedeagus slightly longer than sheathing mediobasal lobes, wide and blunt api-
cally. Hypoproct entire, blunt apically, or with slight, shallow notch, setose and setulose. Cerci
separated by a deep notch, setose and setulose.
Larva (third instar) (Fig 9A–9C).Light to dark yellowish-orange. Cylindrical-ovate. Integu-
ment covered by rounded verrucae. Antennae 1.5–2.0 times as long as wide. Cephalic apodeme
considerably longer than head capsule (Fig 9A). Spatula (Fig 9A and 9H) long shafted and
bidentate; shape of teeth and distance between them highly variable; when teeth farther apart,
sometimes with minute additional projection between them. Sternal papillae without setae;
pleural and dorsal papillae with long setae. On each side of spatula 3–4 asetose lateral papillae
grouped together, and one asetose ventral papilla somewhat farther away (Fig 9B). Terminal
abdominal segment with 2–3 setose papillae on each side (Fig 9C). All specimens obtained in
the present study had 5 lateral papillae on each side; number of terminal papillae varied among
individuals from different host plants: those from Chrysanthemum spp. and Anthemis
rascheyana had 2 papillae on each side, those from A.tinctoria had 3, and those from A.born-
muelleri had either 2 or 3.
Pupa (Fig 4A and 4B).Light to vivid orange. Antennal bases enlarged, forming straight
bidentate horns, tapered and pointed ventroapically. Vertex with long and thin cephalic seta
on each side, situated on conspicuous bulge. Face without apparent papillae. Prothoracic spira-
cle long and slender. Abdominal segments covered by acute spicules.
Distribution. Europe and circum-Mediterranean.
Fig 7. Female abdomen. a. Ozirhincus anthemidis, lateral; b. O.anthemidis, ovipositor, lateral; c. O.anthemidis,7
th
tergite; d. O.hungaricus,7
th
tergite; e. O.
longicollis,7
th
tergite; f. O.millefolii,7
th
tergite. Al–Apical lamella, Dlp–Dorsolateral plate, Lgs–Lateral group of setae on eighth segment. Scale bars = 0.1
mm, except for Fig 28 = 0.5 mm.
doi:10.1371/journal.pone.0130981.g007
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 14 / 29
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 15 / 29
Material examined. The type series of O.anthemidis includes males, females, and pupal
exuviae that have been kept in ethanol vials since their collection. This material was mounted
on permanent microscope slides in euparal for the purpose of the present study, and used for
the designation of the following types: LECTOTYPE: ♀, Germany, Werlau, 1895, EH Rübsaa-
men, reared from Anthemis tinctoria, (115e). The lectotype is mounted on a permanent micro-
scope slide in euparal, is in fair condition, and deposited in ZMHB. PARALECTOTYPES: 1♂,
2 exuviae, Germany, Werlau, 1895, EH Rübsaamen (115e) (ZMHB) (same data as lectotype;
both exuviae on same slide); 2♀,2♂, 4 exuviae, Germany, Oberheimbach, 17.viii.1906, EH
Rübsaamen (115b) (all exuviae on same slide) (ZMHB); 3♀,3♂, Germany, no locality or date
given, EH Rübsaamen (115a) (ZMHB).
OTHER MATERIAL EXAMINED: ex Chrysanthemum coronarium:3♀,2♂, Israel, Herze-
liya, 8.v.2009, A. Freidberg; 27♀,20♂, 14 larvae, Israel, Herzeliya, 19.iv.2012, A. Freidberg (2♀,
2♂ZMHB, 2♀,2♂SMNS, 1♀,1♂ZFMK, others TAUI); 4♀,3♂, Israel, Kefar Hahoresh, 28.
iv.2012, N. Dorchin; 4♀,4♂(on slides), 12♀,12♂(pinned), Israel, Hadera, 11.iii.2013, N.
Dorchin and I. Hayon; 1♀,6♂, Israel, Ziqim, 4.iv.2013, N. Dorchin.
Ex Chrysanthemum segetum:4♀,11♂, Israel, Dan, 10.iv.2014, N. Dorchin.
Ex Anthemis bornmuelleri:5♂, 13 larvae, Israel, Ma’agar Bental, 14.v.2012, N. Dorchin;
19♀,21♂, 7 larvae, Israel, Ma’agar Bental, 25.v.2012, N. Dorchin (2♀,2♂ZMHB, others
TAUI); 3♂, Israel, Ma’agar Bental, 12.v.2014, N. Dorchin.
Ex Anthemis rascheyana:1♀,4♂, 5 larvae, Israel, Mt. Hermon, 1750m, 24.v.2012, N.
Dorchin; 8♀,7♂, 6 larvae, Israel, Mt. Hermon, 1780m, 6.vi.2012, N. Dorchin.
Ex Anthemis retusa:9♀,4♂, 5 larvae, Israel, Nahal Nizzana, 12.v.2013, N. Dorchin.
Ex. Anthemis tinctoria:5♀, Israel, Newe Ativ, 15.v.2012, N. Dorchin; 5♀,6♂, Israel, Newe
Ativ, 25.v.2012, N. Dorchin; 8♀,2♂, 12 larvae, Israel, Newe Ativ, 6.vi.2012, N. Dorchin.
Biology. The phenology of O.anthemidis varies within its distribution range and in rela-
tion to its host plants. In dry Mediterranean regions (Middle East and North Africa), the main
activity period is in spring, whereas in continental Europe adults are active also in summer.
Adults of the overwintered generation emerge in Europe from mid-March to late April and
those of the subsequent generation between August and October [11]. In Israel, adults associ-
ated with Chrysanthemum coronarium emerge in March from infested achenes in the ground
from the previous year, and can be seen hovering over and standing on developing inflores-
cences, where females lay their eggs into individual flowers. Larvae develop and soon pupate
inside the achenes, preventing them from developing seeds. Adults of the subsequent genera-
tion emerge already in April, in contrast to the much slower development of larvae in Europe.
Adults reared in May may represent either late-developing individuals of the second generation
or those of a third generation. Infested achenes are slightly inflated and enlarged compared to
normal achenes, and the larvae or pupae fill them completely making it hard to dissect them
from the achenes without damaging them. Females of the last generation lay eggs in the flow-
ers, and the hatching larvae develop to third instars but do not pupate. Instead, they enter a dia-
pause inside the achenes that drop to the ground when the plants have dried up, and will
emerge as adults the following year. The life history of populations associated with Anthemis
spp. in Israel is essentially similar except that the main activity period is in late rather than
early spring. Diapause takes place in the summer through winter months in Mediterranean
regions, or only during the autumn and winter in continental Europe.
Fig 8. Male abdomen, Ozirhincus anthemidis.a. Post abdomen and terminalia, lateral; b. Terminalia, dorsal, setation shown onright gonopod; c.
Terminalia, lateral, one gonopod removed; d. Terminalia, ventral, showing mediobasal lobes and aedeagus; e. Gonostylus, dorsal (top), ventral (bottom).
