On the identity of Tettigonia krugeri Massa from Libya with some remarks on variability of Tettigonia species (Orthoptera: Tettigoniidae; Tettigoniinae)

Abstract

Morphological and genetic data allowed the authors to resurrect the name Tettigonia krugeri Massa, 1998 as a valid species. It is currently known only from two specimens (one male and one female) collected in Cirenaica (Libya).

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ZOOTAXA
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Accepted by D. Rentz: 30 Jul. 2019; published: 22 Aug. 2019 343
Zootaxa 4658 (2): 343–354
https://www.mapress.com/j/zt/
Copyright © 2019 Magnolia Press Article
https://doi.org/10.11646/zootaxa.4658.2.8
http://zoobank.org/urn:lsid:zoobank.org:pub:FD1EB81B-729F-4D4A-95EB-88BCBFC9B054
On the identity of Tettigonia krugeri Massa from Libya with some remarks
on variability of Tettigonia species (Orthoptera: Tettigoniidae; Tettigoniinae)
BRUNO MASSA1 & MARCELLO TAGLIAVIA2,3
1Department of Agriculture, Food and Forest Sciences, University of Palermo, Viale Scienze Bd 5A, 90128 Palermo, Italy
E-mail: bruno.massa@unipa.it
2IAS-CNR, Via del Mare, 3—Torretta Granitola—91021, Italy
3Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale Sci-
enze Bd 16, 90128 Palermo, Italy. E-mail: marcello.tagliavia@cnr.it, marcello.tagliavia@unipa.it
ORCID: 0000-0002-0960-2919
Abstract
Morphological and genetic data allowed the authors to resurrect the name Tettigonia krugeri Massa, 1998 as a valid
species. It is currently known only from two specimens (one male and one female) collected in Cirenaica (Libya).
Key words: Morphology, Genetics, Valid species, Palaearctic region
Introduction
Tettigonia viridissima (Linnaeus, 1758) has a very wide distribution which covers the Palaearctic and pars of Orien-
tal regions, where it is one of the commonest bushcrickets. According to Rhee (2013) the tegmina of T. viridissima
surpass the hind femora, at rest, the front of the male stridulatory apparatus is rounded but sharp apically. The cerci
of the male are almost two times longer than the styli and the inner teeth are located within the basal one-third. The
ovipositor is weakly curved and does not reach behind the wingtips. However, the length of wings may be variable;
for example, many species of Tettigonia were described from North-West Africa, based on their size and the length
of fore wings: T. vaucheriana Pictet, 1888, T. maroccana Bolívar, 1893, T. lozanoi (Bolívar, 1914), T. longealata
Chopard, 1937. Both Pinedo (1985) and Grzwacz et al. (2017) have observed the existence of an overlap between
populations, with extreme examples ranging from a slender body shape with long wings to a stout body with long
or short wings, sometimes living close together. On the base of biomolecular, morphological and acoustic data,
Grzwacz et al. (2017) established that T. vaucheriana, T. maroccana, T. lozanoi, T. longealata are synonymous with
T. viridissima; in particular the authors did not find differences in the shape of male cerci and genitalia, and female
subgenital plate. According to Grzwacz et al. (2017), in the genus Tettigonia song differences between species
(mainly in sympatric taxa) may express in different syllable arrangement and repetition rate, echeme length, and
duty cycle. In Tettigonia viridissima song recognition based on temporal clues has been shown to be more compli-
cated, and females evaluate the pause within disyllabic echemes and respond only to the species-specific echeme
structure (Schul 1998).
Grzwacz et al. (2017) included within the group of the North-West African Tettigonia synonymous with T.
viridissima also T. krugeri Massa, 1998 described from Cirenaica (North-East Libya), basing their synonymy on
the transitive property (short-winged species with characters similar to those of the other north African Tettigonia
examined by previous authors). The aims of this paper are to show differences existing between T. krugeri and T.
viridissima sensu lato to resurrect the status of valid species to the former.
Material and methods
Many specimens of different species of West-Palaearctic Tettigonia (included the types of T. krugeri) were exam-

