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Journal of Ornithology

, Volume 160, Issue 3, pp 721–731 | Cite as

Phenotypic and genetic characterization of the East Siberian Willow Warbler (Phylloscopus trochilus yakutensis Ticehurst, 1935) in relation to the European subspecies

  • Kristaps SokolovskisEmail author
  • Max Lundberg
  • Miriam Liedvogel
  • Diana Solovyeva
  • Susanne Åkesson
  • Mikkel Willemoes
  • Staffan Bensch
Open Access
Original Article

Abstract

Long-distance migrants with transcontinental breeding ranges are of particular interest for the study of local adaptation and geographic differentiation in birds. We compared phenotypes and genotypes between Far East Siberian Willow Warblers Phylloscopus trochilus yakutensis Ticehurst, 1935 with the European subspecies P. t. trochilus Linnaeus, 1758 and P. t. acredula Linnaeus, 1758. We found significant differences in mean body size and plumage colour, but intra-population variation overlapped extensively between the European and Siberian populations. We used stable isotope composition in winter-grown flight feathers as a proxy for wintering sites and found differences between all three subspecies, indicating different wintering grounds. Out of four nuclear loci analyzed (three of which are known to be substantially divergent between the European subspecies), none allowed to seperate East Siberian yakutensis from North Scandinavian acredula. Hence, neither phenotypic traits nor the currently available genetic resources provide diagnostic criteria for confidently assigning individual Willow Warblers to a particular subspecies. Despite extensive overlap in phenotypes and genotypes, we propose that the subspecies names can still be used as biogeographical references to the three Willow Warbler populations that differ in migration strategies. We propose to use yakutensis for Willow Warblers breeding east of the Ural Mountains that presumably initiate autumn migration towards the southwest or west, in contrast to the genetically most similar acredula that start autumn migration towards the southeast or south. Future field studies are needed to elucidate whether the longitudinal variation in phenotype is a cline, or whether a clear contact zone between these subspecies can be identified.

Keywords

Willow Warbler Phylloscopus trochilus yakutensis Subspecies Genetic structure Phenotypic variation Clock gene Stable isotopes 

Zusammenfassung

Vergleichende phänotypische und genetische Charakterisierung der ostsibirischen ( Phylloscopus trochilus yakutensis Ticehurst, 1935) und europäischen Fitis-Unterarten

Langstreckenzieher mit trans-kontinentalem Verbreitungsgebiet eigenen sich hervorragend zur Untersuchung lokaler Anpassung und geographische Differenzierung. Hier vergleichen wir Phänotyp und Genotyp der ostsibirischem Fitis-Unterart (Phylloscopus trochilus yakutensis Ticehurst, 1935) mit den europäischen Unterarten P. t. trochilus Linnaeus, 1758 und P. t. acredula Linnaeus, 1758. Unsere Ergebnisse zeigen signifikante Unterschiede der mittleren Körpergröße, sowie Gefiederfärbung, wobei die Bandbreite beider Messgrößen zwischen den Unterarten stark überlappen. Die zur Bestimmung der jeweiligen Überwinterungsgebiete genutzte stabile Isotopenkomposition der im Winter gemauserten Schwungfedern weist klare Unterschiede zwischen allen drei Unterarten auf. Zur populationsgenetischen Beschreibung wurden vier genomische Loci genutzt (wovon drei klare Unterschiede zwischen beiden europäischen Unterarten zeigen). Die resultierende populationsgenetische Struktur gruppiert die ostsibirische Unterart yakutensis klar zusammen mit der nordskandinavischen Unterart acredula. Unsere Ergebnisse erlauben weder auf Ebene des Phänotyps, noch auf Ebene des Genotyps eine klare diagnostische Abgrenzung der drei Unterarten. Trotz extensiver Überlappung der hier genutzten genotypischen und phänotypischen Marker halten wir dennoch weiter die Verwendung der bestehenden Nomenklatur als biogeographische Referenz im Kontext der verschiedenen von den Unterarten genutzten Zugrouten für sinnvoll. Um yakutensis von der genetisch eng verwandten Unterart acredula abzugrenzen empfehlen wir yakutensis für Fitispopulationen mit Brutgebiet östlich des Urals die im Herbstzug vermutlich in südwestlicher oder westlicher Richtung aufbrechen zu verwenden. Weiterführende Untersuchungen sind nötig um zu klären, in wie weit die beobachteten Unterschiede zwischen yakutensis und acredula bezüglich Längengrad einen graduellen Verlauf oder eine abrupte Änderung zwischen den Unterarten aufweisen.

