Dtsch. Entomol. Z. 67 (1) 2020, 13-18 | DOI 10.3897/dez.67.49741

yee
BERLIN

Biology of two European Tenthredo species
(Hymenoptera, Tenthredinidae) feeding on Gentiana

Andrew Liston’, Ewald Altenhofer*, Romana Netzberger?, Marko Prous!“

Senckenberg Deutsches Entomologisches Institut, Eberswalder St. 90, 15374 Miincheberg, Germany

Etzen 39, 3920 Grof Gerungs, Austria

Innerzaun 26/2, 3321 Kollmitzberg, Austria

Department of Zoology, Insitute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, 51014 Tartu, Estonia

BR WN

http://zoobank.org/24D3ACCC-AD2B-4726-9E01-908549F3E54A

Corresponding author: Andrew Liston (aliston@senckenberg.de)

Academic editor: Ralph Peters # Received 30 December 2019 # Accepted 6 February 2020 Published 21 February 2020

Abstract

Very few sawflies using Gentianaceae as larval host plants have been recorded. We identified larvae collected in Austria on Gentiana
asclepiadea L. as Tenthredo atra Linnaeus, 1758 and 7: propinqua Klug, 1817. If its current taxonomic circumscription as a single
species 1s accepted, 7? atra is a highly polyphagous species, whereas 7’ propinqua may be more specialised: Gentiana asclepiadea is its
first recorded host. We sequenced plant DNA from the head of one 7! propinqua larva, which confirmed that it had been feeding on this
plant. This is the first recorded use of G. asclepiadea by sawfly larvae. Larvae are illustrated, and identification characters are described.

Key Words

Gentianaceae, host plants, larvae, sawflies, Symphyta, Tenthredinoidea

Introduction

The study of the immature stages of sawflies, including
the identification of their larval hosts, has a long tradi-
tion in Europe, reaching back to the pioneering studies of
Réaumur (1740). Despite many advances since then, we
still know little or nothing about the biology of some taxa.
Here, we fill one of these gaps by documenting the host
plant association of two 7enthredo species with a member
of the Gentianaceae, a plant family which has hitherto
seldom been mentioned as a host of sawflies.

Recently, DNA sequencing has proved itself as a potent
tool for the identification of sawfly larvae (e.g. Shinohara
et al. 2017; Prous et al. 2019). In this study we used DNA
sequences to identify one of the sawfly species. Com-
pared to the traditional method of rearing an adult from
a larva and determining the adult using morphological
characters, sequencing can provide an identification re-
sult much more quickly, and the risk is avoided of all in-

dividuals dying before they reach maturity, in which case
no identification will be obtained. We also demonstrate
that DNA sequencing can be used to identify or confirm
the host of a larva, using DNA extracted from the larva.
This is especially useful for larvae that were collected,
for example by sweeping, without any clear indication of
what they were feeding on, and might also help to iden-
tify the hosts of species of Tenthredininae which do not
feed on the plant species in which eggs are laid, as report-
ed by Chevin (2009) for some Macrophya species.

Methods

Nearly fully grown larvae of two Tenthredo species
were beaten from, or detected visually on Gentiana
asclepiadea L. in the Gesause National Park, Styria,
Austria, by E. Altenhofer and R. Netzberger in 2016
and 2019. Some larvae were kept by EA for rearing, and

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

14

others were preserved in 95% ethanol. Identification of
Tenthredo atra Linnaeus, 1758 is based on the morphol-
ogy of adults and larvae. Tenthredo propinqua Klug,
1817 was identified by genetic sequences obtained
from a larva. Total DNA was extracted from the head of
one 7! propinqua larva (DEI-GISHym12639), and one
mitochondrial (1087 bp of CO1) and two nuclear gene
fragments (1654 bp of NaK and 2543 bp of POL2) were
sequenced (methodology as in Prous et al. 2019). To
test which host plant the larva had been feeding on, we
used the same larval extract to amplify a plastid region
between trnL 5’ exon and trnF using primers c and f
(Taberlet et al. 1991). The region, which turned out to
be 816 bp, contains two variable introns and the trnL
3' exon and was sequenced with primers c, d, e, and f
(Taberlet et al. 1991). The sequences have been depos-
ited in the GenBank (NCBI) database (accession num-
bers MN856146—MN856149).