Aed–Aedeagus, Cer–cercus, Dl–Dorsal part of mediobasal lobe, Gs–Gonostylus, Gx–Gonocoxite, Vl–Ventral part of mediobasal lobe. Scale bars = 0.1 mm.
doi:10.1371/journal.pone.0130981.g008
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 16 / 29
Fig 9. Larvae. a. O.anthemidis, head; b. O.anthemidis, spatula and associated papillae; c. O.anthemidis, terminal abdominal segment; d. O.hungaricus,
terminal abdominal segment. e. Spatula and associated papillae, O.hungaricus; f. Spatula and associated papillae, O.longicollis; g. Spatula and associated
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 17 / 29
Remarks. Rübsaamen [9] repeated Loew’s[26] rearings from Tripleurospermum ino-
dorum and Anthemis arvensis, as well as from A.tinctoria, but noticed that he was getting two
types of adults from the latter two hosts: some with a rather short proboscis and some with the
typical long proboscis mentioned by Loew for his Clinorhyncha chrysanthemi (currently Ozir-
hincus longicollis). Rübsaamen wondered how Loew had not noticed the two different popula-
tions, and speculated that he did not manage to rear the short-snouted individuals for some
reason. He correctly attributed the long-snouted adults to Clinorhyncha chrysanthemi, noting
that they had 10 antennal flagellomeres as opposed to 11–12 in the short-snouted specimens,
and described the latter as a new species–Clinorhyncha anthemidis (currently Ozirhincus
anthemidis).
Ozirhincus anthemidis is easily distinguishable from all other species in the genus by its
short proboscis, short fourth palpal segment, and 11–12 antennal flagellomeres, as opposed to
8–9inO.millefolii and 10 in O.longicollis.Ozirhincus hungaricus, the only other species in the
genus with a regular number of 11–12 antennal flagellomeres, has a much longer proboscis
and is found on different host plants.
Ozirhincus anthemidis is a very common species throughout Europe and the Mediterranean
region. It was previously known only from Anthemis hosts [12], [13], but in the present study
we found that it is also very common on Chrysanthemum coronarium and C.segetum (and it
was the only species we reared from those plants). Host plants that were reported for O.anthe-
midis by Möhn [12] but not verified in the present study are Anthemis austriaca,A.cretica (as
A.montana), A.kotschyana, and A.cotula.Anthemis arvensis is listed here as a verified host
plant based on the information in Rübsaamen’s original description of O.anthemidis.
Ozirhincus dalmaticus and O.kabylensis were described by Möhn [12] as belonging to the
‘anthemidis group’based on small differences in the larval spatula and chaetotaxy, but were
later synonymized under O.anthemidis by Skuhravá [13], with whom we concur.
Ozirhincus hungaricus Möhn 1968
Ozirhincus hungaricus Möhn 1968: 55
Ozirhincus hispanicus Möhn 1968
Ozirhincus parvus Möhn 1968
Characters as in O.anthemidis except for the following:
Host plants. Tanacetum vulgare,Tripleurospermum inodorum, and Tanacetum
corymbosum.
Adult. Head (Fig 3B): Occiput extending forward at expense of eye area in frontal view;
eye bridge about 4 facets long. Antenna: number of flagellomeres 11–12 in both sexes
(n = 100♀,89♂); flagellomeres short-cylindrical to almost quadrate in female, more elongate
in male; first two flagellomeres usually partially to entirely fused, apical flagellomere slightly
tapered, sometimes appear as if ‘budding’to form the beginning of a small additional segment.
Palpus: fourth segment 1.3–2.0 times longer than third. Frontoclypeal membrane greatly
extended, with many strong setae on each side. Labrum long, triangular: at least 4 times as long
as width at base, tapering and strongly setose on apical quarter. Labella more than 10 times as
long as wide, somewhat concave medially, tapered apically, with several strong setae towards
apex and densely setose along entire medial surface (Fig 6B).
papillae, O.millefolii; h. Variation of spatula shape in O.anthemidis; i. Variation of spatula shape in O.hungaricus.Lp–Lateral papillae, Sp–Sternal papilla,
Vp–Ventral papilla. Scale bars = 0.1 mm.
doi:10.1371/journal.pone.0130981.g009
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 18 / 29
Thorax: Anepimeron with a group of 10–20 setae. Wing length: 1.34–1.73 mm in males
(n = 38), 1.28–1.67 mm in females (n = 47).
Female abdomen: Elongate sclerites of tergite 8 (Fig 7D) each with wide anterior area, usu-
ally with rounded rather than pointed dorsal and ventral extensions and wide posterior area
connected by long, narrow band; each sclerite with trichoid sensillum on anterior third of nar-
row band, and 8–10 posterior setae on widened posterior area. Eighth tergite 1.64–3.34 times
as long as seventh tergite (n = 46). Ovipositor 4.13–7.77 times as long as eighth tergite (n = 46).
Larva. (third instar). Integument covered by rounded verrucae. Antennae 2–3 times as
long as wide. Cephalic apodeme considerably longer than head capsule. Spatula (Fig 9E and 9I)
long-shafted and bidentate; teeth widely separated by concave to straight gap, sometimes with
minute additional projection between them. Sternal papillae without setae; pleural and dorsal
papillae with long setae; on each side of spatula 4 asetose lateral papillae grouped together, and
one asetose ventral papilla somewhat farther away (Fig 9E). Eighth abdominal segment with 3
setose papillae on each side (Fig 9D).
Pupa (Fig 4C and 4D).Antennal bases enlarged, bulging, with tiny tapered tip at center of
bulge.
Distribution. Widespread in Europe to Siberia.
Material examined. HOLOTYPE: Larva, Hungary, Ménesi, Nagyboldogasszony-útja, 21.
viii.1949, dissected by E. Möhn from Tanacetum corymbosum. The holotype is the left speci-
men of two larvae mounted under the same cover glass on a permanent microscope slide in
euparal, and is deposited in the SMNS. The second larva on the same slide is currently labeled
as a paratype.
PARATYPE: 1 larva, same data as holotype (SMNS).
OTHER MATERIAL EXAMINED: ex Tanacetum vulgare:1♀,1♂, Czech Republic, Mora-
via, Břeclav, 1.ix.1958, M. Skuhravá (Skuhravá collection, as O.tanaceti); 3♀,2♂, Hungary,
Szentendre, 5.viii.1988, M. Skuhravá (in ethanol); 6♀,8♂(in ethanol), UK, London, vi.2005, B.
Wurzell; 12♀,9♂(on slides), 1♀,1♂(in ethanol), Germany, NRW, Ägidienberg, 11.viii. 2011,
N. Dorchin; 25♀,21♂, 13 larvae (on slides), 11♀,14♂(in ethanol), 4♀(pinned), Germany,
NRW, Wahner Heide, 11.viii. 2011, N. Dorchin, (1♀,1♂on slides ZFMK, others TAUI); 12♀,
11♂(on slides), 6♀,10♂(in ethanol), 6♀(pinned), Germany, NRW, Leverkusen, 14.viii. 2011,
E. Diehl; 5♀,4♂, the Netherlands, Ede, central station, 25.viii.2011, E. Dijsktra (in ethanol).
Ex Tripleurospermum inodorum:1♀,1♂, UK, Surrey, Send, Woodhill, 20.viii.2011, KM
Harris and N. Dorchin; 23♀,11♂, 12 larvae, Germany, Leverkusen, 30.viii.2011, E. Diehl (1♀,
1♂ZFMK, 1♀,1♂ZMBH, others TAUI).
Biology. The life history of this species is similar to that of O.longicollis. An ovipositing
female and infested achenes of the main host plant, Tanacetum vulgare, are shown in Fig 2A–
2D.