Introduction

Species with continent-wide breeding ranges typically show less diversity at higher than lower latitudes. For instance, the Chipping Sparrow (Spizella passerina) has five geographically structured mitochondrial lineages in total, but only two occurring at high latitudes (Carson and Spicer 2003; Martin and Tewksbury 2008). Despite occupying wide breeding ranges, several migratory species winter in relatively narrow and species- or even population-specific areas. For example, the Northern Wheatear (Oenanthe oenanthe) winters exclusively in sub-Saharan Africa, even though it breeds across the entire Holarctic (Bairlein et al. 2012). This pattern of restricted wintering ranges and much wider breeding distribution is best explained by rapid postglacial expansions from common refugia (Milá et al. 2006), combined with existing constraints on establishing new wintering areas. What makes these cases exceptionally interesting is that fast range expansions must have proceeded in parallel with locally adapted evolutionary changes in migration direction and timing. For example, the Common Rosefinch (Carpodacus erythrinus) colonized Scandinavia from central Russia only about 100 years ago (Pavlova et al. 2005), but keeps wintering in the same confined location in southern Asia. Hence, to end up in the same wintering quarters, Scandinavian Rosefinches must have changed their autumn migration direction from south to east southeast (Stach et al. 2016).

Similarly, the breeding range of the Willow Warbler spans from the coast of the Atlantic to the coast of the Pacific Ocean, with wintering grounds in tropical Africa for all subspecies (Shirihai and Svensson 2018). The Willow Warbler is currently classified in three subspecies; the nominal trochilus breeding in central and western Europe, acredula in northern Scandinavia/eastern Europe and yakutensis in eastern Siberia (Ticehurst 1938 mentioned in Williamson 1976). The general appearance of Willow Warblers is typical for a “leaf warbler” (Phylloscopidae), but shows substantial variation across its breeding range: a typical trochilus has a bright yellow breast, yellow under-wing and under-tail coverts, and yellow chin and supercilium (Shirihai and Svensson 2018). In comparison, the subspecies acredula is on average longer-winged and has less yellow in the plumage, with a pale yellow supercilium and faintly streaked breast. The Siberian subspecies yakutensis is described as yet longer-winged with an even paler plumage, with hardly any trace of yellow (Williamson 1976; Shirihai and Svensson 2018). Shirihai and Svensson (2018) sketched the contact zone between trochilus and acredula relatively close to the hybrid zones characterized by Bensch et al. (2009). One notable difference, however, is that the populations breeding in southern Norway were assigned to acredula by Shirihai and Svensson (2018), whereas Bensch et al. (2009) assigned these to trochilus.

The breeding biology and phenotypic variation in European acredula and trochilus has been studied in great detail (Brock 1910; Tiainen 1983; Lawn 1984; Jakobsson 1988). Variation in plumage and size within the European subspecies is extensive, precluding subspecies identification of most, but the extreme individuals (Williamson 1976; Shirihai and Svensson 2018). Variation in genotypes and phenotypic traits in trochilus and acredula shows sharp clines across a narrow and well-defined contact zone in central Scandinavia, and a much wider diffuse cline east of the Baltic Sea (Bensch et al. 2009). This geographic distribution pattern suggests that the species expanded from a single glacial refugium and gradually evolved into the present phenotypes known as trochilus and acredula while colonizing Scandinavia from two directions (Bensch et al. 2009). However, robust population history inferences will require comprehensive data from the entire breeding range of the species, including populations from its easternmost breeding range. Our knowledge to date about the phenotypic characteristics of populations from the eastern range, which undertake the longest migratory journeys (Sokolovskis et al. 2018), is based almost exclusively on specimens from museum collections.

Here we characterize a yakutensis population from its extreme northeast breeding range based on morphology, genetic markers and migratory behaviour. We evaluate our novel results in the context of traits from the well-characterized European subspecies to discuss the phylogeography, population structure and taxonomy of this model species of migration research.