Material examined

The abbreviation SDEI refers to the insect collection of
the Senckenberg Deutsches Entomologisches Institut
(SDEI), Muncheberg, Germany.

Tenthredo atra Linnaeus, 1758

Austria: Styria: Gesduse, Kroisalm, 47.60N 14.63E,
900 m, 26.08.2016, 3 females reared from larvae on
Gentiana asclepiadea (emerged May 2017), specimens
were overlooked after emergence, and are in very poor
condition, i.e. fragmented, with diverse parts gummed
on one card (DEI-GISHym12664), and 1 larva, leg.
E. Altenhofer (SDEI). Gesaéuse, E Admont, 47.58N
14.62E, 11.09.2019, 10 larvae on Gentiana asclepia-
dea, leg. E. Altenhofer (SDEI). The last of these lar-
vae entered the ground to overwinter on 21.09.2019.
Gesduse, Hartelsgraben, 47.59N 14.73E, 23.08.2019,
larva on Gentiana asclepiadea, photographic record by
R. Netzberger (Fig. 1).

Tenthredo propinqua Klug, 1817

Larvae:

Austria: Styria: Gesause (E Admont), between Gstat-
terboden and Hochscheibenalm, 47.58N 14.62E, 600-—
1150 m, 15.09.2019, 10 larvae on Gentiana asclepiadea,
leg. E. Altenhofer (2 larvae in SDEI [DEI-GISHym 12639,
12640], others retained by EA for rearing).

Imagines:

Ukraine: 1 male (DEIGISHym20102), Jablunitsa, Ber-
kut, 48.72N 24.37E, 840 m, 08.07.2004, leg. E. Heibo
(SDEI). 1 male (DEIGISHym20103), Jablunitsa, Berkut,
48.72N 24.37E, 840 m, 06.07.2004, leg. E. Heibo (SDE).
1 male (DEIGISHym20104), Jasinja, Tatariv, 48.37N
24.56E, 710 m, 03.07.2004, leg. E. Heibo (SDEI). 1 fe-
male (DEIGISHym20105), Jablunitsa, Berkut, 48.72N
24.37E, 840 m, 06.07.2004, leg. E. Heibo (SDEI).

dez.pensoft.net

Andrew Liston et al.: Tenthredo larvae on Gentiana

Austria: 1 female (DEIGISHym17738), Carinthia,
Eisenkappel 10km E, St Margarethen, 46.46N 14.66E,
28.06.1993, leg. L. Behne (SDEI).

Tenthredo scrophulariae Linnaeus, 1758

The larvae illustrated in Figs 6, 7 were photographed by
Henri Savina in France, Ari¢ge, Aulus-les-Bains, 42.80N
1.33E, respectively on 08.09.2007 and 30.09.2007. Host:
Scrophularia sp.

Results

Tenthredo atra
Figs 1, 2

Notes. TJenthredo atra has already been associated by
various authors with larval hosts in many higher plant
taxa. Taeger et al. (1998), in a summary of these records,
mentioned the families Brassicaceae, Caprifoliaceae,
Lamiaceae, Plantaginaceae, and Solanaceae. There are
also records of larvae of 7 atra feeding on Asteraceae
(Pschorn-Walcher and Altenhofer 2006), Betulaceae and
Salicaceae (Loth 1913), Ranunculaceae (Conde 1934),
Rosaceae (Kangas 1985), and Urticaceae (Pschorn-Wal-
cher and Altenhofer 2000). It is not clear whether records
from Menyanthes trifoliata (Menyanthaceae) and Sedum
telephium (Crassulaceae), which are sometimes named
as hosts of 7. atra (e.g. Taeger et al. 1998), really refer
to this species, or respectively to the closely related 7°
moniliata Klug, 1817 and T: ignobilis Klug, 1817. Tae-
ger et al. (1998) mentioned some additional plant taxa
on which oviposition by 7: atra has been observed but
which have not been proved to be hosts of the larvae. Our
identification of the larvae from Gentiana as T: atra ac-
cepts the premise that the name refers to only one, highly
polyphagous species. However, a wide morphological
variability, most obvious in the colour pattern of 7. atra
imagines, might indicate that more than one species are
currently grouped under this name.

