Forensic entomology uses the larval and pupal developmental stages of insects sampled on a corpse to estimate a minimum post-mortem interval (PMImin) of the corpse (Amendt et al. 2004, 2007). This requires i) detailed and accurate knowledge of the developmental rate of the species of forensic interest under different temperature conditions (Charabidze 2012), and ii) identification tools by which the different immature insect stadia can be identified (Catts 1992). Blowflies (family Calliphoridae) are among the most common insects found on dead bodies shortly after death. The species differ in their developmental times and have therefore a high potential for the accurate estimation of the PMImin. Unfortunately, several forensically important blow fly species can hardly be distinguished morphologically, especially in the larval and pupal stages (e.g. Catts 1992). To improve the success and reliability of identifications, a number of molecular techniques and tools have been explored to identify forensically important species (Wells and Stevens 2008, reviewed in Jordaens et al. in press).

Currently, the most popular molecular method for organismal identification is DNA barcoding, which was promoted by Hebert et al. (2003a, b) as a standardized molecular identification tool for all animals. It refers to establishing species-level identifications by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene, the “DNA barcode”, into a taxonomically unknown specimen and performing comparisons with a reference library of barcodes of well-identified species. COI barcodes (and other fragments of COI) indeed have been successfully applied in the identification of many calliphorid species (e.g. Wallman and Donnellan 2001, Wells and Sperling 2001, Nelson et al. 2007, Wells and Williams 2007, Harvey et al. 2008, Desmyter and Gosselin 2009, DeBry et al. 2013). Yet, COI fails to unambiguously discriminate among several calliphorid species pairs (e.g. Nelson et al. 2007, see also the Discussion) and the use of alternative identification tools (e.g. other genes) could be necessary to acquire correct identifications.

The monophyly of Calliphoridae has been questioned for many years (e.g. Griffiths 1982) and paraphyly or polyphyly was suggested by a morphology-based parsimony analysis (Rognes 1997). Nonmonophyly was also found in a molecular phylogenetic analysis of the Calyptratae with Calliphoridae being polyphyletic with respect to the Tachinidae and Rhinophoridae. Within this ‘calliphorid-tachinid-rhinophorid’ clade, the subfamily Chrysomyinae was para- or polyphyletic (Kutty et al. 2010). The Chrysomyinae comprises two tribes, Chrysomyini and Phormiini, of which the Phormiini has three genera (Table 1). Phormia regina (Meigen, 1826) (black fly) is the only species in the monotypic genus Phormia. It is a Holarctic blow fly species that is commonly found on human or animal faeces (Coffey 1966) and that is frequently found on corpses. It therefore serves as a primary species to estimate the PMImin (e.g. Byrd and Allen 2001). Further, the species also plays an important role in secondary myasis in cattle (e.g. Francesconi and Lupi 2012) and is used in maggot therapy (Knipling and Rainwater 1937).

Phormia regina is a highly mobile species that is abundant in North American areas with cool spring and fall temperatures and in warmer areas, but then at higher altitudes (Hall 1948, Brundage et al. 2011). The developmental time of Phormia regina seems highly variable and could be influenced by a number of environmental variables (Kamal 1958, Greenberg 1991, Anderson 2000, Byrd and Allen 2001, Nabity et al. 2007, Núñez-Vázquez et al. 2013). Using amplified fragment length polymorphisms (AFLP), Picard and Wells (2009) studied the population genetic structure of N American Phormia regina and found that the N American populations were panmictic but with significant temporal genetic differences within populations, even over short periods of time. They therefore suggested that part of the variation in developmental times and growth curves that was observed in laboratory studies is not only due to local environmental (i.e. laboratory) conditions, but also to differences in the genetic composition of the laboratory stocks. This finding is important for forensic sciences since it shows that forensically relevant ecological data from one population (i.e. from a forensic case) cannot be extrapolated to other populations (i.e. to other forensic cases). Interestingly, Desmyter and Gosselin (2009) found a 4.2% sequence divergence at a 304 bp COI fragment between N American and W European specimens. Subsequently, Boehme et al. (2012) found a similar sequence divergence (range: 3.5%–4.31%) at the COI barcodes between N American and W European Phormia regina specimens.

