Phylogeography of the willow tit in the Palaearctic
The results clearly show that there is no phylogeographic structure in the mitochondrial DNA (mtDNA) of the sampled populations of the willow tits through the Palaearctic and the same holds true when the populations are pooled according to the distribution ranges of the subspecies. The reason for this genetic panmixia may be a result of a combination of several ecological and genetic factors, including a relatively homogeneous habitat available throughout the distribution range, lack of obvious geographical barriers, high gene flow and a large long-term effective population size. These are discussed in the following sections in more detail.
The willow tit is found in coniferous forests (Fennoscandia, Siberia) as well as in mixed and broadleaved forests (western and central Europe). These habitats are abundant as a more or less continuous belt throughout the distribution range of the species, from the Atlantic coast of British Isles to the Pacific coast. In Europe the edge of the distribution range is east of the Pyrenees and north of Greece and includes the whole Alps. In Asia, the several mountainous regions do not seem to have had any marked effects on the distribution and actually the willow tits have been recorded to breed at high elevations (up to 2400 m in Japan, Harrap & Quinn, 1996 and even up to at least 2900 m in China, J. Martens, personal observation). This certainly has affected the dispersal capacity and enabled high gene flow even over the Urals and the Altai mountains. However, whether the present gene flow is still high can be questioned, because the borders of the song types today are sharp (Martens et al., 1995) and the differences in morphological traits like coloration and size of the subspecies contradict high present gene flow.
Both estimates of the effective female population size, the long-term and the coalescent estimate were large. Especially the large long-term Nef indicates that there have not been any strong population bottlenecks in the recent history of the willow tit, although suitable forest habitats during the Weichselian glaciation were restricted in the eastern Palaearctic and were almost absent from the western Palaearctic. However, south of Mongolia, there were regions covered with forest vegetation (Adams, 1997), which the species could have inhabited.
It takes approximately 4 N generations to reach reciprocal monophyly of mitochondrial haplotypes in a stable-sized population and even longer if the population is expanding (Avise et al., 1984). Given the large long-term female effective population size of 148 000 in willow tits, it would take about 1 300 000 years for the monophyly to occur in a stable population (assuming a generation time of 2.26 years and a substitution rate of 2% between lineages/Myr) and the coalescent time (estimated from 0.5 × 108 × p, where p is the mean pairwise genetic distance; Avise et al., 1988) is only 570 000 years. Evidently the willow tit population has expanded in size and range after the Ice Ages. This means that the time to monophyly would be even longer. If a population is less than 0.5 N generations old, it is highly likely that there are many mitochondrial lineages present which predate the age of the population (Avise et al., 1984). In other words, it is possible that although dispersal would be restricted and differentiation of subspecies would be in progress, not enough time has passed for monophyletic groups of subspecies to occur in a mitochondrial gene tree. The problems and approaches of distinguishing between the coalescence time of the haplotypes and the population divergence time are discussed also by Edwards & Beerli (2000), where the authors point out that multilocus and multispecies studies should be accomplished in order to estimate population divergence times.
It has been suggested that colonization of new areas from ‘a leading edge’ (Hewitt, 1996) would reduce genetic variation and might even lead to a fairly homogenous population structure because of sequential bottlenecks. This indicates that the areas with lower diversity are more distant from the refuges than the areas with higher diversity (Fry & Zink, 1998). Decrease of nucleotide diversity with increasing latitude has been documented from some widespread bird species in the Nearctic (red-winged blackbird Agelaius phoeniceus, Ball et al., 1988 and Fig. 3a in Fry & Zink, 1998) and Palaearctic (greenfinch Carduelis chloris, Meriläet al., 1997). In willow tits there was no correlation between latitude and nucleotide diversity, instead we found a trend of increasing nucleotide diversity towards east. This combined with the large long-term effective population size (indicating lack of recent bottlenecks) and the palaeovegetation maps reconstructed to present vegetation cover about 18 000 years ago (Adams, 1997) suggests that during the last Ice Ages the willow tit could have occupied fairly large regions in the south-eastern Palaearctic. Thus they may have colonized the present western distribution area from the east. This scenario is supported by a study by Martens et al. (1995), who concluded that different territorial songs of the willow tit may have developed and radiated in the eastern Palaearctic region. To some extent the nucleotide diversity values followed the cline described for the subspecies in the salicarius group, but because of the lack of morphological measures of any kind from the individuals sampled this cannot be tested nor confidently stated.
