Variability and fluctuating asymmetry of mid tibial spurs in Eucremastus Szépligeti ( Hymenoptera , Ichneumonidae , Cremastinae )

The presence of one or two spurs on the mid tibia has been widely used as a diagnostic character in many taxa, but it has been discovered to be a highly variable character in the Cremastinae genus Eucremastus. Variation in the number of mid tibial spurs and occurrence of asymmetry in this genus was studied. The anomalies observed in this character are classified into three categories. Other non-cremastine genera traditionally characterized by having a single mid tibial spur were studied, but no anomalies were found. The taxonomic implications of this variation are discussed.


Introduction
Fluctuating asymmetry refers to the random deviation from perfect symmetry in bilateral traits (Van Halen 1962, Parsons 1990).It is more frequent in sexually selected traits, and it has been proposed as an important force for sexual selection (Moller and Pomiankowski 1993).Fluctuating asymmetry in morphological traits has been suggested to be the result of environmental (Silva et al. 2009) or genetic stress (Smith et al. 1997) during ontogeny, although this relationship remains unclear (Bjorksten et al. 2000).
When asymmetry affects morphological characters with taxonomic importance, its diagnostic value is reduced since it gives rise to different states of that character, sometimes in the same individual, and contributes to potentially misleading taxonomic descriptions and identifications.
The number and relative size of mid tibial spurs in Ichneumonidae has been considered a relatively invariant taxonomic character.The presence of a single mid tibial spur has been used in the diagnostic description of a tribe, Exenterini, of the subfamily Tryphoninae (Townes 1969a), but mostly at the generic level, e.g.Metopius Panzer (Townes 1971, Gauld et al. 2002), Acerataspis Uchida (males) (Townes 1971) (Metopiinae); Anomalon Panzer (Gauld 1997), Liopterna Townes, Calcaneum Townes and Ophionellus Westwood (Townes 1971) (Anomaloninae), or even at species level in Ophioninae (Gauld and Mitchell 1978).
As a general rule, the Cremastinae, a cosmopolitan group rather common in dry open areas (Gauld 2000) have two spurs on both the mid and hind tibiae.However, there is one genus, Eucremastus Szépligeti, which is characterized by having only one mid tibial spur (Fig. 1) (Townes 1971).Some authors (Ceballos 1921, Sedivy andNarolsky 2001) pointed out the presence of two spurs in several specimens of E. manni (Tschek).Moreover, E. villiersi (Benoit) form turkmeniensis was described as having two distinct mid tibial spurs in the female (Narolsky 2001), which highlights how variable the presence of one or two spurs is in this genus.Nevertheless, the occurrence of asymmetry in this character has not been reported, but it was found in some specimens of E. manni and E. collaris Narolsky from different collections.
These circumstances suggest not only that the presence of one or two spurs on the mid tibiae of Eucremastus may be a more variable feature that was considered until now, but also that this genus may present fluctuating asymmetry associated with this character.The aim of this paper is to demonstrate the variability of this character particularly in Eucremastus but also in other genera characterized by having a single mid tibial spur, and to investigate the occurrence of fluctuating asymmetry in this genus.

Studied material of this genus belongs to the following institutions:
CEUA University of Alicante, Alicante, Spain.