Because high COI sequence divergences are often indicating species level differentiation (e.g. Hebert et al. 2003a, b), the strong COI differentiation between N American and W European Phormia regina specimens calls for a taxonomic re-assessment. We therefore studied DNA sequence variation in mitochondrial and nuclear DNA, and examined morphological differentiation between N American and W European populations of Phormia regina to i) provide additional DNA barcodes for Phormia regina, ii) examine molecular differentiation between N American and W European specimens in other genes, and iii) assess whether the COI differentiation is correlated with morphological differentiation. The taxonomy of Phormia regina is then re-evaluated in the light of these results.

Desmyter and Gosselin (2009) and Boehme et al. (2012) found a mean sequence divergence of approximately 4% within a 304 bp and the barcoding COI region between N American and W European Phormia regina, respectively. We confirmed this COI divergence with newly sequenced material. Such a strong divergence at COI is common among insect species (e.g. Park et al. 2011a, b, Webb et al. 2012, Ng’endo et al. 2013). Moreover, we here show a similar degree of divergence at two other mtDNA genes, viz. COII (3.7%) and cyt b (5.3%). The ‘within-continent’ divergence in Phormia regina was very low (0.2-0.5% for the three genes) and comparable to the intraspecific differentiation of other Chrysomyinae (0.5% for COI, 1.4% for COII, 0.3% for cyt b). Hence, the high between-continent mtDNA differentiation, and low within-continent mtDNA divergence may hint at a taxonomic difference between the N American and W European populations. In order to evaluate this suggestion, we included all publicly available GenBank sequences from species of the subfamily Chrysomyinae for the four mtDNA and two nDNA gene fragments that we sequenced. The combined study of mtDNA and nDNA has proven valuable to disentangle the taxonomy of other calliphorid species (e.g. Nelson et al. 2007, Sonet et al. 2012).

On the one hand, our results show that the mean p-distance of other intrageneric interspecific comparisons (COI: 5–6.8%, COII: 4.8-5.9%, cyt b: 4.6%, 16S (251 bp): 5.9%), or among other Chrysomyinae species in general (COI: 6.6%, COII: 4.6%, cyt b: 7.9%, 16S (251 bp): 3.8%), are higher than the mean p-distances between N American and W European Phormia regina at the four mtDNA fragments (COI: 4%, COII: 3.7%, cyt b: 5.3%, 16S: 0.6%). For cyt b the NA-EU differentiation in Phormia regina is higher than that observed within other Chrysomyinae species (0.3%) yet still below the minimum interspecific p-distance (7.3%). On the other hand, for COI and COII, the NA-EU differentiation in Phormia regina is higher than the intraspecific differentiation in other Chrysomyinae species and well within the range of interspecific p-distances within Chrysomyinae. Yet, the low interspecific p-distance between some Chrysomyinae species may be due to misidentifications or may be the result of a natural process (e.g. hybridization, incomplete lineage sorting). Likewise, the high intraspecific variation within some species may be indicative of cryptic diversity (see further).

North American and W European Phormia regina were not differentiated at both nDNA fragments, and at the mtDNA 16S (< 1%), whereas interspecific p-distances in Chrysomyinae in general are substantial for ITS2 (8.5%) and 16S (3.8%). Moreover, the NA-EU differentiation in Phormia regina at these genes was even lower than the minimum intraspecific differentiation within other Chrysomyinae. This suggests that the variation at these genes in Phormia regina is intraspecific variation. Finally, we could neither detect color differences in 11 external characters, nor in the general shape of the male copulatory organs between N American and W European specimens. Evidently, a statistical analysis of more specimens (from a wider range of the species’ distribution) is necessary to reliably assess within and among population variation at these (and eventually other) morphological characters. For the time being, we consider the high differentiation at COI, COII and cyt b, but the low (16S, nDNA) or lack of (morphological) differentiation, as indicative of substantial intraspecific mtDNA sequence divergence, rather than as a species-level differentiation.

Our findings may have important implications for the use of Phormia regina in forensic and other scientific fields. Indeed, it has been suggested that the high variation in developmental times and growth curves of Phormia regina (e.g. Byrd and Allen 2001 and references therein) is partly due to differences in the population genetic structure (Picard and Wells 2009) and that therefore ecological data obtained from one population should not be generalized or extrapolated to other populations (Byrne et al. 1995). Interestingly, Marchenko (2001) reports a mean accumulated degree-days (from egg to adult) of 148 °C (lower development temperature: 11.4 °C) for Russian/Lithuanian Phormia regina, whereas a mean accumulated degree-days of 162 °C (lower development temperature: 11.16 °C) was found for N American Phormia regina (Yves Braet, unpublished preliminary results). Hence, the strong mtDNA divergence between N American and W European Phormia regina requires a sound comparison of the ecology of populations from both continents, especially since Phormia regina is a key species in the study of the physiology and neurology of insect feeding (e.g. Haselton et al. 2009, Larson and Stoffolano 2011, Ishida et al. 2012). Moreover, if locally diverged populations differ in their developmental biology, then this may affect the estimate of PMImin.