Methods

Sampling

The fieldwork was carried out in north-eastern Siberia in the Chaun River delta (68.80°N, 170.61°E) during June and July 2016. Territorial Willow Warbler males were lured into mist nets with song playback. The following morphological measurements were taken: total length of the bill to head; tarsus max length (to closest 0.1 mm), length of wing chord [“flattened wing” method as in Svensson (1992)], and whole tail length to closest 0.5 mm. Body mass was recorded with a Pesola LightLine balance, to the nearest 0.1 g. KS (sole data collector from Chaun) calibrated field measurements with SB to ensure that the data set from Chaun was comparable to data previously collected from Scandinavia. We combined all four morphological measurements into a principal component analysis (PCA) with R (version 3.5.1) package factoextra (Kassambara 2015). All phenotypic traits were also analysed individually using one-way analysis of variance (ANOVA), with subspecies as the explanatory factor, to test whether differences were significant. We then used Tukey’s honestly significant difference (HSD) test to see which of the subspecies were different (Tukey 1949).

Following a previously developed standard protocol (Bensch et al. 2009), plumage colour was scored by comparing all captured birds to three stuffed reference specimens representing the diversity in colouration of phenotypes across Scandinavia (Suppl. Figure 1). Plumage colour was scored from one to nine, where one translates to not even a hint of yellow and nine to bright yellow. All morphological and colour measurements taken for newly collected yakutensis were analysed with reference to the data set of trochilus and acredula presented in Bensch et al. (2009). In this study all birds classified as trochilus were sampled on breeding territories in Denmark and Sweden south of 60°N latitude, whereas birds classified as acredula were sampled on breeding territories in Finland, Norway and Sweden north of 65°N latitude (Suppl. Table 1).

We collected the innermost primary feather from every captured individual and preserved these in separate paper envelopes for stable isotope analysis. For DNA analysis we collected 20 μl of blood from each individual using a glass capillary tube, by puncturing the brachial wing vein with a syringe needle. The blood was stored in SET buffer (0.015 M NaCl, 0.05 M Tris, 0.001 M of EDTA, pH 8.0) at room temperature until brought to the lab for permanent storage at − 20 °C. Willow Warbler is not included in Red Data Book of Russian Federation therefore no special permits were needed for our study. Metal rings were provided by Institute of Biological Problems of the North according to agreement with Bird Ringing Centre of Russia.

We did not analyse vocalization because our extensive field observations suggest that the Willow Warbler song is virtually undifferentiated throughout its vast breeding range and would unlikely contribute a clear characteristic to add pattern for classifying subspecies.

Information about sample sizes and geographic origins of all samples used for the statistical comparisons are listed in Supplementary Table 1.

DNA extraction and genotyping

DNA was extracted from blood samples following an ammonium acetate protocol (as described in Richardson et al. 2001). Willow Warblers were genotyped at the following four loc ion; chromosome 1 (an intron of the NBEA gene by polymerase chain reaction (PCR) and Sanger sequencing as in Liedvogel et al. 2014), chromosome 3 (a biallelic marker “ww1” using PCR—RFLP as in Bensch et al. 2002), chromosome 4 (protein coding region of the Clock gene of one exonic poly-glutamine motif that varies in length, by PCR-fragment length analysis as in Johnsen et al. (2007) and chromosome 5 [intron of C11orf41 gene by PCR and Sanger sequencing as in Liedvogel et al. (2014)]. On chromosome 4 we used a candidate gene approach to compare the number of glutamine (Q, encoded as CAA or CAG) repeats, a classical proxy for Clock gene diversity, following a protocol described in Johnsen et al. (2007). We sent PCR products of 36 yakutensis, 140 acredula and 47 trochilus samples to “Uppsala Genome Centre” for fragment length analysis of this variable Clock region. The number of repeats scored from the fragment length analyses were confirmed by Sanger sequencing of five randomly chosen Siberian Willow Warblers over the entire poly-Q repeat, allowing to precisely match the number of glutamine repeats to fragment length. To ensure that the analysed poly-Q region was not duplicated in the Willow Warbler genome, we blasted a consensus sequence of five sequences obtained from ABI sequencer (279 bp) with BLASTN 2.5.0 (Zhang et al. 2000) to a Willow Warbler genome assembly published in Lundberg et al. (2017). This sequence aligned within the annotated interval of the Clock gene. The genomic loci on chromosomes 1, 3 and 5 were chosen because they have been previously shown to differ between trochilus and acredula (Lundberg et al. 2013; Liedvogel et al. 2014). The locus on chromosome 4 (segment of the Clock gene) was included because of its putative role in migration timing (Saino et al. 2013).