The larvae from Gentiana asclepiadea (Figs 1, 2) are
in general appearance not distinguishable from larvae of
T. atra from other hosts, nor from the larvae of the relat-
ed T. moniliata on Menyanthes trifoliata (Conde 1934;
Liston personal observations). Lorenz and Kraus (1957)
did not examine larvae of 7. atra, and their description
is based on those of Cameron (1882) and Carpentier
(1888). Lorenz and Kraus (1957) did not mention the
faint, oblique, darker dorso-lateral stripes shared by the
larvae from Gentiana, the larva described by Cameron
(1882; as 7. dispar Klug, 1817 from Succisa pratensis),
and larvae of 7. moniliata examined by Liston. Note also
that Carpentier’s (1888) description, as 7? dispar, is of
larvae from Menyanthes trifoliata, and may therefore
refer to TZ. moniliata, but he did not mention any body
markings. On the other hand, the larva of TJenthredo
ignobilis, another species in the complex with 7° atra,

Dtsch. Entomol. Z. 67 (1) 2020, 13-18

15

Figures 1, 2. 7enthredo atra \arvae, nearly full-grown, on Gentiana asclepiadea (photographed respectively on 23.08.2019,
11.09.2019). Photos: R. Netzberger (1), E. Altenhofer (2).

possibly differs from 7 atra in lacking the oblique body
markings (Liston 2015: fig. 9).

Larvae of the later instars feed mainly on the leaves;
they feed from the edge, leaving irregular holes. Inflo-
rescences are also sometimes consumed, at least under
rearing conditions (Fig. 2). The largest full-grown larvae
are similar in size to those of 7? propinqua, 1.e. somewhat
over 20 mm long.

Tenthredo propinqua
Figs 3-5

Notes. A mitochondrial CO1 sequence from one larva
(DEI-GISHym12639) corresponded closely (maximum
divergence 0.5%) with sequences from 7! propinqua
imagines (DEI-GISHym20102, DEI-GISHym20103,
DEI-GISHym20104, DEI-GISHym20105, DEI-

dez.pensoft.net

16

Andrew Liston et al.: Tenthredo larvae on Gentiana

Figures 3—7. Larvae of Tenthredo propinqua and scrophulariae. 3-5. Tenthredo propinqua, from Gentiana asclepiadea. Arrows
indicate some of the black dorso-median markings, which on the abdomen occupy the width of a single annulet; 3. Preserved in
ethanol (wax dissolved); 4. Alive, with wax coating (photographed on 11.09.2019); 5. Head, preserved in ethanol. 6, 7. Zenthredo
scrophulariae larvae, on Scrophularia species. Arrows indicate some of the black dorso-median markings, which on the abdomen
extend across two annulets at least in part; 6. Half-grown (photographed on 08.09.2007); 7. Nearly full-grown (photographed on
30.09.2007). Photos: A. Liston (3, 5), E. Altenhofer (4), H. Savina (6, 7).

GISHym17738). Nuclear sequences (NaK and POL2)
are available only for the specimen sequenced here (DEI-
GISHym12639). The sequenced plastid trnL-trnF region
(816 bp) from the larval DNA extract confirmed Gentiana
as the host. The closest (99-100% similarity) according to
NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
were four species of Gentiana, among them G. asclepiadea.
A shorter G. asclepiadea sequence in GenBank (accession
AB453085, 387 bp) was identical to our sequence, while a
longer one (AJ580515) differed by three substitutions and
one deletion over the length of 781 bp (because of apparent-
ly numerous sequencing errors at the 3’ end of AJ580515,
21 bp of that sequence were excluded from the comparison).

dez.pensoft.net

Gentiana asclepiadea is the first recorded host plant
of the hitherto unknown larva of 7? propinqua, which is a
close relative of 7. scrophulariae Linnaeus, 1758. These
species have long been known to strongly resemble each
other in the morphology of their imagines, but they are
distinguishable using some colour characters (Enslin
1912). Their larvae are also closely similar in general ap-
pearance (see the description of a 7? scrophulariae larva
by Lorenz and Kraus 1957 and below). However, 7! scro-
phulariae has a very different host plant spectrum, which
consists mainly of Scrophularia and Verbascum species,
but sometimes Buddleja species (Muche 1962), all of
which belong to the Scrophulariaceae.