Intraspecific mtDNA divergence in other Chrysomyinae species is sometimes also high, viz. 4.3% for COI in Chrysomya megacephala, and 2.2%, 2.6% and 3.7% for COII in Chrysomya megacephala, Chrysomya semimetallica and Chrysomya rufifacies, respectively. Whereas these high intraspecific divergences may be due to hybridization/introgression or incomplete lineage sorting, they may also point to misidentifications. Obviously these issues are problematic if DNA barcoding of animals is only based on COI, as advocated by Hebert et al. (2003a, b). For instance, three Chrysomya megacephala specimens (KC135924, KC139925, KC135926) have a remarkably high p-distance of 8% with the other Chrysomya megacephala haplotypes and it would be advisable to re-identify these specimens. Also Chrysomya semimetallica shows much more intraspecific sequence variation (mean p-distance = 0.011 ± 0.003) as compared to other Chrysomyinae species but at the same time the species has a low mean interspecific p-distance with Chrysomya albiceps (p-distance = 0.017 ± 0.004).

Although there is no doubt that COI is a useful tool for the identification of forensically important Chrysomyinae species (Wells and Sperling 2001, Nelson et al. 2007, Wells and Williams 2007, Desmyter and Gosselin 2009, Boehme et al. 2012) not all species can be identified with COI. For instance, there is very low mean interspecific p-distance of 0.006 ± 0.002 between Chrysomya megacephala (excluding the three aforementioned haplotypes), Chrysomya cabrerai, Chrysomya saffranea and Chrysomya pacifica (the first two even share a haplotype) (see also Harvey et al. 2008). Therefore, other genes (or gene fragments) might help to overcome the shortcomings of the sole use of COI as molecular identification tool. We here showed that also COII may be a good DNA barcode marker in the Chrysomyinae. Indeed, the mean interspecific p-distance at COII is 4.6%, whereas the mean intraspecific distance is much lower (1.4%). Yet, the amount of Chrysomyinae COII data that is currently available in public libraries such as GenBank (194 sequences representing 108 haplotypes from 20 species), is rather limited compared to the amount of COI data (339 sequences representing 180 haplotypes from 36 species) (Table 1). Moreover, the problems inherent to misidentifications and introgression also apply to COII (or any other DNA marker). For instance, Chrysomya megacephala FJ153270 shares a haplotype within the Chrysomya rufifacies clade, and Chrysomya rufifacies FJ839395 shares a haplotype within the Chrysomya megacephala clade. Also other species share haplotypes such as Chrysomya semimetallica and Chrysomya latifrons. The other two mtDNA fragments (cyt b and 16S) cannot yet be evaluated as DNA barcode markers because of insufficient sequence data (cyt b: 32 sequences representing 20 haplotypes of five species; 16S: 39 sequences representing nine haplotypes of six species) (Table 1), but both have been shown to discriminate sufficiently between other dipteran species of forensic interest (Vincent et al. 2000, Li et al. 2010).

So far, the forensically important species within the Chrysomyinae belong to the genera Chrysomya, Cochliomyia, Paralucilia, Protophormia and Phormia. A number of COI reference datasets of these species are available (e.g. Wallman and Donnellan 2001, Wells and Sperling 2001, Nelson et al. 2007, Wells and Williams 2007, Harvey et al. 2008, Desmyter and Gosselin 2009, Boehme et al. 2012) and they seem to work well to identify most forensically important species. Yet, it is important to also include species without a clear forensic interest in (local) reference databases because this will improve the assessment of species boundaries which, in turn, may help to reach a stable taxonomy.

In conclusion, we observed substantial differentiation between N American and W European Phormia regina at the mtDNA genes COI, COII and cyt b, but not at the 16S rDNA and the nDNA genes ITS2 and 28S. Moreover, we neither detected any morphological differentiation between specimens from both continents. We therefore consider the strong mtDNA divergence between specimens from both continents as intraspecific variation. This differentiation has to be taken into account when using Phormia regina in forensic casework or physiological studies. Finally, the use of COII as a DNA barcode marker in the Chrysomyinae seems to perform as good as the standard COI barcode region.