All Sanger sequencing was carried out using a BigDye sequencing kit (Applied Biosystems, Austin, TX, USA), following standard protocols, on an ABI PRISM 3100 sequencer (Applied Biosystems, FL, USA) at the Department of Biology at Lund University. Primers and PCR conditions are listed in Supplementary Table 2. Sequences have been deposited at NCBI as PopSet: 1485777242 (chromosome 1) and PopSet: 1485777195 (chromosome 5).

Molecular analysis

We used Geneious 6.1.8 (Kearse et al. 2012) and Aliview (Larsson 2014) to curate and trim sequences. The generated yakutensis sequences were aligned to existing reference sequences of trochilus and acredula (Suppl. Table 1). We used DnaSP Ver. 5.10.01 (Librado and Rozas 2009) to calculate Tajima’s D and Fst and MEGA7 (Kumar et al. 2016) to calculate nucleotide diversity (π). We inferred haplotypes using the PHASE algorithm implemented in DnaSP with default settings. NETWORK 5.0.0.1 (Bandelt et al. 1999) was then used to calculate median-joining haplotype network. We used Geneious 6.1.8 microsatellite analysis external plugin 1.4.0 to infer Clock allele lengths. We calculated Fst for poly-Q genotypes with Fstat 2.9.3.2 (Goudet 1995).

Stable isotope analysis

Stable isotope ratios for δ 13C and nitrogen δ 15N were analysed for the innermost flight feather at the Stable Isotope Service Lab, Department of Biology, Lund University, Sweden (www.biology.lu.se/services/instrumental-chemistry). Stable isotope analyses were carried out for 20 male Willow Warbler males from the Chaun delta and ten males from southern Sweden sampled in the same year (2016). We analysed material (6 to 8 mg) from the tip of the innermost primary feather as described in Bensch et al. (2006a). We compared the samples collected in 2016 from yakutensis (n = 20) and trochilus (n = 10) with data in Bensch et al. (2006a) from breeding range samples of acredula (n = 100) and trochilus (n = 100) as well as winter samples of actively moulting birds collected from multiple countries in western (n = 46), eastern (n = 26) and southern (n = 27) Africa.

Results

Morphometrics and plumage

Male Willow Warblers sampled in Chaun were larger than conspecifics from Scandinavia (Figs. 1 and 2, Suppl. Tables 3 and 4). This was most prominent for tail length (one-way ANOVA, F2,622 = 65.056, P < 0.001; Tukey’s HSD test, alpha = 0.05) and wing length (one-way ANOVA, F2,622 = 67.524, P < 0.001; Tukey’s HSD test, alpha = 0.05). Mean wing length was 69.9 mm (SD 1.3) for trochilus, 71.0 mm (SD 1.4) for acredula and 72.2 mm (SD 1.6) for yakutensis. In the entire data set, tail length strongly correlated with wing length (Pearson’s r = 0.66, P < 0.05). Tarsus length did not differ between the subspecies (one-way ANOVA, F2,622 = 0.1169 P = 0.889). Bill-head length differed significantly between the subspecies (one-way ANOVA, F2,622 = 27.267 P < 0.001). Post hoc Tukey’s HSD test suggested that bill-head lengths of acredula was significantly longer compared to trochilus and yakutensis. However, we advise caution when interpreting the results as the observed difference is within possible measurement error. The breast colour scores differed significantly between all three subspecies (one-way ANOVA F2,622 = 162.43 P < 0.001). Although all yakutensis trapped in Chaun were grey/white on the breast with the yellow restricted exclusively to the under-wing coverts and auxiliaries, similarly grey birds do also occur at low frequency within the range of acredula (Fig. 3).
Fig. 1

Principal component analysis of Willow Warbler morphology (males only). trochilus n = 300, acredula n = 291, yakutensis n = 34. (See eigenvalues in Suppl. Table 3 and variable loadings in Suppl. Figure 2)