Dtsch. Entomol. Z. 67 (1) 2020, 13-18

Differences in the pattern of black markings may
enable 7’ propinqua larvae to be distinguished from 7
scrophulariae, but a larger number of 7’ propinqua lar-
vae should be checked, to confirm that the differences
are consistent. In 7? propinqua, each of the medio-dorsal
black spots on the abdominal segments occupies only the
width of single annulet (Figs 3, 4), whereas in 7. scrophu-
lariae, these spots occupy parts or the whole width of two
annulets (Figs 6, 7). The position of the corresponding
spots on the thorax is, however, similar in both species.
At least in the later instars of 7. scrophulariae \arvae, the
position of these markings is thought to be stable: com-
pare Fig. 6 (half-grown) with Fig. 7 (nearly full-grown).
After each moult, the integument of the larvae of both
species temporarily lacks the covering of white wax and
has a greenish ground colour. The head colour pattern of
T. propinqua (Fig. 5) is the same as described by Lorenz
and Kraus (1957) for 7. scrophulariae. In practice, the
identity of their host plant should be sufficient to distin-
guish larvae of these species.

The feeding habits of Zenthredo propinqua larvae are
similar to those of 7° atra, i.e. irregularly shaped parts of
the leaf-blade are consumed from the edge. But, unlike
for 7: atra, we did not observe feeding on the inflores-
cences by 7. propinqua. The largest full-grown larvae
are 22—25mm long, which 1s about the same as given by
Lorenz and Kraus (1957) for 7) scrophulariae.

Discussion

As far as we are aware, neither Gentiana nor any oth-
er member of the Gentianaceae has previously been re-
corded as a larval host of a sawfly, except by Wang et
al. (2015), who studied in China the effect of florivory
by larvae on Halenia elliptica D. Don; they referred the
larvae to as an undescribed species of Tenthredinidae.
Otherwise, the only reported interaction between a saw-
fly species and a species of Gentianaceae involves vis-
its to the inflorescences of Frasera speciosa Douglas ex
Griseb. by the Nearctic 7Zenthredo erythromera Provanch-
er, 1885 (Norment 1988).

Tenthredo propinqua is a rather rarely collected spe-
cies (Ritzau 1998), whose known distribution compris-
es south-eastern Europe, Turkey, and the Transcaucasus
(Lacourt 1999). Although the eastern part of the range
of Gentiana asclepiadea is more or less congruent with
that of 7. propinqua, the sawfly has not yet been re-
corded further west than Berchtesgaden (Bavaria, Ger-
many), although the plant is widespread in Switzerland
and occurs as far west as northern Spain (Zajac and
Pindel 2011). Tenthredo propinqua has been consid-
ered to be to some extent endangered or even locally
extinct, at least in the Alps of eastern Bavaria on the
north-western edge of its range (Ritzau 1998; Liston et
al. 2012). In the future, we should be able to more ef-
fectively assess its distribution and conservation status
by searching for its larvae.

Le

Approximately 400 species of Gentiana occur world-
wide in Eurasia, North Africa, the Americas, and eastern
Australia, but South-East Asia is a hotspot of diversity
of this genus, with 248 species known from China alone,
whereas only 27—29 species occur in Europe (Ho and
Pringle 1995; Mel’nyk et al. 2014). Because China also
possesses a very rich fauna of 7enthredo species (Wei et
al. 2006), it is possible that Gentiana is more widely used
there as a host plant by these sawflies than it 1s in Europe.

