Assessing genetic and morphological variation in populations of Eastern European Lucilia sericata ( Diptera : Calliphoridae )

The population structures of different species of Calliphoridae fl ies are highly diverse at different locations. We investigated populations of the Eastern European L. sericata using chaetotaxy and eight microsatellite loci. Our results strongly indicate that a panmictic population of L. sericata exists in the area studied, possibly with a high rate of intra-population gene fl ow. Analysis of chaetotaxy also supports the panmictic population hypothesis. * Equal contributors. INTRODUCTION Lucilia sericata Meigen, 1826 (Diptera: Calliphoridae), or the common green bottle fl y, is widespread and usually abundant. This species is forensically important, and often used for estimating time since death (Postmortem Interval) (Karabey & Sert, 2014). Moreover, their larvae are facultative parasites, capable of developing in body tissues of mammals, which causes myiasis (Nelson & Rice, 1956; Hall & Wall, 1995). This disease often affects sheep, causing signifi cant economic loss and making L. sericata an important veterinary pest (French et al., 1994). The population structures of different species of fl ies belonging to the family Calliphoridae are very diverse. Some species are panmictic over a wide geographic range, as is Phormia regina Meigen, 1826 (Diptera: Calliphoridae) in USA (Picard & Wells, 2009; Jordaens et al., 2013). Some appear to have distinct well separated populations, for example Lucilia cuprina Wiedemann, 1830 (Diptera: Calliphoridae) in Australia (Clarke & McKenzie, 1987). Different types of population structure may exist even in one species of Calliphoridae: e.g., Cochliomyia hominivoEur. J. Entomol. 115: 192–197, 2018 doi: 10.14411/eje.2018.017


INTRODUCTION
Lucilia sericata Meigen, 1826 (Diptera: Calliphoridae), or the common green bottle fl y, is widespread and usually abundant.This species is forensically important, and often used for estimating time since death (Postmortem Interval) (Karabey & Sert, 2014).Moreover, their larvae are facultative parasites, capable of developing in body tissues of mammals, which causes myiasis (Nelson & Rice, 1956;Hall & Wall, 1995).This disease often affects sheep, causing signifi cant economic loss and making L. sericata an important veterinary pest (French et al., 1994).
The population structures of different species of fl ies belonging to the family Calliphoridae are very diverse.Some species are panmictic over a wide geographic range, as is Phormia regina Meigen, 1826 (Diptera: Calliphoridae) in USA (Picard & Wells, 2009;Jordaens et al., 2013).Some appear to have distinct well separated populations, for example Lucilia cuprina Wiedemann, 1830 (Diptera: Calliphoridae) in Australia (Clarke & McKenzie, 1987).Different types of population structure may exist even in one species of Calliphoridae: e.g., Cochliomyia hominivo-

M icrosatellite analysis
Eight pairs of primers were designed by us using Websat software ( Martins et al., 2009) and the L. cuprina genome sequence ( Anstead et al., 2015).One pair of primers was adapted from F lorin & Gyllenstrand (2002) (Table 2).
Amplifi cations for this study were done using a HS-Taq Kit (Evrogen, Moscow, Russia).
Forward primers were labelled on 5' end with TAMRA, FAM or R6G fl uorescent marker.Product length was measured by capillary gel-electrophoresis on an Applied Biosystems Genetic Analyzer 3500 (Thermo Fisher Scientifi c Inc, Waltham, MA, USA).The results were then processed using GeneMarker software ( Hulce et al., 2011).

Population genetic analysis
We determined whether putative genetic segregation exists in the samples studied using STRUCTURE 2.0 software and by implementing the Bayesian algorithm for detecting population structure (Pritchard et al., 2000).Further we used an improved method described by Evanno et al. (2005) to detect the proper number of clusters (K).Allele frequencies were calculated using Chaetotaxy, on the other hand, is a method based on set of characters widely used in the morphological approach to systematics, evolution and population studies.We compared the results of the chaetotaxy study with those of the genetic analysis.

Specimen collection
Adult fl ies of L. sericata were collected on decaying organic matter at seven locations (Table 1, Fig. 1) and stored in 95% ethanol, in a freezer at −20°С.SPAGeDi software (Hardy et al., 2003).Effective population size was estimated using NeEstimator software (Do et al., 2014).

Morphological survey
The characters analyzed included: number of hairs on the posterior slope of the humeral callus behind the basal setae (see 1 in Fig. 2А, В), number of hairs on the edge of the notopleuron behind the posterior notopleural seta (see 2 in Fig. 2А, В), number  of setulae on the "quadrat" between the discal setae and anterior margin of scutellum (Fig. 2С).These characters were adapted from a study on L. sericata, L. cuprina and their hybrids (Williams & Villet, 2014).Statistical analysis was performed using STATISTICA 8.0 software (TIBCO Software Inc, Palo Alto, CA, USA).