Fig. 2

Comparison of tail length, wing length, bill and head length and tarsus length across all three Willow Warbler subspecies (males only). Dashed lines show mean values. trochilus n = 300, acredula n = 291, yakutensis n = 34

Fig. 3

Breast colour of all three Willow Warbler subspecies. 1 refers to absolute lack of yellow colour, whereas 9 refers to a yellow breast. Only males are included. trochilus n = 300, acredula n = 291, yakutensis n = 34

Stable isotopes

Feathers of yakutensis that had been moulted during the previous winter showed the highest δ 15N values (mean 12.30 ± 1.79, one-way ANOVA F2,217 = 101.18, P < 0.001, HSD-Tukey test with alpha = 0.05) and the lowest δ 13C values (mean − 21.64 ± 1.85, one-way ANOVA F2,217 = 5.9, P = 0.003, HSD-Tukey test with alpha = 0.05) compared to Scandinavian subspecies (Fig. 4). Overall, the δ 13C and δ 15N signatures of yakutensis were more similar to the signatures of Willow Warbler feathers moulted in southern Africa than to feathers moulted in western or eastern Africa (Fig. 4).
Fig. 4

δ 13C and δ 15N ratios from Willow Warblers caught at breeding and wintering grounds. The isotope signatures for trochilus (n = 100), acredula (n = 100) and Africa (West n = 47, East n = 26, South = 27) from Bensch et al. (2006a), yakutensis (n = 20)

Genotypes

We extracted DNA for genotyping samples from 36 yakutensis. While DNA extraction was successful for all individuals, clear genotyping results were obtained for all 36 birds for markers on chromosomes 3 (PCR–RFLP method) and 4 (fragment lengths analysis), but only for 26 individuals for the locus of chromosome 1, and 14 from the locus on chromosome 5. Most of these failures to successfully genotype those loci can likely be explained by the high density of indels in the Willow Warbler genome that creates many frame shifts and makes Sanger sequencing notoriously difficult in this species (Lundberg et al. 2017).

Across all the Willow Warbler subspecies, the sequenced region on chromosome 1 (214 bp) carried 19 SNPs (of which nine were singletons), 23 haplotypes and a nucleotide diversity (π) of 0.007. The sequenced fragment on chromosome 5 (338 bp) had 21 SNPs (of which five were singletons), 26 haplotypes and a π of 0.01. All three subspecies showed negative values of Tajima’s D for both loci although only significantly negative (− 2.19) for yakutensis on chromosome 1 (Suppl. Table 5).

For both Sanger sequenced loci, there was a clear separation between haplotypes primarily occurring among trochilus and haplotypes shared between acredula and yakutensis (Fig. 5). The pairwise Fst values were high (> 0.6) with significant difference between trochilus and each of the other subspecies, but there were no differences (Fst< 0.01) between yakutensis and acredula (Table 1).
Fig. 5

Haplotype networks for loci on chromosome 1 (a) and chromosome 5 (b). Each circle represents a unique haplotype. Colours show frequency of each subspecies carrying each haplotype. green: trochilus, blue: acredula, red: yakutensis. The size of the circle is proportional to the frequency of the haplotype and branch lengths are proportional to divergence between haplotypes. trochilus n = 28(chr1)/24(chr5), acredula n = 12(chr1)/28(chr5), yakutensis n = 58(chr1)/32(chr5)

Table 1

Pairwise Fst values for all three subspecies for all four loci investigated

Locus

Acr vs tro

Acr vs yak

Tro vs yak

Total

Chrom 1

0.625

0.008

0.587

0.547

Chrom 3

0.468

0.268

0.906

0.511

Chrom 4

0.002

0.008

0.010

0.001

Chrom 5

0.621

0.009

0.729

0.565

Detailed account of sample size and origin in Suppl. Table 6

Acr acredula, tro trochilus, yak yakutensis

For the bi-allelic locus on chromosome 3, 35 of the 36 yakutensis were homozygous for the “northern allele”, with only one individual carrying a heterozygous genotype. The latter individual was also heterozygous for the marker on chromosome 5. On chromosome 1, it was homozygous for the commonest acredula/yakutensis (northern) haplotype. The overall Fst for the locus on chromosome 3 was 0.51, with the lowest value between acredula and trochilus: 0.27 (Table 1).