Acknowledgements

The Museum fiir Naturkunde, Berlin, generously paid
for open access publication of this work. Henri Savina
(Toulouse, France) kindly permitted us to reproduce his
images of larvae of 7enthredo scrophulariae. Erik Hei-
bo (Lierskogen, Norway) provided tissue samples of 7.
propinqua. Dustin Kulanek and Katja Kramp (SDEI,
Muncheberg) did some of the lab work. Akihiko Shino-
hara and Spencer Monckton reviewed the manuscript and
suggested many worthwhile improvements.

References

Cameron P (1882) A Monograph of the British Phytophagous Hy-
menoptera. (7enthredo, Sirex and Cynips, Linné.). Volume 1. The
Ray Society, London, 1-340. [21 pls] https://doi.org/10.5962/bhl.
tithe.59527

Carpentier L (1888) Notes sur quelques larves de tenthrédides.
Mémoires de la Societé Linnéenne du Nord de la France 7: 254—286.

Chevin H (2009) Biologie comparée des especes francaises du genre
Macrophya (Hymenoptera, Symphyta, Tenthredinidae). Bulletin des
Naturalistes des Yvelines 36: 44—50.

Conde O (1934) Ostbaltische Tenthredinoidea, I. Teil. Korrespondenz-
blatt des Naturforscher-Vereins zu Riga 61: 168-198.

Enslin E (1912) Die Tenthredinoidea Mitteleuropas. Deutsche Entomol-
ogische Zeitschrift [1912](Beiheft 1): 1-98. https://doi.org/10.1002/
mmnd.48019120209

Ho T, Pringle JS (1995) Gentianaceae: Gentiana Linnaeus. In: Wu Z,
Raven PH (Eds) Flora of China, Volume 16 — Gentianaceae through
Boraginaceae. Science Press and Missouri Botanical Garden Press,
Beijing and St. Louis, 15-93.

JK (1985)

Palkane-Seuran Julkaisuja 5: 1-113.

Kangas Palkaneen Sahapistiaisfauna 1953-1983.

Lacourt J (1999) Répertoire des Tenthredinidae ouest-paléarctiques
(Hymenoptera, Symphyta). Mémoires de la Société Entomologique
de France 1-432.

Liston AD (2015) New records and host plants of Symphyta (Hymenop-
tera) for Germany, Berlin and Brandenburg. Contributions to En-
tomology. Beitrage zur Entomologie 65(2): 383-391. https://www.
contributions-to-entomology.org/article/view/1890/188

Liston AD, Jansen E, Blank SM, Kraus M, Taeger A (2012) Rote Liste
und Gesamtartenliste der Pflanzenwespen (Hymenoptera: Symphy-
ta) Deutschlands. Stand Marz 2011. In: Binot-Hafke M, Balzer S,
Becker N, Gruttke H, Haupt H, Hofbauer N, Ludwig G, Strauch

M (Eds) Rote Liste gefahrdeter Tiere, Pflanzen und Pilze Deutsch-

dez.pensoft.net

18

lands. Wirbellose Tiere Teil 1. Naturschutz und Biologische Vielfalt,
Bonn-Bad Godesberg 70 (3): 489-556.

Lorenz H, Kraus M (1957) Die Larvalsystematik der Blattwespen (Ten-
thredinoidea und Megalodontoidea). Abhandlungen zur Larvalsys-
tematik der Insekten 1: 1-389.

Loth N (1913) Verzeichnis der im Gebiete des rheinischen Schieferge-
birges und in einem Teile der niederrheinischen Tiefebene vorkom-
menden Tenthrediniden. Berliner entomologische Zeitschrift 58:
46-95.

Mel’nyk V, Drobyk NM, Twardovska MO, Kunakh V (2014) Karyology
of European species of genus Gentiana L. In: Rybezynski JJ, Davey
MR, Mikula A (Eds) The Gentianaceae- Volume 1: Characterization
and Ecology. Springer, Berlin and Heidelberg, 219-230. https://doi.
org/10.1007/978-3-642-54010-3_7

Muche WH (1962) Neue Wirtspflanze fiir A//antus scrophulariae L. En-
tomologische Nachrichten 6(2): 24.