Remarks. Möhn [12] described O.hungaricus,O.parvus, and O.hispanicus as belonging
to the ‘tanaceti group’based on characters of the larvae alone, but these species were later syn-
onymized under O.tanaceti by Skuhravá [13]. The larvae of all species in the ‘tanaceti group’
have 4 lateral papillae on each side of the spatula and 3 terminal papillae on each side of the
anus. Because the holotype larva of Ozirhincus hungaricus survived in a recognizable condition
while those of O.parvus and O.hispanicus did not, we reinstate O.hungaricus as the name of
the common species associated with Tanacetum vulgare in Europe, and synonymize the other
two species under it. Tanacetum corymbosum is listed here as a confirmed host because the
holotype of O.hungaricus had been found in that plant [12].
In the present study we found that, in addition to T.vulgare,O.hungaricus also develops in
Tripleurospermum inodorum, which it sometimes shares with O.longicollis. When the two spe-
cies occur together on that host plant, they can be distinguished from each other easily by: the
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 19 / 29
number of antennal flagellomeres (10 in O.longicollis vs. 11–12 in O.hungaricus); the charac-
teristic constrictions of the male flagellomeres in O.longicollis, where they are girdled by the
circumfila loops (Figs 3E,5D and 5E); the clear difference in the shape of the pupal antennal
horns (Fig 4E vs. Fig 4C); and by the following combination of characters in the third-instar
larva: the teeth of the spatula in O.longicollis are usually closer together than in O.hungaricus
(compare Fig 9F and 9E); O.longicollis has 3 lateral papillae on each side of the spatula and 2
terminal papillae on each side of the anus, as opposed to 4 lateral papillae and 3 terminal papil-
lae on each side in O.hungaricus.
Ozirhincus longicollis Rondani 1840
Ozirhincus longicollis Rondani 1840: 16
Clinorhyncha chrysanthemi Loew 1850
Clinorrhyncha crassipes Winnertz 1853
Clinorrhyncha tanaceti Kieffer 1889 –new synonym
Clinorrhyncha leucanthemi Kieffer 1898
Characters as in O.anthemidis except for the following:
Host plants. Tripleurospermum inodorum,Anthemis arvensis,A.bornmuelleri,A.cotula,
A.pseudocotula,A.rascheyana,Leucanthemum vulgare,Tanacetum balsamita,T.coccineum,
T.parthenium,T.poteriifolium,T.vulgare.
Adult. Head (Fig 3D and 3E): Occiput extending forward at expense of eye area in frontal
view; eye bridge about 4 facets long. Antenna: number of flagellomeres 10 in both sexes
(n = 57♀,36♂); flagellomeres short-cylindrical to almost quadrate in female, more elongate in
male (Fig 5E); first two flagellomeres usually partially to entirely fused, apical flagellomere
slightly tapered, sometimes appears as if ‘budding’to form the beginning of a small additional
segment; male flagellomeres characteristically constricted in mid-section by circumfila loops
(Figs 3E and 5D). Palpus: fourth segment usually 1.3–1.6 times longer than third. Frontoclypeal
membrane greatly extended, with many strong setae on each side. Labrum long-triangular: at
least 4 times as long as width at base, tapering and strongly setose on apical quarter. Labella
more than 10 times as long as wide, somewhat concave medially, tapered apically, with several
strong setae towards apex and densely setose along entire medial surface.
Thorax: Anepimeron with a group of 5–15 setae. Wing length: 1.13–1.57 mm in males
(n = 19), 1.18–1.64 mm in females (n = 22).
Female abdomen: Elongate sclerites of tergite 8 (Fig 7E), each with wide anterior area with
pointed dorsal and ventral extensions and wide posterior area connected by long, narrow band;
each sclerite with trichoid sensillum on anterior part of narrow band, and 4–6 posterior setae
on widened posterior area. Eighth tergite 1.53–2.51 times as long as seventh tergite (n = 22).
Ovipositor 3.90–7.24 times as long as eighth tergite (n = 12).
Larva. (third instar). Integument covered by rounded verrucae. Antennae 2–3 times as
long as wide. Cephalic apodeme considerably longer than head capsule. Spatula (Fig 9F) long
shafted and bidentate; teeth separated by narrow gap. On each side of spatula 3 asetose lateral
papillae grouped together, and one asetose ventral papilla somewhat farther away. Eighth
abdominal segment with 2 setose papillae on each side.
Pupa (Fig 4E).Light to vivid orange. Antennal bases enlarged, tapered into short bi-dentate
horns; lateral lobe tapered, median lobe rounded.
Distribution. Europe, Israel. Probably circum-Mediterranean.
Material examined. The type of Ozirhincus longicollis Rondani is considered lost, based
on an exhaustive study of Rondani’s collection by Gagné and Solinas [27]. Given that this is the
type species of Ozirhincus, we hereby designate a neotype for it in order to clarify the
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 20 / 29
application of the name O.longicollis Rondani and the generic concept of Ozirhincus as a
whole. Although the host plant from which O.longicollis was described in Italy is unknown,
the morphological description given by Rondani is distinctive, and the species has been
reported since from various locations and host plants throughout Europe [9–11], [26]. Based
on this information, we designate the neotype from Germany, from the first host plant with
which this species has been associated [26].
NEOTYPE: ♀, Germany, Leverkusen, 30.viii.2011, E. Diehl, reared from Tripleurospermum
inodorum. The neotype is mounted on a permanent microscope slide in euparal and deposited
in TAUI.
OTHER MATERIAL EXAMINED: 1♀, UK, Surrey, Wisley, 19.vi.1926, HF Barnes, ex Leu-
canthemum vulgare (as ‘Oxeye Daisy’), BMNH(E) 1633265; 9♀,2♂, UK, RHS Wisley, 13.
vii.1953, HF Barnes, ex. Tanacetum coccineum (as Chrysanthemum coccineum), Tanacetum
parthenium (as Chrysanthemum parthenium), and Tanacetum poteriifolium (as Chrysanth-
mum cassium), BMNH(E) 1633190, 1633192, 1633228–9, 1633232–5, 1633240–1, 1633243–4;
1♀, UK, Lincolnshire, Barnack station, near Stamford, HF Barnes, ex Leucanthemum vulgare
(as ‘wild Oxeye Daisy’), BMNH(E) 1633239; 13♀,2♂, UK, Hertsfordshire, Bayfordbury, HF
Barnes, ex Tanacetum poteriifolium (as Chrysanthemum cassium), Tanacetum balsamita (as
Chrysanthemum balsamita), and ‘Chrysanthemum sp.’(most probably Tanacetum sp.),
BMNH(E) 16331989, 1633-201-7, 1633224–5, 1633242, 1633248, 1633249–251; 1♂, Czech
Republic, Bohemia, Dolni Poćenice, 13.viii.1956, M. Skuhravá, ex Anthemis arvensis (Skuhravá
collection, as O.anthemidis); 1♀,1♂, Czech Republic, Bohemia, Davle, 7.vii.1958, M. Skuh-
ravá, ex Anthemis arvensis (Skuhravá collection, as O.anthemidis); 3♀,2♂, Czech Republic,
Petrovice, 22.vii.1964, M. Skuhravá, ex Anthemis cotula (in ethanol); 1♀, Czech Republic,
Bohemia, ŹameÆl, 16.viii.1964, M. Skuhravá, ex Tripleurospermum inodorum (as Matricaria
inodora) (Skuhravá collection, as O.anthemidis); 2♀, Czech Republic, Bohemia, ZámeÆl, 7.