For the Clock gene (chromosome 4) we found five alleles corresponding to variable numbers of glutamine repeats (9 to 12). All five alleles were present in all three Willow Warbler subspecies at similar frequencies (Fig. 6). The predominant genotype in all populations was homozygous for the most common allele with glutamine repeat length 11 (“11/11”) and the Fst value across all populations was 0.001. The level of heterozygosity (0.30 in trochilus, 0.33 in acredula and 0.19 in yakutensis) did not differ from what is expected under Hardy–Weinberg Equilibrium (P > 0.05 Suppl. Table 6).
Fig. 6

Frequency of Clock genotypes (chromosome 4) in all Willow Warbler subspecies. n = 47 for trochilus, 140 for acredula and 36 for yakutensis

Discussion

In this study we found P. t. yakutensis to be genetically inseparable from P. t. acredula. Meanwhile, the subspecies differ on average in size and plumage colouration.

As predicted from earlier work (Ticehurst 1936 in Williamson 1976; Shirihai and Svensson 2018), Willow Warblers from the north-eastern edge of the breeding range are larger compared to the western and north-western edge of the range, with the wing being the single best trait discriminating between all three subspecies. Since yakutensis are migrating longer distances than acredula and trochilus (Sokolovskis et al. 2018) this pattern matches Seebohm’s rule, predicting that populations with longer migration routes have longer wings (Seebohm 1901). Unlike traits that directly facilitate flying (i.e., wing and tail length), the traits that are more exclusively associated with foraging (i.e., tarsus and bill to head length) did not differ between the Willow Warbler subspecies. Similarly, in Eurasian Blackcaps (Sylvia atricapilla), wing length differed between populations that migrate different distances, while tarsus did not (Rolshausen et al. 2009).

In Chaun, the plumage of Willow Warblers showed little variation, with grey upperparts, snow-white underparts, and yellow restricted to auxiliaries. In Scandinavia, the typical grey/white acredula phenotype can be found at low frequency within the breeding range of on average more yellow trochilus and vice versa. However, we neither caught nor visually observed any distinctly yellow trochilus type coloured individuals in north-eastern Siberia suggesting that grey/white yakutensis phenotype is not just a morph. We speculate that the frequency of yellow birds shows a gradual cline across the continent and decreases from west to east but we need additional samples to confirm it.

In contrast to other phenotypic traits, the migratory behaviour separates the European Willow Warbler subspecies relatively clearly, with little variation within populations but with clearly defined contact zones, referred to as migratory divides (Bensch et al. 2009). Birds breeding in the range of trochilus migrate to western Africa in autumn, whereas birds from the range of acredula migrate to eastern and southern Africa, supported by ring recoveries (Hedenström and Pettersson 1987), stable isotopes (Bensch et al. 2009) and tracking data for trochilus (Lerche-Jørgensen et al. 2017). In the Scandinavian contact zone, clines in morphology and migratory behaviours are largely overlapping however east of the Baltic, these are displaced from each other by 1500 km. Hence, in Southern Finland and the Baltic countries, Willow Warblers look mainly like trochilus at similar latitudes, but migrate like acredula. The birds breeding east of the Ural Mountains, presently classified as P. t. yakutensis have been supposed to migrate to southern Africa (Alerstam et al. 2003). Compared to isotope signatures of Willow Warbler feathers in active moult across Africa, the isotope composition of birds from Chaun suggested wintering locations in southern Africa. However, our recent geolocator study of three birds from Chaun revealed wintering areas in southeast Africa (Sokolovskis et al. 2018). It is important to note that the isoscape of Africa is very heterogeneous and assigning any single sample to correct geographic origin is difficult if not impossible (Hobson et al. 2012). The isotope study of Bensch et al. (2006a) did not include samples from this exact part of Africa, and because geolocator data are lacking for acredula, we do not yet know to what extent acredula and yakutensis might share wintering areas. The differences we found suggest that Willow Warblers from Chaun winter in a region with different δ13C and δ 15N values than the Scandinavian acredula.