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344 · Zootaxa 4658 (2) © 2019 Magnolia Press
ined and their peculiar characters were photographed with a Nikon Coolpix 4500 digital camera, mounted on a Wild
M3 or on a Optech SL Stereomicroscope, and photos were integrated using the freeware CombineZP (Hadley 2008).
In particular the following characters were photographed: male cerci, male subgenital plate, male stridulatory file
below the left tegmen, male mirror on the right tegmen, titillators, and female subgenital plate. The following spe-
cies were considered: Tettigonia krugeri Massa, 1998 [according to Grzwacz et al. (2017) synonym of T. viridissima
(L., 1758)], T. viridissima (L., 1758), T. vaucheriana (Pictet, 1888) [according to Grzwacz et al. (2017) synonym
of T. viridissima (L., 1758)], Tettigonia cantans (Fuessly, 1775), T. silana Capra, 1936, T. balcanica Chobanov &
Lemonnier-Darcemont, 2014, T. hispanica (Bolívar, 1893), T. c. caudata (Charpentier, 1845), T. armeniaca Tarbin-
sky, 1940, and T. longispina Ingrisch, 1983.
DNA extraction and amplification. DNA extraction was performed from the museum holotype and allotype of
T. krugeri combining an initial step of manual grinding by micropestel and alkaline lysis (as described in Tagliavia
et al. 2016), followed by DNA column purification as reported in Tagliavia et al. (2011). In particular, the alka-
line lysis allowed to avoid the automatic grinding step; the addition of the lysis/binding buffer of DNA extraction
columns (Illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare Life Sciences), together with the
column purification, allowed to avoid phenol/chloroform extraction, ensuring high quality DNA from all samples.
A PCR and gel band purification column kit was chosen instead of a genomic DNA purification kit because of the
expected average molecular weight of DNA present into museum stored samples, in order to improve the overall
yield.
DNA was used as PCR template to amplify the internal transcribed spacer, namely ITS1, of the nuclear ribo-
somal gene cluster. According to Wang et al. (2015), the ITS1 was chosen as target region, which was found supe-
rior to ITS2 primers.
For nuclear ITS1 region, PCR amplification was carried out using the primers 18S-28S (5’-TAGAGGAAGTA-
AAAGTCG-3’) (Weekers et al. 2001) and ITS-R1 (5’-CATTGACCCACGAGCC-3’) (Ullrich et al. 2010), whereas
the primers ITS2-28S (5’-GGATCGATGAAGAACG3’) and 28S-18S (5’-GCTTAATTCAGCGG-3’) (Weekers et
al. 2001) were used for ITS2 amplification.
Forward primers were added with the M13F sequence (5’-TTGTAAAACGACGGCCAGT-3’) at their 5’ end,
in order to employ the M13F sequence as starting point for DNA sequencing.
PCR reactions were performed in a 50 µl volume, containing 1X PCR buffer, 750 nM for each primer, 10 mM
of each dNTP, 1µL Phire HotStart II DNA Polymerase (Thermofisher), and 10–50 ng of DNA.
A two-steps touchdown amplification program was used, including the following thermal profile: initial de-
naturation at 98 °C for 30”, followed by seven cycles consisting in an initial denaturation step at 98 °C for 7”, an
annealing step at 58 °C for 15” and an elongation step at 72 °C for 20”; the same scheme was applied in the follow-
ing 35 cycles, were the annealing temperature was reduced to 50 °C. A final elongation step at 72 °C for 2 minutes
completed the reaction.
Amplicons were analyzed by agarose (1.8%) gel electrophoresis in TAE 1X (40mM Tris-Acetate, 1mM EDTA,
pH 8) containing GelRed (Biotium).
DNA sequencing and sequences analysis. PCR products were purified using Illustra GFX PCR DNA and Gel
Band Purification Kit (GE Healthcare Life Sciences) following the manufacturer’s instructions and sequenced (us-
ing M13 primer) by Sanger method by Eurofins Genomics sequencing service.
DNA sequences were trimmed (in order to analyze high quality sequences only) and manually edited, then a
Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi) was carried out.
Results and discussion
Morphology. Subgenital plate of the male. T. krugeri differs both in the length of styli and cerci from T. viridissima;
styli are about two third of cerci and the inner spine of cerci is placed about at the half length of cerci (length of cerci:
4.8 mm according to Massa 1998; with a more precise measure: 4.6 mm) (Fig. 1). This character is certainly very
peculiar in T. krugeri and this (with the shortness of tegmina) is probably the reason for which Giglio-Tos (1923)
identified the male specimen as T. caudata. T. viridissima has a typical subgenital plate of the male with two paral-
lel keels ending with long styli, whose length is about half that of cerci; these are characterized by an inner spine
placed about within the basal one-third. The inner spine is generally not much marked, but sometimes may be more