v.1965, M. Skuhravá, ex Leucanthemum vulgare (in ethanol); 2♀,2♂, Czech Republic, Rybná,
11.v.1965, M. Skuhravá, ex Leucanthemum vulgare (in ethanol); 2♀,3♂, 2 larvae, Germany,
Leverkusen, 30.viii.2011, E. Diehl, ex Tripleurospermum inodorum (same data as neotype); 4♀,
4♂, UK, Surrey, Send, Woodhill, 20.viii.2011, KM. Harris and N. Dorchin, ex Tripleurosper-
mum inodorum;11♀,11♂, 4 larvae, Israel, Ma’agar Bental, 14.v.2012, N. Dorchin, ex Anthemis
bornmuelleri (1♀,1♂ZMBH, 1♀,1♂SMNS, 1♀,1♂NHMW, 1♀,1♂ZFMK, others TAUI); 1
larva, Israel, Mt. Hermon, 1750m, 24.v.2012, N. Dorchin, ex Anthemis rascheyana;2♀,1
♂,
Israel, Ma’agar Bental, 25.v.2012, N. Dorchin, ex Anthemis bornmuelleri;2♀, Israel, Hermon,
1780m, 6.vi.2012, N. Dorchin, ex Anthemis rascheyana;3♀,7♂, Israel, Ma’agar Bental, 22.
v.2014, A. Freidberg, ex Anthemis bornmuelleri;6♀,6♂, Israel, En Timrat 0.5km S, 4.iv.2015,
N. Dorchin and U. Dorchin, ex Anthemis pseudocotula (in enthanol).
Biology. The life history of this species is similar to that of O.anthemidis. In the present
study it was most abundant on Tripleurospermum inodorum and Anthemis bornmuelleri, from
which adults emerged at the same time as those of O.hungaricus and O.anthemidis, respec-
tively. The association of O.longicollis with the several Tanacetum spp. that are mentioned
above is based on examination of material reared by HF Barnes [10] and housed at the BMNH,
material received from the private collection of E. Dijkstra, and on Kieffer’s original description
of O.tanaceti.
Remarks. Rondani [7] described Ozirhincus longicollis without a host plant association,
but mentioned that the proboscis is long, tapered, and held perpendicular to the head. He
named the genus after this character, and the species after the long neck. Loew [26] later
described Clinorhyncha chrysanthemi, also noting that the proboscis is very long and is bent
under the head against the thorax, but he did not give a reason for its separation from O.longi-
collis. The two species were later synonymized by Möhn [11].
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 21 / 29
Two other species synonymized by Möhn [11] under O.longicollis in the same work are
Clinorrhyncha leucanthemi and C.crassipes. The types of these species are lost, but information
in subsequent publications [26], [9], [10], as well as our own findings support Möhn’s synon-
ymy. Winnertz [28] described C.crassipes without an associated host but mentioned that it had
a long proboscis and 10 antennal flagellomeres in the male. This description places it clearly in
O.longicollis. Kieffer [29] described C.leucanthemi from Leucanthemum vulgare (as Chrysan-
themum leucanthemum) without any further information, but detailed information was given
in Barnes et al. [10], who reared it from the same host plant. Barnes et al. show the long probos-
cis and long fourth segment of the palp, and state that the species has 10 antennal flagello-
meres. Again, this combination of characters fits only with O.longicollis.
Finally, O.tanaceti, described by Kieffer [30] from Tanacetum vulgare, is also synonymized
here under O.longicollis. As in the above-mentioned cases, the type series of O.tanaceti is con-
sidered lost, hence our decision is based on Kieffer’s original description and our own findings.
In that description, Kieffer emphasized the fact that all 14 specimens he examined had 10
antennal flagellomeres, a character that fits only O.longicollis, based on our examination of 189
specimens from T.vulgare from the UK, Germany, Austria, Czech Republic, and the Nether-
lands. A single female we reared from T.vulgare, which had 10 antennal flagellomeres, was
found to be O.longicollis based on DNA sequencing, corroborating our conclusion that Kief-
fer’s specimens, upon which he based the description of O.tanaceti, actually constituted a series
of O.longicollis individuals. This finding also confirms our conclusion that flagellomere num-
ber is a reliable character in Ozirhincus.
In the present study we found that O.longicollis often occurs together with O.hungaricus on
Tripleurospermum inodorum. In this scenario, one can tell the two species apart by the differ-
ences described above under O.hungaricus. When O.longicollis occurs in the same host plants
with O.anthemidis, the two species can be distinguished easily by their adult and pupal charac-
ters as described above under O.anthemidis.
Ozirhincus millefolii (Wachtl 1884)
Clinorrhyncha millefolii Wachtl 1884: 161
Clinorhyncha filicis Felt 1907
Clinorhyncha karnerensis Felt 1908
Characters as in O.anthemidis except for the following:
Host plants. Achillea millefolium,Achillea ptarmica.
Adult. Head (Fig 3C): Occiput extending forward at expense of eye area in frontal view;
eye bridge 2-facets long. Palpus: segment four usually 1.2–1.7 times longer than segment three.
Frontoclypeal membrane greatly extended, with many strong setae on each side. Labrum long
triangular: at least 4 times as long as width at base, tapering and strongly setose on apical quar-
ter. Labella at least 4 times as long as wide, somewhat concave medially, tapered apically, with
several strong setae towards apex and densely setose along entire medial surface. Antenna (Fig
5F): number of flagellomeres 8, occasionally 9 in both sexes (n = 49♀,32♂); number occasion-
ally differs between antennae of same individual; flagellomeres cylindrical, barrel-shaped,
about 1.3 times as long as wide in female, 1.7 times as long as wide in male; apical flagellomere
slightly tapered.
Thorax: Brownish-orange, covered by black and white scales creating three longitudinal
black stripes along dorsum separated by thinner white stripes; pleura with white scales. Anepi-
meron with a group of 5–18 setae. Empodia about as long as bend in claw. Wing length: 1.06–
1.44 mm in males (n = 17), 1.14–1.44 mm in females (n = 21). Halter white.
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 22 / 29
Female abdomen: General color brownish-orange covered by a mixture of reddish-brown
scales speckled with black scales and narrow transverse strip of white scales along posterior
margin. Sclerites of tergite 8 (Fig 7F) strongly pigmented, with wide anterior area narrowing
gradually towards posterior, with 3–7 setae on widened posterior section. Eighth tergite 1.46–
2.16 times as long as seventh tergite (n = 21). Ovipositor 5.05–7.04 times as long as eighth ter-
gite (n = 18). Dorso-lateral plate on cercal segment with 10–14 strong, straight setae.
Larva (third instar). Antennae about twice as long as wide. Spatula (Fig 9G) bidentate, with
narrow gap between teeth; shaft pigmentation evanescent posteriorly. On each side of spatula 4
asetose lateral papillae, one of which farther laterally than others. Eighth abdominal segment
with 2 setose papillae on each side.
Pupa (Fig 4F).Antennal bases form short bi-dentate horns; lateral lobe slightly longer than
median lobe; both tapered, pointed ventroapically. Horns widely separated on vertex by hori-
zontal ridge.
Distribution. Widespread in Europe to Siberia. Introduced into and presently widespread
in North America.