Using whole-genome resequencing, Lundberg et al. (2017) reported the presence of two putative inversions on chromosomes 1 and 5, harbouring most of the fixed differences between acredula and trochilus (in this study we sequenced parts of both these differentiating regions). Lundberg et al. (2017) also used a roughly 4000-SNP array to genotype approximately 1000 birds across their European breeding range, and also included four birds from far Eastern Siberia (Anadyr 64.71°N, 177.32°E) and four from Western Siberia (Yekaterinburg 56.97°N, 60.28°E) in the analysis. The combined genotypes of these Siberian birds from the range of yakutensis turned out to be inseparable from acredula. Our present study supports Lundberg et al. (2017) results by confirming that acredula and yakutensis are genetically extremely similar and differences, if any, are very small. The sequencing approach presented in this study allowed us to look for more variation in yakutensis that is not captured by the Lundberg et al. (2017) array, which was designed from SNPs that had originally been found in a contrast between acredula and trochilus. Yet, the same haplotypes seem to exist in acredula and yakutensis. Repeat length variation (e.g., microsatellites) was not included in the SNP array used by Lundberg et al. (2017). One of the four genetic markers investigated in our study (Clock) shows length polymorphism. This locus was included in our analyses because previous studies on songbirds have found the number of glutamine repeats in the Clock gene to be correlated with migration distance and breeding latitude (Caprioli et al. 2012; Saino et al. 2015; Bazzi et al. 2016). Because the Willow Warbler populations differ substantially in these traits, we expected them to show differences in repeat length at the Clock gene. However, the size ranges and frequencies of different Clock alleles were almost identical across all three subspecies (Fst = 0.001). These results reject the hypothesis of an association between Clock repeat length and migration timing, distance or breeding latitude, at least in this species.

It has been estimated that there are 5.5 described subspecies for every continental bird species even though there are only 1.9 discrete mitochondrial lineages for the same species (Zink 2004). The three Willow Warbler subspecies all share the same mitochondrial haplotypes (Bensch et al. 2006b), which points out that mitochondria is not always informative for detecting very recent differentiation. In addition to the three subspecies discussed in this paper, minor variation in wing length has previously been used to describe P. t. fitis from Germany and P. t. eversmanni from south-western Siberia (Salomonsen 1928). Haffer and Tiainen (in Glutz von Blotzheim 1991) call eversmanni a morph and question the separation of Willow Warblers in subspecies, but nevertheless acknowledges the existence of geographic structure in phenotype. More recent sources, such as Clements checklist of birds of the world (Clements et al. 2018) and Shirihai and Svensson (2018), agree on the validity of the three Willow Warbler subspecies that we discuss in the current paper. It is worth mentioning that the curators at one of the largest existing bird skin collections, the Zoological Museum of Moscow State University, still distinguish P. t. fitis and P. t. eversmanni alongside trochilus, acredula and yakutensis based on thorough analysis of morphological differences (Yaroslav Redkin pers. comm.). Future research combining these yet unpublished results with genomic analyses is something to strive for.

If a strong phylogenetic signal is found when using a limited set of genetic markers, such as in the Arctic Warbler (Phylloscopus borealis) (Alström et al. 2018), it clearly supports a taxonomic split. Alternatively, failing to find evidence of a population structure when examining a limited set of genetic markers, does not mean that no genetic difference exists. Even very small and potentially overlooked local genomic differences can lead to strikingly different phenotypes [e.g., Corvus corone corone and C. c. cornix (Poelstra et al. 2014)]. Even though our study used candidate markers derived from full genome comparisons, genome assemblies are never perfect and small differences can easily be missed. Commonly used neutral markers (e.g., majority of microsatellites) will mostly show differences among populations that occur because of drift. It is possible that any given subtle differentiation in phenotype is a result of recent point mutation and increases in frequency due to natural selection. In such case, even with a modern set of genomic tools, differences might be very difficult to find. Nevertheless, the true evolutionary history of each population is encrypted in genes, but still very difficult to extract. Examples of mismatching phenotypic and genetic structures in birds are accumulating (Harris et al. 2017; Schweizer et al. 2018); therefore, it is important to use detailed information of phenotypes together with genotypes in taxonomy. A serious revision of all currently acknowledged avian subspecies, integrating characteristics from various disciplines including genomics, tracking technology, and comparative morphology, remains an important task for modern ornithology because most of them have been described based on a handful of morphological characters only.