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prominent and backward pointed (Figs. 2, 3, 5, 6, 7); only in one specimen from Jordan the spine was bifid (Fig. 8).
Other species of Tettigonia differ from T. viridissima, because they have cerci much shorter, their length being just
one third longer than styli (e.g., T. silana: Fig. 4).
Subgenital plate of the female. The subgenital plate of T. viridissima has two raised converging keels; at the
apex a V-shaped concavity is present, which may be more or less deep. The variability is shown in the Figs. 10–14
and 20 (Fig. 10 shows the subgenital plate of a syntype of T. vaucheriana, synonymized with T. viridissima by
Grzwacz et al. 2017); the female subgenital plate of T. krugeri lies within this variability (Fig. 9). In this respect also
T. silana has a similar female subgenital plate (Fig. 17), while those of T. cantans (Fig. 15), T. caudata (Fig. 16) and
T. armeniaca (Fig. 18) have two short parallel central keels and the apex of the subgenital plate is differently shaped.
The female subgenital plate of T. hispanica (Fig. 19) is rather short and its hind tips appear very different from T.
silana (compare Figs. 17 and 19); the latter was considered subspecies of T. hispanica by Harz (1969), while it was
reinstated as valid species by Fontana & Odé (1999).
Stridulatory file of the the male. The stridulatory file of T. krugeri is straight and does not exceed 85 pegs (Fig.
21), while that of T. viridissima is generally a little arched and consists of 110–120 pegs (see Figs. 22–26; Fig. 22
shows the stridulatory file of a syntype of T. vaucheriana, synonymized with T. viridissima by Grzwacz et al. 2017);
in T. caudata and T. armeniaca pegs are about 90 (Figs. 27, 28), in T. cantans they are generally between 90 and
110 (Figs. 29, 30), while in T. balcanica they are over 104 (Fig. 32) (see also Heller 1988, Chobanov et al. 2014).
Interestingly, the stridulatory file of T. longispina (Fig. 31) from Sardinia is similar to that of T. krugeri and consists
of about 85 pegs.
Mirror of right tegmen of the male. The mirror of T. viridissima is nearly pentagonal, with the left side rounded;
this character is very similar both in T. krugeri, T. longispina and T. cantans (Figs. 33–36 and 39–40), while in T.
armeniaca and T. caudata (Figs. 37–38) the mirror is narrower in the half posterior part compared to those of previ-
ous species.
Titillators of the male. We cannot compare titillators of T. krugeri with those of other species, because they were
extracted from the male specimen before the description of the new species and later were lost (cf. Massa 1998).
However, the titillators of the genus Tettigonia are only of two types: a) long, thin, pointed and out-curved (Figs. 41,
42); b) short, stout, more or less straight, with two apical outcurved tips (Figs. 43–46).
Genetic Analysis. In order to get further insights about relationships between T. krugeri and other Tettigonidae,
including T. viridissima, DNA analyses were carried out using ITS1 as target region. In fact, such region has been
reported highly informative and preferable to ITS2 (Wang et al. 2015), so that it is considered a better DNA barcode
than ITS2 for eukaryotic species.
A 465bp region of high-quality sequence was selected after trimming and editing from the entire ITS1 sequence
for subsequent bioinformatics analyses.
In particular, sequence was submitted to Basic Local Alignment Search Tool (BLAST) and challenged with the
sequences in GenBank.
Unexpectedly, the highest identity value was with Anterastes burri (89.3%), whereas only 79.4% and 79% of
identity resulted with T. cantans and T. viridissima, respectively.
Noteworthy, most differences between analyzed sequences consisted of insertions or deletions of several con-
secutive nucleotides, which makes highly unlike that observed differences could result from random sequencing
errors, that would result an overestimation of the distances.
The aforementioned results, showing identity differences exceeding 10% (up to 21%) in the considered locus,
made unnecessary further bioinformatics analyses that would have aimed to a more fine assessment of the actual
species separation.
Nevertheless, further genetic analyses involving a larger number of loci, either nuclear or mitochondrial, would
be desirable and helpful to better understand the actual phylogenetic relationships between T. krugeri and other
related species.
Conclusions
Morphological and genetic data allow to resurrect the name Tettigonia krugeri Massa, 1998 as a valid species,
currently known only from two specimens (one male and one female) collected in Cirenaica and preserved in the
Museo Civico di Storia Naturale ‘G. Doria’ of Genoa.

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FIGURES 1–4. Male subgenital plate, styli and cerci of: 1) Tettigonia krugeri Massa, 1998 (holotype, Libya, Cirene, VI.1935);
2) T. vaucheriana (Pictet, 1888) [synonym of T. viridissima (L., 1758)] (syntype, Morocco); 3) T. viridissima (North East
Greece); 4) T. silana Capra, 1936 (South Italy, Calabria, Sila 18.VI.2010).