Material examined. The type series of Ozirhincus millefolii (Wachtl) could not be found
in the Natural History Museum in Vienna (NHMW), where it was supposedly deposited
(Peter Sehnal, pers. comm.) and is considered lost. We therefore designate a Neotype for it as
follows: NEOTYPE: ♀, Germany, NRW, Ägedienberg, 11.viii.2011, N. Dorchin, reared from
Achillea millefolium. The neotype is mounted on a permanent microscope slide in euparal and
deposited in TAUI.
OTHER MATERIAL EXAMINED: (all from Achillea millefolium): 2♀, Germany, Remagen,
14.v.1907, collector not specified (NHMW); 2♀,1♂, collection details not given, from Mik col-
lection (NHMW); 1♀,1♂, UK, Slough, viii.1938, HF Barnes, BMNH(E) 1633185–6; 1♀,1♂,
Czech Republic, Silesia, Roudno, 30.vii.1958, M. Skuhravá (Skuhravá collection); 2♀,2♂, Aus-
tria, Waldviertel, Pargatstetten, 700m a.s.l., 2.ix.1991, Marcela Skuhravá (in ethanol); 5♀,3♂
(on slides), 3♀(pinned), Germany, NRW, Ägedienberg, 11.viii.2011, N. Dorchin (same data as
neotype) (1♀and 1♂ZMBH, others TAUI); 6♀,3♂, 4 larvae, Germany, NRW, Wahner
Heide, 11.viii.2011, N. Dorchin; 7♀,11♂(on slides), 4♀,2♂(pinned), Germany, NRW, Lever-
kusen, 14.viii.2011, E. Diehl (2♀,2♂ZFMK, others TAUI); 5♀,6♂, UK, Ripley, Surrey, Gravel
Pits, 01.viii.2011, KM. Harris; 17♀,4♂,USA, PA, Lewisburg, 15.vii.2012, N. Dorchin.
Biology. The life history of this species is similar to that of O.longicollis. Its main host
plant is Achillea millefolium, from which it can be reared in great numbers in July-August in
Europe and the USA.
Remarks. This species was described in detail by Wachtl [31] from Austria, with compari-
son to Clinorrhyncha chrysanthemi, but not to Ozirhincus longicollis, which was placed in a dif-
ferent genus at the time. It was probably introduced into North America with the seeds of its
main host plant, Achillea millefolium, during colonial times [4], and was first recognized there
by Felt [32], who described it as Clinorhyncha filicis, and again in 1908 as C.kanerensis [33].
These two species were later synonymized under O.millefolii by Gagné [5].
Ozirhincus millefolii is usually smaller than other Ozirhincus spp., the scale covering on its
thorax and abdomen is largely reddish-brown rather than strictly black-and-white, and it is
easily distinguishable from the remaining three species by having only 8–9 antennal flagello-
meres, and by the position of the antennal horns in the pupa, which are widely splayed. Möhn
[12] listed Santolina chamaecyparissus as a host plant based on larvae he found in dried herbar-
ium material from France but this plant genus was not verified as a host in the present study.
Ozirhincus trichatus Möhn was described from Achillea fragrantissima based on three lar-
vae, which were extracted from herbarium material originally collected in Syria, near Damas-
cus [12]. Achillea fragrantissima is found along the eastern Mediterranean region, from Syria,
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 23 / 29
through Jordan and Israel to Egypt, as well as in the Arabian Peninsula. It is a common and
widespread plant in Israel, and we have sampled it many times at different localities and dates
in an effort to rear Ozirhincus from it, without success. We therefore suspect that the identifica-
tion of the host plant might have been erroneous. Because morphological characters of the lar-
vae are generally unreliable for distinguishing among Ozirhincus species, it is currently
impossible to verify that O.trichatus is a valid species rather than a synonym of O.millefolii,
which has been recorded by Möhn [12] from numerous Achillea species. In view of these facts,
we make O.trichatus a nomen dubium until adults are reared from A.fragrantissima that can
be identified to species with certainty.
Discussion
Species delimitation and host-plant ranges in Ozirhincus have been unclear for over a century,
mainly due to complex host associations and the fact that the only revision of the genus [11],
[12] was based on the mostly uninformative larvae. As noted by Barnes et al. [10], resolving the
taxonomic issues in Ozirhincus necessitated a thorough comparative study of specimens from
a wide array of host plants. In the present work we combined such a study with molecular
methods in order to infer the phylogeny of the genus and provide, for the first time, discrete
characters for recognizing the species within it. We found that Ozirhincus includes four clearly
defined species that can be distinguished from each other based on morphological characters
of adults and pupae. Our molecular data supported the morphological findings and corrobo-
rated our taxonomic conclusions. It is noteworthy that the most useful morphological charac-
ter we found is the number of antennal flagellomeres, which is generally considered an
unreliable character in the supertribe Lasiopteridi (to which Ozirhincus belongs) [22]. While in
other Lasiopteridi flagellomere numbers may vary among individuals within the same species
(e.g., [6], [34–38]), we showed that in Ozirhincus flagellomere numbers are consistent within a
species and are diagnostic.
The most conspicuous morphological character of Ozirhincus is the elongate proboscis,
although we do not know what function it serves. While it is assumed that the short-lived
adults of most cecidomyiids do not feed [4], some are known to consume nectar or pollen from
flowers, and may effect pollination (e.g., [39], [40]). This is in contrast to cecidomyiid species
that effect pollination by using flowers as oviposition sites rather than a food source for the
adults (e.g., [41–43]). In the case of Ozirhincus, because the females spend some time on the
inflorescences when ovipositing into flowers, they end up carrying some pollen grains on their
body, and possibly contribute towards pollination, but we do not know if they actually feed on
the flowers by inserting their proboscis into them. If the adults do feed on nectar or pollen, one
might expect that the length of their proboscis will correlate with the length of the flowers of
their hosts, but this is not the case. Ozirhincus anthemidis, whose main hosts are Chrysanthe-
mum species, has the shortest proboscis in the genus, whereas O.millefolii and O.longicollis
have much longer proboscises, yet they are associated with the shorter flowers of Achillea and
Anthemis species. Determining if and how Ozirhincus adults use the flowers of their host plants
as a food source requires more study.
Ozirhincus, which evolved for larval development in achenes of Anthemideae host plants,
can be regarded as an offshoot of the closely related genus Lasioptera. Möhn [12] argued that a
short proboscis, as seen in O.anthemidis, is the ancestral state in the genus, because it repre-
sents a transitional state between this genus and Lasioptera. The molecular analysis conducted
in the present study, while clearly supporting the validity of four species in Ozirhincus, was
unable to resolve the phylogenetic relationships among them, and ancestral states reconstruc-
tion does not offer insight into the evolution of proboscis size. Nevertheless, a position of the
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 24 / 29
Table 2. Samples used for analysis of the COI and 16S mitochondrial genes, with GenBank accession numbers.
ID on tree Taxon Collecting
location
Host plant Collecting
date
Morphological
voucher ID
DNA voucher
ID
GenBank
Acc. # COI
GenBank
Acc. # 16S
Baldratia sp.Baldratia sp. Israel: Mizpe
Yeriho
Suaeda asphaltica 16.ii.2014 178092 KP399947
Careopalpis
sp.
Careopalpis
sp.