Norment CJ (1988) The effect of nectar-thieving ants on the reproduc-
tive success of Frasera speciosa (Gentianaceae). American Midland
Naturalist 120(2): 331-336. https://doi.org/10.2307/2426005

Prous M, Liston A, Kramp K, Savina H, Vardal H, Taeger A (2019)
The West Palaearctic genera of Nematinae (Hymenoptera, Ten-
thredinidae). ZooKeys 875: 63-127. https://doi.org/10.3897/zook-
eys.875.35748

Pschorn-Walcher H, Altenhofer E (2000) Langjahrige Larvenaufsam-
mlungen und Zuchten von Pflanzenwespen (Hymenoptera, Sym-
phyta) in Mitteleuropa. Linzer biologische Beitrage 32(1): 273-327.

Pschorn-Walcher H, Altenhofer E (2006) Neuere Larvenaufsammlun-
gen und Zuchten von mitteleuropaischen Pflanzenwespen (Hyme-
noptera, Symphyta). Linzer biologische Beitrage 38(2): 1609-1636.

Réaumur RAF de (1740) Troisieme Mémoire. Et le premier sur les
Mouches a quatre ailes. Des fausses Chenilles, & des Mouches a sci-
es, dans lesquelles elles se transforment. In: Réaumur RAF de 1740:
Mémoires pour servir a |’ Histoire des Insectes. Tome Cinquiéme.

dez.pensoft.net

Andrew Liston et al.: Tenthredo larvae on Gentiana

Suite de I’Histoire des Mouches a deux ailes, & |’ Histoire de plu-
sieurs Mouches a quatre ailes, s¢avoir, des Mouches a scies, des Ci-
gales, & des Abeilles. Imprimerie Royale, Paris, 87-144.

Ritzau C (1998) Tenthredo propinqua Klug, 1817 neu fir Deutschland
(Hymenoptera: Tenthredinidae). In: Taeger A, Blank SM (Eds)
Pflanzenwespen Deutschlands (Hymenoptera, Symphyta). Kom-
mentierte Bestandsaufnahme. Goecke & Evers, Keltern, 43-44.

Shinohara A, Hara H, Kramp K, Blank SM, Kameda Y (2017) Bird
droppings on chestnut leaves or sawfly larvae: DNA barcodes veri-
fy the occurrence of the archaic Megaxyela togashii (Hymenoptera,
Xyelidae) in Hokkaido, Japan. Zootaxa 4221(2): 220-232. https://
doi.org/10.11646/zootaxa.4221.2.6

Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for
amplification of three non-coding regions of chloroplast DNA.
Plant Molecular Biology 17: 1105-1109. https://doi.org/10.1007/
BF00037152

Taeger A, Altenhofer E, Blank SM, Jansen E, Kraus M, Pschorn-Wal-
cher H, Ritzau C (1998) Kommentare zur Biologie, Verbreitung und
Gefahrdung der Pflanzenwespen Deutschlands (Hymenoptera, Sym-
phyta). In: Taeger A, Blank SM (Eds) Pflanzenwespen Deutschlands
(Hymenoptera, Symphyta). Kommentierte Bestandsaufnahme.
Goecke & Evers, Keltern, 49-135.

Wang L, Meng L, Luo J (2015) Florivory Modulates the Seed
Number-Seed Weight Relationship in Halenia elliptica (Genti-
anaceae). The Scientific World Journal 2015: 1-7. https://doi.
org/10.1155/2015/610735

Wei M, Nie H, Taeger A (2006) Sawflies (Hymenoptera: Symphyta) of
China - Checklist and Review of Research. In: Blank SM, Schmidt
S, Taeger A (Eds) Recent Sawfly Research: Synthesis and Prospects.
Goecke & Evers, Keltern, 505-574.

Zajac A, Pindel A (2011) Review of the Willow Gentian, Gentiana as-
clepiadea L. Biodiversity 12(3): 181-185. https://doi.org/10.1080/1
4888386.2011.628247