RESULTS
All the loci used in the present study were highly polymorphic (Table 3).The STRUCTURE simulation indicated no signifi cant correlation between locations and potential clusters (Fig. 3).The most likely number of clusters was one, which clearly indicates the absence of any population structure.
The effective population size was estimated as "infi nite".The authors of the original software suggest that this result indicates an absence of evidence of genetic drift due to a fi nite number of parents in the samples.
Signifi cant variability was recorded in the chaetotaxy characters.Using two-way ANOVA, we detected sexual dimorphism based on a difference in the number of setulae on the scutellum in males and females (Table 4).Pairwise comparisons were performed using the Tukey test (Fig. 4).No groups with distinct differentiation were found for any of the three characters analyzed.Furthermore, we detected no correlation between sampling location and the chaetotaxy characters studied.

DISCUSSION
Results of our genetic analysis indicate that there is a panmictic population of L. sericata in the area studied, with putatively a high rate of gene fl ow within this population.We assume that the ability of L. sericata to migrate long distances and their high fertility outweighs existing genetic drift and effects of geographical distances.The assumption that genetic drift is relatively negligible is also supported by the results of the effective population size estimations, i.e. the "infi nite" number of breeders.However, it is also possible that overlapping generations or a sampling error could have led to similar results ( Waples et al., 2014).The chaetotaxy analysis also strongly supports our panmictic population hypothesis.
In previous studies, evidence of L. sericata panmixy in part of the range of this species in the United States is reported by Picard & Wells ( 2010).No correlation between location and population structure was established.However, fl ies coming to bait over a short period of time were closely related.In earlier work, Stevens and Wall also failed to demonstrate signifi cant genetic differences between geographically separated worldwide populations of L. sericata ( Stevens & Wall, 1997).
A detailed study of populations of Fletcherimyia fl etcheri Aldrich, 1916 (Diptera: Sarcophagidae) revealed signifi cantly different results, yet F. fl etcheri belongs to the same superfamily, Oestridae ( Rasic & Keyghobadi, 2012).This species oviposit less than 10 eggs and the larvae develop inside the leaves of an insectivorous plant, Sarracenia purpurea.However, it is not surprising that with such a strong connection with their habitat and such a low fertility populations of F. fl etcheri have a distinct structure even over small ranges, as isolation by distance is reported over  Considering the examples mentioned above, we conclude that the population structure of fl ies is highly infl uenced by aspects of their biology, especially fertility, migration capacity, dependence on food sources and abundance of food.In the case of L. sericata our fi ndings, as well as earlier studies, indicate a tendency towards panmixy in different parts of the world.This should be taken into account in future research on this species, as well as in forensic and veterinary studies.
from leg muscle following the protocoldescribed by G alinskaya et al. (2016).

Fig. 1 .
Fig. 1.Map showing the sites where Lucilia sericata were collected, indicated by black dots.Grey dots indicate some of the larger cities.

Fig. 3 .
Fig. 3. STRUCTURE simulation of four clusters.Each vertical line represents the possibility of assigning a given specimen to one of the colour-coded clusters.Localities are indicated below the fi gure.

Fig. 4 .
Fig. 4. Results of the Tukey test pairwise comparisons of the distribution of chaetotaxy traits among samples of Lucilia sericata from different locations.Y-axis shows number of setulae on "quadrat" between discal setae and anterior margin of scutellum for A, number of hairs on edge of notopleuron behind posterior notopleural seta for B, and number of hairs on posterior slope of humeral callus behind basal setae for C. Small letters above bars indicate statistical signifi cance; the same letter indicates the absence of statistical difference between locations.

Table 1 .
Geographic locations, dates of collection and numbers of Lucilia sericata collected.

Table 2 .
Microsatellite primers used in the genetic analysis of Lucilia sericata.

Table 3 .
Microsatellite loci analyzed in the genetic survey of Lucilia sericata.

Table 4 .
Two-way ANOVA of the distribution of chaetotaxy traits among samples of Lucilia sericata from different locations."Location*Sex" is an interaction term, showing non-additive effect of the combination of two factors.F stands for the ratio of between-group variation to within-group variation.P-value is the probability of rejecting the true null hypothesis.Red colour indicates values suffi cient for rejection.
distances of 10-26 km.I n comparison, the distance sufficient for isolation for populations of Musca domestica Linnaeus, 1758 (Diptera: Muscidae) is estimated as more than 100 km(C hakrabarti et al., 2010).It should be noted that M. domestica is polyphagous and oviposits about as many eggs as L. sericata (100-150 in M. domestica compared to 150-200 in L. sericata).