Israel: Enot
Zuqim
Suaeda fruticosa 2.iii.2014 178909 KP399948
Stefaniola sp.Stefaniola sp. Israel: Nahal
Zeruya
Suaeda fruticosa 16.ii.2014 178094 KP399949
Lasioptera
carophila
Lasioptera
carophila
Israel: Kefar
Hahoresh
Phoeniculum
vulgare
12.x.2009 133693 ZFMK-DNA-
0155668040
KP399946
Her12-5 Ozirhincus
anthemidis
Israel:
Herzeliya
Chrysanthemum
coronarium
19.iv.2012 137023–137025 ZFMK-DNA-
0100405620
KP399913 KR338912
Her12-6 Ozirhincus
anthemidis
Israel:
Herzeliya
Chrysanthemum
coronarium
19.iv.2012 137023–137025 ZFMK-DNA-
0100405619
KP399916 KR338915
Her12-7 Ozirhincus
anthemidis
Israel:
Herzeliya
Chrysanthemum
coronarium
19.iv.2012 137023–137025 ZFMK-DNA-
0100405618
KP399919 KR338916
KH12-8 Ozirhincus
anthemidis
Israel: Kefar
Hahoresh
Chrysanthemum
coronarium
28.iv.2012 137026–137027 ZFMK-DNA-
0100405617
KP399922 KR338918
KH12-9 Ozirhincus
anthemidis
Israel: Kefar
Hahoresh
Chrysanthemum
coronarium
28.iv.2012 137026–137027 ZFMK-DNA-
0100405616
KP399925 KR338919
KH12-10 Ozirhincus
anthemidis
Israel: Kefar
Hahoresh
Chrysanthemum
coronarium
28.iv.2012 137026–137027 ZFMK-DNA-
0100405615
KP399927 KR338921
NeA12-11 Ozirhincus
anthemidis
Israel: Newe
Ativ
Anthemis tinctoria 25.v.2012 153147 ZFMK-DNA-
0100405614
KP399929 KR338923
NeA12-12 Ozirhincus
anthemidis
Israel: Newe
Ativ
Anthemis tinctoria 25.v.2012 153147 ZFMK-DNA-
0100405613
KP399931 KR338924
NeA12-13 Ozirhincus
anthemidis
Israel: Newe
Ativ
Anthemis tinctoria 25.v.2012 153147 ZFMK-DNA-
0100405612
KP399933 KR338926
Bu12-14 Ozirhincus
anthemidis
Israel: Mt.
Hermon
Anthemis
rascheyana
25.v.2012 137030–137031 ZFMK-DNA-
0100404594
KP399934 KR338927
Bu12-15 Ozirhincus
anthemidis
Israel: Mt.
Hermon
Anthemis
rascheyana
25.v.2012 137030–137031 ZFMK-DNA-
0100405599
KP399935 KR338928
Bu12-16 Ozirhincus
anthemidis
Israel: Mt.
Hermon
Anthemis
rascheyana
25.v.2012 137030–137031 ZFMK-DNA-
0100405600
KP399936 KR338929
Niz13-16 Ozirhincus
anthemidis
Israel: Nahal
Nizzana
Anthemis retusa 12.v.2013 151802 ZFMK-DNA-
0155668041
KP399937 KR338930
Niz13-17 Ozirhincus
anthemidis
Israel: Nahal
Nizzana
Anthemis retusa 12.v.2013 151802 ZFMK-DNA-
0155668042
KP399939 KR338932
Ben12-18 Ozirhincus
anthemidis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405602
KP399940 KR338933
Niz13-18 Ozirhincus
anthemidis
Israel: Nahal
Nizzana
Anthemis retusa 12.v.2013 151802 ZFMK-DNA-
0155667693
KP399941 KR338934
Ben12-19 Ozirhincus
anthemidis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405603
KP399942 KR338935
Ben12-20 Ozirhincus
anthemidis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405604
KP399943 KR338936
Ben12-21 Ozirhincus
anthemidis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405605
KP399944 KR338937
WH11-3 Ozirhincus
hungaricus
Germany:
Wahner Heide
Tanacetum vulgare 11.viii.2011 137016, 137018,
137022
ZFMK-DNA-
0100405216
KP399906 KR338907
Lev11-4 Ozirhincus
hungaricus
Germany:
Leverkusen
Tanacetum vulgare 14.viii.2011 137020, 137021 ZFMK-DNA-
0100405217
KP399909 KR338909
Aeg11-5 Ozirhincus
hungaricus
Germany:
Aegidienberg
Tanacetum vulgare 11.viii.2011 137017, 137019 ZFMK-DNA-
0100405218
KP399912 KR338911
(Continued)
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 25 / 29
Table 2. (Continued)
ID on tree Taxon Collecting
location
Host plant Collecting
date
Morphological
voucher ID
DNA voucher
ID
GenBank
Acc. # COI
GenBank
Acc. # 16S
UKS11-7 Ozirhincus
hungaricus
UK: Send,
Woodhill
Tripleurospermum
inodorum
20.viii.2011 137043–137044 ZFMK-DNA-
0100405220
KP399918
Lev13-10 Ozirhincus
hungaricus
Germany:
Leverkusen
Tripleurospermum
inodorum
30.viii.2011 137046, 151804 ZFMK-DNA-
0155667731
KP399928 KR338922
Lev13-11 Ozirhincus
hungaricus
Germany:
Leverkusen
Tripleurospermum
inodorum
30.viii.2011 137046, 151804 ZFMK-DNA-
0155668036
KP399930
UKP13-7 Ozirhincus
hungaricus
UK:
Papercourt
Lock
Tanacetum vulgare 18.viii.2011 153704 ZFMK-DNA-
0155667692
KP399920
UKP13-8 Ozirhincus
hungaricus
UK:
Papercourt
Lock
Tanacetum vulgare 18.viii.2011 153704 ZFMK-DNA-
0155668033
KP399923
UK11-6 Ozirhincus
longicollis
UK: Send,
Woodhill
Tripleurospermum
inodorum
20.viii.2011 137043–137044 ZFMK-DNA-
0100404551
KP399915 KR338914
Lev11-8 Ozirhincus
longicollis
Germany:
Leverkusen
Tripleurospermum
inodorum
30.viii.2011 137046, 151804 ZFMK-DNA-
0100405221
KP399921 KR338917
Ben13-1 Ozirhincus
longicollis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
25.v.2012 137034 ZFMK-DNA-
0155622342
KP399902 KR338904
Ben13-2 Ozirhincus
longicollis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
25.v.2012 137034 ZFMK-DNA-
0155622343
KP399905 KR338906
Ben13-3 Ozirhincus
longicollis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
25.v.2012 137034 ZFMK-DNA-
0155622344
KP399908 KR338908
UK13-4 Ozirhincus
longicollis
UK: Send,
Woodhill
Tripleurospermum
inodorum
25.viii.2011 153248 ZFMK-DNA-
0155622345
KP399911 KR338910
UK13-9 Ozirhincus
longicollis
UK: Send,
Woodhill
Tripleurospermum
inodorum
20.viii.2011 137043, 137044 ZFMK-DNA-
0155668034
KP399926 KR338920
UK13-12 Ozirhincus
longicollis
UK: Newlands
Corner
Leucanthemum
vulgare
15.ix.2011 153149 ZFMK-DNA-
0155668037
KP399932 KR338925
Ben12-17 Ozirhincus
longicollis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405601
KP399938 KR338931
Ben12-22 Ozirhincus
longicollis
Israel: Ma'agar
Bental
Anthemis
bornmuelleri
14.v.2012 137036 ZFMK-DNA-
0100405606
KP399945 KR338938
Lev11-1 Ozirhincus
millefolii
Germany:
Wahner Heide
Achillea millefolium 11.viii.2011 137010, 137011,
137013
ZFMK-DNA-
0100405227
KP399900 KR338902
US12-1 Ozirhincus
millefolii
USA: PA,
Lewisburg
Achillea millefolium 15.vii.2012 133890 ZFMK-DNA-
0100405633
KP399901 KR338903
Lev11-2 Ozirhincus
millefolii
Germany:
Leverkusen
Achillea millefolium 14.viii.2011 137012 ZFMK-DNA-
0100405215
KP399903 KR338905
US12-2 Ozirhincus
millefolii
USA: PA,
Lewisburg
Achillea millefolium 15.vii.2012 133890 ZFMK-DNA-
0100405634
KP399904
US12-3 Ozirhincus
millefolii
USA: PA,
Lewisburg
Achillea millefolium 15.vii.2012 133890 ZFMK-DNA-
0100405622
KP399907
Aeg12-4 Ozirhincus
millefolii
Germany:
Aegidienberg
Achillea millefolium 11.viii.2011 137007 ZFMK-DNA-
0100405621
KP399910
UK13-5 Ozirhincus
millefolii
UK: Ripley,
Gravel Pitts
Achillea ptarmica 2.viii.2011 153150 ZFMK-DNA-
0155622346
KP399914 KR338913
UK13-6 Ozirhincus
millefolii
UK: Ripley,
Gravel Pitts
Achillea ptarmica 2.viii.2011 153150 ZFMK-DNA-
0155622347
KP399917
Lev11-9 Ozirhincus
millefolii
Germany:
Leverkusen
Achillea millefolium 15.viii.2011 ZFMK-DNA-
0100405222
KP399924
The identification of specimens was done by ND. Morphological vouchers are kept at TAUI, and DNA vouchers are kept at ZFMK.
