Species are often regarded as basic units of evolution and correct species delimitation serves as a backbone in most biological studies (Mayr 1982; Roe and Sperling 2007). However, the number of described species is a small portion of the estimated extant number and there is a need for an increased ability to identify and discriminate species (Blaxter 2004; Silva-Brandao et al. 2009). For the last three decades, mitochondrial DNA has been extensively used (Ballard and Whitlock 2004) and proven to be an important tool in species delimitation as it possesses biological properties making it suitable as a marker for molecular biodiversity (Moore 1995; Hebert et al. 2003).
A universal system for rapid, inexpensive species identification applicable for any life stage, DNA barcoding, has been proposed by Hebert et al. (2003). The ambition behind DNA barcoding is identification by sequencing of short standardized gene regions in order to assign unknown individuals to species and to enhance the discovery of new species. The assumptions underlying DNA barcoding are that every species have sets of unique barcode sequences and hence constitutes monophyletic clades and that genetic variation between species exceeds the variation within species (Hebert et al. 2003). Nevertheless, there are examples of deep intraspecific divergences in mtDNA, also in sympatric populations of animal groups such as birds (Omland et al. 2000; Johnsen et al. 2010; Hogner et al. in press), beetles (Schulenburg et al. 2002; Avtzis et al. 2008), and spiders (Chang et al. 2007). There are several possible explanations for high intraspecific variation. First, this pattern may reflect the presence of cryptic species. The exploration of cryptic species within the Skipper butterfly, Astraptes fulgerator, performed by Hebert et al. (2004) is a well-known example. By combining DNA barcoding with information about ecology and morphology of A. fulgerator, at least 10, largely sympatric cryptic species were revealed (but see Brower 2006). Second, demographic effects like isolation will cause differentiation between isolated populations by the accumulation of mutations over time. The differentiation may then reflect early stages of speciation. Secondary admixture of allopatrically evolved populations will in many cases result in gene trees with pronounced phylogenetic gaps between branches (Avise 2000). However, haplotype loss due to genetic drift (i.e., lineage sorting) will over time make a population monophyletic for a single gene lineage (Beebee and Rowe 2004). As lineage sorting is more prominent in small populations, the number of haplotypes maintained in a population is a function of current and historic effective population sizes. In closely related species, allele fixation often fails to complete and they will in these cases share ancestral polymorphisms resulting in discordance between gene trees and species trees (Moore 1995; Beebee and Rowe 2004). For nuclear DNA, when reproductive barriers do not evolve in allopatry and if secondary contact is obtained, variation must be maintained by factors opposing gene flow (e.g., geography and ecology). This is because gene flow will homogenize the nuclear genome over time (Futuyma 2005). Third, introgression by hybridization between closely related species can cause mtDNA to show a different gene genealogy than most other genes in the species in question. As the gene genealogy resulting from introgression is very similar to that expected by ancestral polymorphism and incomplete linage sorting (Ballard and Whitlock 2004), distinguishing between isolation and ancient hybridization can be very difficult. Finally, interpretation of mitochondrial genetic diversity may be hampered by the presence of heritable endoparasitic symbionts and in some cases result in incongruence between nDNA and mtDNA (Linares et al. 2009). Among the most widespread are bacteria from the genus Wolbachia (Alphaproteobacteria: Rickettsiales) (Russell et al. 2009). It has long been recognized that endoparasitic symbionts are prevalent among arthropods and that these organisms may have an important role in arthropod evolution as they can cause a number of reproductive alterations in their host, the most common being cytoplasmic incompatibility (Rousset et al. 1992; Werren 1997; Hurst et al. 1999; Hurst and Jiggins 2005; Narita et al. 2009). Male-killing parthenogenesis and feminization of genetic males are other alterations documented in arthropods (Rousset et al. 1992; Grandjean et al. 1993; Werren et al. 1995; Werren 1997; Jiggins 2003; Hurst and Jiggins 2005). The effects of inherited symbionts can be mistaken as evidence for population structure and admixture, as an mtDNA genealogy with deep internal branches could be the result of multiple selective sweeps from different Wolbachia strains, rather than a population being large and old or because of secondary admixture (Hurst and Jiggins 2005). Nevertheless, analysis and comparison of sequence data from both mtDNA and nDNA should help distinguishing between demographic effects and indirect selection on mtDNA by parasitic bacteria in an infected population (Rokas et al. 2001; Raychoudhury et al. 2010).