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FIGURES 5–8. Male subgenital plate, styli and cerci of: 5) Tettigonia viridissima (South Italy, Calabria, Mouth of Crati river
19.VI.2010); 6) T. viridissima (Italy, Sicily, Madonie 6.VII.2002); 7) T. viridissima (Italy, Sardinia, Macomer 10.VII.1946); 8)
T. viridissima (Jordan, Ajlun 25.V.1999; inset: particular of the right cercus).

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FIGURES 9–14. Female subgenital plate of: 9) Tettigonia krugeri (allotype, Libya, Merg); 10) T. vaucheriana (synonym of
T. viridissima) (syntype, Morocco); 11) T. viridissima (Greece, Crete, Kasos Is. 3.VI.2009); 12) T. viridissima (Italy, Calabria,
Rogliano 21.VII.1978); 13) T. viridissima (Turkey, Dacta peninsula 10.V.2010); 14) T. viridissima (Italy, Sardinia, Macomer
10.VII.1946).

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FIGURES 15–20. Female subgenital plate of: 15) Tettigonia cantans (Fuessly, 1775) (Italy, Tuscany, Secchieta 8.VIII.2010);
16) T. c. caudata (Charpentier, 1845) (Italy, Alps, Trentino Alto Adige 31.VII.1968); 17) T. silana (South Italy, Calabria, Sila
18.VI.2010); 18) T. armeniaca Tarbinsky, 1940 (Armenia, Kosrov Reserve 24.VI.2006); 19) T. hispanica (Bolívar, 1893)
(Spain); 20) T. viridissima (Armenia, Hartsavan 5.VII.2010).

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FIGURES 21–32. Stridulatory file below the male left tegmen of: 21) Tettigonia krugeri (holotype, Libya, Cirene, VI.1935);
22) T. vaucheriana (synonym of T. viridissima) (syntype, Morocco); 23) T. viridissima (Armenia, Vanand 19.VI.2012); 24)
T. viridissima (Italy, Sardinia, Macomer 10.VII.1946); 25) T. viridissima (Italy, Sicily, Madonie 6.VII.2002); 26) T. viridis-
sima (Central Italy, Molise, Campobasso); 27) T. c. caudata (Italy, Alps, Trentino Alto Adige 31.VII.1968); 28) T. armeniaca
(Armenia, Karabagh Mts. 23.VI.2018); 29) T. cantans (Italy, Campania, Acerno 30.VII.1978); 30) T. cantans (Austria, Linz
12.VII.1992); 31) T. longispina Ingrisch, 1983 (Italy, Sardinia, Bruncu Spina 20.VIII.1999); 32) T. balcanica Chobanov &
Lemonnier-Darcemont, 2014 (Bulgaria, Predela pass: after Chobanov et al. 2014).

IDENTITY OF TETTIGONIA KRUGERI Zootaxa 4658 (2) © 2019 Magnolia Press · 351
Acknowledgements
Roberto Poggi, Maria Luisa Tavano and Giuliano Doria (Museo Civico di Storia Naturale ‘G. Doria’ of Genoa)
facilitated the study of specimens preserved in their museum, Augusto Cattaneo provided specimens from Greece
and Turkey, Marcus Kalashian from Armenia, Paolo Fontana and Filippo M. Buzzetti sent some photographs or
specimens preserved in the Museo Civico di Rovereto; we thank them very much.
FIGURES 33–36. Mirror on the male right tegmen of: 33) Tettigonia krugeri (holotype, Libya, Cirene, VI.1935); 34) T. vauche-
riana (synonym of T. viridissima) (syntype, Morocco); 35) T. viridissima (Italy, Sardinia, Macomer 10.VII.1946); 36) T. long-
ispina (Italy, Sardinia, Bruncu Spina 20.VIII.1999).

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FIGURES 37–40. Mirror on the male right tegmen of: 37) Tettigonia armeniaca (Armenia, Karabagh Mts. 23.VI.2018); 38) T.
c. caudata (Italy, Alps, Trentino Alto Adige 31.VII.1968); 39) T. cantans (Italy, Campania, Acerno 30.VII.1978); 40) T. cantans
(Austria, Linz 12.VII.1992).

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FIGURES 41–46. Titillators of males of: 41) Tettigonia longispina (Italy, Sardinia, Bruncu Spina 20.VIII.1999); 42) T. viridis-
sima (Italy, Calabria, Rogliano 21.VII.1978); 43) T. cantans (Italy, Campania, Acerno 30.VII.1978); 44) T. cantans (Italy, Tren-
tino Alto Adige, Val di Sole 30.VII.1996); 45) T. silana (South Italy, Calabria, Sila 18.VI.2010); 46) T. armeniaca (Armenia,
Karabagh Mts. 23.VI.2018).
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