doi:10.1371/journal.pone.0130981.t002
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 26 / 29
short-snouted O.anthemidis at the base of the tree would be the most parsimonious inference,
with the very long snout of O.longicollis representing a derived state. Morphologically, Ozir-
hincus is much closer to Lasioptera than to other Lasiopterini, as has also been confirmed by
our molecular data. However, it is not clear if Ozirhincus evolved from Asteraceae-feeding spe-
cies within the biologically diverse Lasioptera (currently with 130 species) because most of the
relevant Lasioptera species are associated with different Asteraceae tribes. Of the six Lasioptera
species that develop in Anthemideae, five are associated with Artemisia, which is not used by
Ozirhincus [1]. The sixth species, L.francoisi Kieffer, was recorded from Achillea millefolium
but we did not rear it in the present study despite repeated collections of this host plant. Fur-
ther large-scale sampling of Lasioptera species was beyond the scope of the present work.
The fact that Ozirhincus is limited to host plants of the tribe Anthemideae is of interest
given that other cecidomyiid genera, such as the large genus Rhopalomyia Rübsaamen, also
appear to prefer this tribe [1]. However, within the Anthemideae, no Ozirhincus species has
ever been reared from Artemisia, a genus that hosts more than 170 Rhopalomyia species [44],
[1]. One of the main goals achieved in the present study, was to clarify the complex pattern of
host use in Ozirhincus, which had been a major cause for confusion and uncertainty in the tax-
onomy of this genus. With the exception of O.millefolii, each Ozirhincus species is now known
to use several host-plant species in more than one genus, and each host-plant genus other than
Achillea is known to support more than one Ozirhincus species. This means that a single sam-
ple of some plants can yield two Ozirhincus species simultaneously, but in such cases the key
and descriptions we provided here make it easy to recognize the species.
This work shows that Ozirhincus species are oligophagous, similar to some species of
Lasioptera (e.g., L.buhri Möhn, L.carophila Löw), but in stark contrast to the mostly monoph-
agous species in the subtribe Baldratiina (e.g., Baldratia,Careopalpis, and Stefaniola)([
6] and
Dorchin, unpubl. data). The extent of oligophagy within Ozirhincus varies, with species cur-
rently known from between 1–4 genera and 3–12 species of host plants. In those species that
exhibit the wider host ranges (O.longicollis and O.anthemidis), we did not find any evidence
for host-race formation, as individuals from different host plants were intermixed in our phylo-
genetic tree. It is noteworthy that the two species with the smaller number of host species are
limited to Europe and the Russian Far East, whereas those with a larger number of host species
extend all the way to the southern Mediterranean region. Not surprisingly, this observation
suggests that having a greater number of host species contributes toward a wider distribution
range.
Acknowledgments
We are grateful to Elise Diehl (Museum Koenig, Bonn) and to Alexandra Brodezki (Dept. of
Zoology, Tel Aviv University) for their invaluable help in field collecting and laboratory work,
to Claudia Etzbauer, Hannah Janssen and Tina Blume (Museum Koenig, Bonn) for help with
the molecular work, to Raymond J. Gagné (USDA, SEL, Washington DC) and Neal L. Evenhuis
(Bishop Museum, Hawaii) for nomenclatorial advice, to Ofer Cohen and Yuval Sapir (Tel Aviv
University) for help in host-plant identifications, and to the following colleagues for lending,
imaging, or donating material from the collections under their care: Hans-Peter Tschorsnig
(Staatlisches Museum für Naturkunde, Stuttgart), Joachim Ziegler (Natural History Museum,
Berlin), Peter Sehnal (Natural History Museum, Vienna), Duncan Sivell (Natural History
Museum, London), Marcela Skuhravá (private collection, Prague), and Eddy Dijkstra (private
collection, Wageningen). We are greatly indebted to Janet Rowley, Hedy Jansen, Jerry Clough,
and CG Magnusson for their kind permission to use their beautiful photos of live Ozirhincus
adults in this publication and in scientific presentations that were based on this work. RJ
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 27 / 29
Gagné, Junichi Yukawa, and Chris Borkent provided helpful comments on an earlier version of
this manuscript. This study was supported by the Israeli Taxonomy Initiative.
Author Contributions
Conceived and designed the experiments: ND JJA KMH LB. Performed the experiments: ND
JJA KMH LB. Analyzed the data: ND JJA. Wrote the paper: ND JJA KMH.
References
1. Gagné RJ, Jaschhof M (2014) Update for a Catalog of the Cecidomyiidae (Diptera) of the World. 3rd
edition, digital version 2. Available: http://www.ars.usda.gov/SP2UserFiles/Place/80420580/Gagne_
2014_World_Cecidomyiidae_Catalog_3rd_Edition.pdf. Accessed 30 December 2014.
2. Yukawa J, Rohfritsch O (2005) Biology and ecology of gall-inducing Cecidomyiidae (Diptera). In:
Raman A, Schaefer CW, Withers TM, editors. Biology, ecology, and evolution of gall-inducing arthro-
pods. Enfield: Science Publishers, Inc. pp. 273–304.
3. Redfern M (2011) Plant Galls. London: Harper Collins Publishers.
4. Gagné RJ (1989) The Plant Feeding Gall Midges of North America. New York: Cornell University
Press.