This study describes the population genetic structure of a sedentary bird species having a Palaearctic distribution range. A plethora of sedentary birds have been studied in America (e.g. chickadees, Gill et al., 1993; fox sparrow Passerella iliaca, Zink, 1994; song sparrow Melospiza melodia, Fry & Zink, 1998; rock ptarmigan Lagopus mutus, Holder et al., 1999, 2000) and some in western Europe (greenfinch Carduelis chloris, Meriläet al., 1997; blue tit Parus caeruleus, Taberlet et al., 1992 and Kvist et al., 1999a; great tit P. majorKvist et al., 1999b), but very little is known so far from the eastern Palaearctic. Existence of divergent intraspecific lineages has been found in many of the sedentary Nearctic bird species although presently no barriers seem to prohibit gene flow. At least some of these intraspecific lineages have been related either to a specific refuges (e.g. rock ptarmigan; Holder et al., 1999) or morphological subspecies (e.g. fox sparrow, Zink, 1996). However, many studies have revealed also species with no obvious phylogeographic structuring, many of those exhibiting shallow mitochondrial trees and indications of recent population expansion (e.g. Ball & Avise, 1992; Seutin et al., 1995; Zink, 1996). In western Europe, the lack of divergent lineages in the greenfinch and the great tit could also be attributed to population growth after a bottleneck during the Weichselian glaciation (Meriläet al., 1997; Kvist et al., 1999b). In southern Europe divergent lineages found from the blue tits, could instead be related to two subspecies and two refuges (Kvist et al., 1999a). In the willow tit no divergent lineages were found, but the high amount of genetic variation found implies that the species has not encountered a similar history than the relative species in Europe, instead the species survived in the east when the conditions became harsh during the Ice Ages.
Phylogenetic status of the songar tit
The phylogenetic status of the songar tit has been controversial, as it is sometimes considered to be a subspecies of the willow tit and sometimes an own species. Voous (1977) placed the songar tit within the willow tit, but stated that the conspecificity has not been proven. Eck (1979, 1980) divided the willow tit into three ‘sectors’, the first including P. m. montanus and the salicarius group, the second including the kamtchatkensis group and the third including the songar tit. He also considered the North American black-capped chickadee to be conspecific with the willow tit. Cramp & Perrins (1993) and Harrap & Quinn (1996) treated the songar tit and the willow tit as separate species, but noted the problematic status. Martens et al. (1995) analysed the territorial songs of the willow and songar tits in the Palaearctic and concluded them to be conspecifics.
Of the four subspecies described for the songar tit (P. m. songarus, P. m. weigoldicus, P. m. affinis and P. m. stoetzneri) we were able to include two in our study. Both, P. m. songarus and P. m. affinis were monophyletic. In addition, there were two mitochondrial lineages in P. m. affinis, one from Gansu and other from Qinghai, China. Also the P. m. songarus individual from Kirghizia differed a lot from the two individuals from Kazakhstan. The songarus–montanus group was monophyletic with a bootstrap support of 97%. The Tajima–Nei distances between P. m. songarus, P. m. affinis and the rest of P. montanus ranged from 3.15 to 4.13% (Table 3), the largest being between P. m. affinis and P. m. songarus. The distances to and between the other species of the Poecile group were roughly twofold. When compared with the distances of the North American chickadee species, estimated from mtDNA (RFLP), the observed distances between subspecies were about half of those estimated between recognized species within the Poecile group (Gill et al., 1993). Genetic distance, estimated from the mitochondrial control region sequences, between two subspecies of the blue tit has been found to be 1.3% (Kvist et al., 1999a), smaller than the distance between the songar and the willow tits.
According to the mitochondrial gene tree and genetic distances both subspecies of songar tit are as distinct from each other as the willow tit from them. On the basis of the phylogenetic species concept (PSC), defined as the smallest diagnosable cluster of individual organisms within which there is a parental pattern of ancestry and descent (e.g. Vogler & DeSalle, 1994), the songar tit cannot be considered as one species. Whether the two subspecies should be treated as subspecies of the willow tit or as two separate species cannot be answered only on the grounds of mtDNA. Martens et al. (1995) proposed on the basis of acoustic characters of the songar tit, that the territorial song of the songarus group points to an origin from the borealis group of the willow tit, and that according to the song characters, the songar tit cannot be considered as an independent species. To conclude, the phylogenetic status of the songar tit cannot be definitely determined, but there are however, indications that it should be considered conspecific with the willow tit.
To summarize, the common ancestors of the willow and songar tits could have existed somewhere in the south-eastern Asia, some 1.5–2 Myr ago (assuming the 2% divergence rate). The split of the ancestral species, into the ‘subspecies of songar tit’ and into the willow tit happened almost simultaneously. As the climate began to warm after the last Ice Ages, willow tit was able to expand all through the Palaearctic, whereas P. m. songarus and P. m. affinis were restricted to eastern Asia, more or less within their presumed areas of origin. The rapid range expansion, population growth and large effective population size of the willow tit are mainly responsible for the observed lack of phylogeograhic patterns in the mtDNA. It is possible that there are genetic differences, other than those studied by us between the willow tit populations, but they may have evolved so recently, simultaneously with the expansion, that they cannot yet be revealed by the mtDNA.