The genus Epirrita constitutes nine species (Scoble 1999), of which three are distributed in Norway (Aarvik et al. 2009). These are the autumnal moth, Epirrita autumnata, pale November moth, Epirrita christyi, and November moth, Epirrita dilutata. E. autumnata (Fig. 1) is distributed from Japan and Manchuria through Mongolia, Siberia, and Caucasus, to Western Europe and from the northern parts of Scandinavia to the Mediterranean (Skou 1984). The subspecies E. autumnata omissa and E. autumnata henshawi are found in North America (Tenow 1972; Scoble 1999). The larvae feed on deciduous trees, especially on birch (Betula), alder (Alnus), and willow (Salix) and have cyclic outbursts with ~9- to10-year intervals (Tenow 1972; Aarvik et al. 2009). In periods with high larvae densities, it can defoliate and seriously harm the mountain birch (Betula pubescens ssp. czerepanovii) forests (Ruohomaki et al. 2000; Jepsen et al. 2008; Yang et al. 2008). As a consequence of the moths' cyclical population dynamics, northern populations of E. autumnata may experience present-day bottlenecks as outbreaks are followed by collapse in population size and subsequent decline in genetic variability. Hence, one might expect to find relatively low levels of genetic variation within this species (Futuyma 1998; Snäll et al. 2004). However, preliminary results from DNA barcoding of Scandinavian moths and butterflies (Lepidoptera) revealed discrepancy between present division to species and sequence divergence in the genus Epirrita (Johnsen, Aarvik & Lifjeld, unpublished data). In particular, high sequence variation clustered in several well-defined haplogroups within sympatric E. autumnata suggested that this might be a complex of cryptic species.
The main aims of this study were to examine the relatively high mtDNA variation found within E. autumnata, describe the degree of sympatry among haplogroups within Norwegian populations of this species and compare the variation at mitochondrial (Cytochrome c Oxidase subunit 1, CO1) and nuclear (Internal Transcribed Spacer 2, ITS2 and Wingless) loci. In particular, we wanted to investigate four possible explanations for high intraspecific mtDNA variation: (1) presence of cryptic species; (2) historic isolation and secondary contact; (3) introgression from a related species; and (4) Wolbachia infections associated with different haplogroups. First, if the high mtDNA diversity reflects cryptic species, we predict congruence between divergence in mtDNA and nDNA sequence data, given that there has been sufficient time for divergence. Second, if the pattern is due to isolation and secondary contact, we predict higher differentiation in mtDNA compared with nDNA because the former has a relatively high evolutionary rate (5–10 times higher than single copy nDNA) (Avise 1986). Furthermore, depending on the amount of time since range expansions and secondary contact, we expect some degree of mtDNA- and nDNA structure, reflecting the demographic history and original geographic distribution of the lineages, again with higher degree of structure in mtDNA. Third, if ancient introgression by hybridization caused the differentiation in E. autumnata mtDNA, the same predictions as for historic isolation with secondary contact will apply. However, if introgression occurred recently, we would expect to find overlapping haplotypes with closely related species (e.g., E. dilutata and/or E. christyi). Finally, if Wolbachia infections have affected the mtDNA variation within this species, we predict an association between infection status and haplogroups and incongruence between mtDNA and nDNA. The samples were screened for Wolbachia infections to evaluate whether Wolbachia might have influenced patterns of mitochondrial diversity in E. autumnata.