5. Gagné RJ (1969) A tribal and generic revision of the Nearctic Lasiopteridi (Diptera: Cecidomyiidae).
Ann Entomol Soc Am 62: 1348–1364. PMID: 5374172
6. Dorchin N, Freidberg A, Mokady O (2004) Phylogeny of the Baldratiina (Diptera: Cecidomyiidae)
inferred from multiple data sources, and evolutionary patterns in plant-galler relationships. Mol Phylo-
genet Evol 30: 503–515. PMID: 15012935
7. Rondani C (1840) Sopra alcuni nuovi generi di insetti ditteri. Memoria Seconda per Servire alla Dittero-
logia Italiana. Parma. 27 pp.
8. Verrall GH (1889) A study in orthography. Wien Entomol Z 8: 121–123.
9. Rübsaamen H (1916) Cecidomyidenstudien IV. Revision der deutschen Oligotropharien und Lasiopter-
arien nebst Beschreibung neuer Arten. Sitz Gesell Nat Fr Berlin 1915: 485–567.
10. Barnes HF, Arnold MK, Heath GW (1962) The gall midges (Diptera: Cecidomyiidae) of wild ox-eye
daisy (Chrysanthemum leucanthemum L.) flowers, with the description of a new species. Trans. Soc.
British Entomol 15: 1–20.
11. Möhn E (1966) Cecidomyiidae = (Itonididae) In: Lindner E (editor). Die Fliegen der palaearktischen
Region II 2, 6 L, Lieferung 269: 1–48.
12. Möhn E (1968) Cecidomyiidae = (Itonididae) In: Lindner E (editor). Die Fliegen der palaearktischen
Region II 2, 6 L, Lieferung 273: 49–96.
13. Skuhravá M (1989) Taxonomic changes and records in Palaearctic Cecidomyiidae (Diptera). Acta
Entomol Bohemos 86: 202–233.
14. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial
cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3: 294–
299.
15. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phyloge-
netic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reac-
tion primers. Ann Entomol Soc Am 87: 651–701.
16. Astrin JJ & Stüben PE (2008) Phylogeny in cryptic weevils: molecules, morphology and new genera of
western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). Invertebr Syst 22: 503–522.
17. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT. Nucl Acids Symp Ser 41: 95–98.
18. Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:
817–818. PMID: 9918953
19. Lanave C, Preparata G, Saccone C, Serio G (1984) A new method for calculating evolutionary substitu-
tion rates. J Mol Evol 20: 86–93. PMID: 6429346
20. Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics
17: 754–755. PMID: 11524383
21. Maddison WP, Maddison DR. 2011. Mesquite: a modular system for evolutionary analysis. Version
2.75. Available at: http://mesquiteproject.org (accessed 22 Apr 2015).
22. Gagné RJ (1994) The Gall Midges of the Neotropical Region. New York: Cornell University Press.
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 28 / 29
23. McAlpine JF, Peterson BV, Shewell GE, Teskey HJ, Vockeroth JR, Wood DM, editors (1981) Manual of
Nearctic Diptera Vol. 1. Research Branch, Agriculture Canada. Monograph No. 27.
24. Rohfritsch O (1997) Morphological and behavioral adaptations of the gall midgeLasioptera arundinis
(Schiner) (Diptera, Cecidomyiidae) to collect and transport conidia of its fungal symbiont. Tijdschr Ento-
mol 140:59–66.
25. Rohfritsch O (2008) Plants, gall midges, and fungi: a three component system. Entomol Exp Appl 128:
208–216.
26. Loew H (1850) Dipt Beitr IV, Posen. 40pp.
27. Gagné RJ, Solinas M (1996) The Rondani Cecidomyiidae (Diptera). Insect Mundi 10: 69–76.
28. Winnertz J (1853) Beitrag zu einer Monographie der Gallmücken. Linn Entomol 8: 154–322.
29. Kieffer JJ (1898) Synopse des cécidomyies d'Europe et d'Algérie décrites jusqu'àce jour. Bull Soc Hist
Nat Metz (2: ) 8: 1–64.
30. Kieffer JJ (1889) Neue Beiträge zur Kenntniss der Gallmücken. Entomol Nach 15: 208–212.
31. Wachtl FA (1884) Eine neue und eine verkannte Cecidomyide. Wien Entomol Z 3: 161–166.
32. Felt EP (1907) New species of Cecidomyiidae. New York State Education Department, New York.
33. Felt EP (1908) Appendix D. pp. 286–422, 489–510, in: 23d report of the State Entomologist on injurious
and other insects of the State of New York 1907. N Y State Mus Bull 124: 5–541.
34. Dorchin N, Freidberg A (2008) The Chenopodiaceae gall midges (Diptera: Cecidomyiidae) of the Na’a-
man salt marsh, Israel. Zootaxa 1937: 1–22.
35. Dorchin N, Freidberg A (2011) The gall midges (Diptera: Cecidomyiidae) of Apiaceae in Israel. Zootaxa
3044: 28–48.
36. Dorchin N, McEvoy MV, Dowling TA, Abrahamson WG, Moore JG (2009). Revision of the goldenrod-
feeding Rhopalomyia (Diptera: Cecidomyiidae) in North America. Zootaxa 2152: 1–35.
37. Gagné RJ, Graney L (2014) Piceacecis (Diptera: Cecidomyiidae), a new genus for a non-native pest of
Norway spruce from Europe and its North American relative. Proc Entomol Soc Wash 116: 378–393.
38. Gagné RJ, Moser JC (2013) The North American gall midges (Diptera: Cecidomyiidae) of hackberries
(Cannabaceae: Celtis spp.). Mem Am Entomol Soc 49: 1–103.
39. Yuan LC, Luo YB, Thien LB, Fan JH, Xu HL, Yukawa J, et al. (2008). Pollination of Kadsura longipedun-
culata (Schisandraceae), a monoecious basal angiosperm, by female, pollen-eating Megommata sp.
(Cecidomyiidae: Diptera) in China. Biol J Linn Soc 93: 523–536.
40. Yukawa J, Sato S, Xu HL, Tokuda M (2011) Description of a new species of the genus Resseliella (Dip-
tera: Cecidomyiidae), a pollinator of Kadsura longipedunculata (Schisandraceae) in China, with com-
ments on its flower-visiting habits. Entomol Sci 14: 297–303.
41. Feil JP (1992) Reproductive ecology of dioecious Siparuna (Monimiaceae) in Ecuador–A case of gall
midge pollination. Bot J Linn Soc 110: 171–203.
42. Luo SX, Chaw S, Zhang D, Renner SS (2010). Flower heating following anthesis and the evolution of
tall midge pollination in Schisandraceae. Am J Bot 97: 1220–1228. doi: 10.3732/ajb.1000077 PMID:
21616873
43. Vislobokov NA, Galinskaya TV, Degtjareva GV, Valiejo-Roman CM, Samigullin TH, Kuznetsov AN,
et al. (2014) Pollination of Vietamese Aspidistra xuansonensis (Asparagaceae) by female Cecidomyiidi
flies: Larvae of pollinator feed on fertile pollen in anthers of anthetic bisexual flowers. Am J Bot 101:
1519–1531. doi: 10.3732/ajb.1400359 PMID: 25253712
44. Jones RG, Gagné RJ, Barr WF (1983) Biology and taxonomy of the Rhopalomyia gall midges (Diptera:
Cecidomyiidae) of Artemisia tridentata Nuttall (Compositae) in Idaho. Contr Am Entomol Inst 21: 1–79.
Systematics and Phylogeny of Ozirhincus
PLOS ONE | DOI:10.1371/journal.pone.0130981 July 2, 2015 29 / 29