ZISP
Zoological Institute of Russian Academy of Sciences, Saint Petersburg, Russia.BMNH Natural History Museum, London, United Kingdom.HNHM Hungarian Natural History Museum in Budapest, Hungary.MNCN Museo Nacional de Ciencias Naturales, Madrid, Spain.
In order to establish the importance of variability, we also studied some material belonging to other Ichneumonidae genera which traditionally have been separated by having a single mid tibial spur.This material is preserved in the following institutions: 1) University of Alicante, Alicante, Spain (CEUA).
We considered the morphological structure to be anomalous when deviations from normality occurred in any specimens belonging to one species, which usually did not possess these anomalies.Normality is the commonest state of the character, and is defined as Type 0, as follows: Type 0: both mid tibiae with a single long spur.
The variability of anomalies found in the studied material was classified into 3 categories, as follows: Type 1: both mid tibiae with two unequal spurs.Type 2: one single spur on a mid tibia and a long spur clearly developed jointly with a very small stump on the other mid tibia (Fig. 2).Type 3: two distinct spurs on a mid tibia (Figs 3 and 5) and a long spur clearly developed jointly with a short stump on the other mid tibia (Figs 4 and 6).
Type 1 is a symmetric anomaly, while types 2 and 3 are asymmetric anomalies.Since anomalies have been found only in two species (i.e. E. manni and E. collaris), we will report only the material belonging to these two species, although some individuals of E. villiersi (Benoit) (11 specimens, including holotype, ZISP) and E. parvipes (Morley) (holotype, BMNH) were also checked.Eucremastus villiersi form turkmeniensis may be considered to represent an anomaly type 1; however, we did not see any material belonging to this form, thus it is not included in the material studied.As for the other non-cremastine genera, we only report data from specimens at the genus level, since most of the material we examined was only sorted to genera.
Fluctuating asymmetry is usually measured as the mean difference between right and left sides (Palmer and Strobeck 1986).However, since we measured the mid tibia spurs asymmetries qualitatively (i.e., type 2 and 3 anomalies), we present the frequency distribution of asymmetry by considering type 2 and 3 anomalies as both sides' asymmetries, respectively, and both type 1 and type 0 anomalies as bilateral symmetries.
Scanning Electron Microscopy studies were done using a Hitachi S-3000N (University of Alicante, Spain) and a LEO 1455VPL (Natural History Museum, United Kingdom), both in low vacuum mode.

Results
In this paper we examined a total of 60 specimens belonging to Eucremastus manni and E. collaris.We found mid tibial spur anomalies in seven specimens.These are detailed below and summarized in Table 1.We also examined 347 specimens belonging to other genera with a single mid tibial spur, but no anomalies were found.
Asymmetries (i.e.type 2 and 3 anomalies) were only found in E. manni.About 90% of E. manni individuals were symmetrical, and asymmetries occurred at very low frequency (Fig. 7).

Discussion
About one in 10 individuals of E. manni had asymmetric spurs, more frequently in females.Since we measured a discrete variable, fluctuating asymmetry could not be tested, but the occurrence of asymmetry at this relatively high proportion would suggest this genus would be particularly sensitive to environmental or genetic stress.Therefore, given the growing interest in the use of fluctuating asymmetry as a measure of developmental instability, and hence of environmental stress during development (Hogg et al. 2001, Silva et al. 2009, amongst others), this character may be used for this purpose by measuring the relative size of each spur on each mid tibia.Despite the apparently low sample size of our study, we must underline that this frequency of anomalies is noteworthy considering that the genus Eucremastus is rather rare in collections.Fluctuating asymmetry has not been measured in Ichneumonidae, although isolated teratologies have been reported (Kerrich 1934, Bordera andTormos 1986).Not only asymmetry but the overall high rate of variation in the presence of one or two mid tibial spurs in some species of Eucremastus invalidates this character for taxonomic diagnosis of this genus, although not in other taxa, since anomalies were absent in other genera that usually have a single mid tibial spur.According to this, we redefine the diagnostic characters of Eucremastus, and provide some features that separate it from related genera (Table 2).Wings moderately darkened.Occipital carina interrupted above.Frons concave.Genal carina joining oral carina very close to base of mandible.Lower tooth of mandible nearly twice as long as upper tooth.Pronotum slightly convex, without a median longitudinal carina.Scutellum rather convex, with a strong lateral carina reaching its apex.Hind femur without tooth beneath.Mid tibia with one or two spurs, when two spurs are present they are of unequal length.Ventral edges of first tergite touching each other, enclosing sternite in its middle.Glymma present, short and oblique.Ovipositor sheath about 1.5 × longer than hind tibia.Ovipositor tip almost straight.

Figures 1- 6 .
Figures 1-6.Mid tibial spurs of Eucremastus manni: 1 mid tibia without anomalies 2 type 2 anomaly in specimen from Turkey, right leg 3-6 type 3 anomalies in specimens from Spain: 3-4 right leg and left leg, respectively, of specimen from Fresneda 5-6 right leg and left leg, respectively, of specimen from Raña del Pocico.The white arrows point to the stump of the spur.

Figure 7 .
Figure 7. Frequency distribution of fluctuating asymmetries of mid tibial spurs in E. manni.Symmetric means both type 0 and anomaly type 1.

Table 1 .
Sample size of examined material and proportions of anomaly occurrence (between brackets), by species and by sex.

Table 2 .
Diagnostic characters to distinguish amongst some related genera in Cremastinae.