Some songbird species with large breeding ranges, such as the Siberian Rubythroat (Calliope calliope), are described as monotypic, while the Black Redstart (Phoenicurus ochruros), for example, has five distinct subspecies within a substantially smaller breeding range (Shirihai and Svensson 2018). Such differences between species in intraspecific variation ask for explanations and having described subspecies is a good starting point (Seebohm in Allen 1884). We agree with Fitzpatrick (2010) that the subspecies concept, although imperfect, is a useful aid when sorting samples and discussing geographic variation. In addition, by having subspecies as an entity means that we can save the well-defined biological entities as species and use subspecies for resolving and debating more ambiguously differentiated patterns. For example, thanks to trinomial taxonomy in the late nineteenth century in North America, 315 taxa were downgraded from full species to subspecies status (Mayr 1982).

A large continuous breeding range and minor clinal differentiation across Eurasia is a common pattern for songbirds [e.g., Bluethroat Luscinia svecica, and goldcrest Regulus regulus (Shirihai and Svensson 2018)]. In such species contrasts, the individuals from the extreme east and west might be apparent (as also illustrated by our study), but drawing a line between two morphs is arbitrary when variation changes over a gradual cline. In this regard, northern Eurasian songbirds might provide an excellent system for investigating early stages of local adaptation that may lead to parapatric speciation in the wild, which has not yet been clearly documented in vertebrates due to methodological difficulties, but might be a common speciation mechanism (Endler 1978), although this is still debated (Price 2008). The Willow Warbler in particular offers an excellent study system in this regard because, of its well-defined secondary contact zone in central Scandinavia, a less distinct primary contact zone in the south-eastern Baltic region (Bensch et al. 2009) and very likely a 6000-km-long gradual cline from northern Scandinavia to the coast of the Pacific ocean (Lundberg et al. 2017).

We encourage other researchers working with birds, even if taxonomy is not their main field, to publish descriptive articles about the life history data of their study species. Wesołowski (2012) has pointed out that recent research trends have severely decreased quality of life history data in published papers. Each species is a unique entity, and generalizations of ecological or evolutionary concepts derived from theoretical models without knowledge of the specific life history nuances can lead to fundamentally wrong yet attractively titled high-profile publications.

We suggest that the taxonomic classification of Willow Warblers into three subspecies can still be used as a biogeographical reference to populations that show average but overlapping differences in phenotypes, but distinctly different migration patterns. Based on our results, we propose to use yakutensis for Willow Warblers breeding east of the Ural Mountains that initiate the autumn migration towards the southwest or west, in contrast to the otherwise most similar acredula that initiate the autumn migration towards the southeast or south from their breeding grounds in northern and eastern Europe. To more firmly locate the contact zone between yakutensis and acredula, which might occur around the Yenisei River (Seebohm 1901; Shirihai and Svensson 2018; Yaroslav Redkin pers. comm.), data from a longitudinal transect across Siberia will be required.

In conclusion, neither size nor plumage nor the molecular markers tested here allowed us to decipher a coherent population structure of the currently recognized Willow Warbler subspecies. However, taken together, stable isotope signatures, ring recoveries and tracking studies suggest that wintering sites and migration routes may serve as subspecies delimiting criteria in this species.

Notes

Acknowledgements

We wish to acknowledge A. G. Voenkov, A. D. Voenkov, E. V. Voenkova, S. Vartanyan, G. Danilov, D. Barykina, A. Mylnikova, S. Ivanov and H. Ris for help with planning and executing field expeditions to Chaun delta. We say special thanks to “Kinross Gold” especially Anna Apoka and Evgeniya Saevich for immense support with logistics that made fieldwork in a location this remote possible.

Funding

KS was supported by a National Geographic young explorer grant (nr: WW-034ER-17) and Mörner scholarship for biological research from Uplands Nation, Uppsala University, SÅ, with Swedish Research Council grant (nr: 621-2013-4361). The project received significant aid from the Centre for Animal Movement Research (CAnMove) financed by a Linnaeus grant (nr: 349-2007-8690) from the Swedish Research Council. MLi was supported by a Feodor Lynen fellowship, Alexander von Humboldt-Foundation. MW was supported by the Danish Council of Independent Research (DFF—6108-00597); SB was supported by Swedish Research Council grant (nr: 621-2013-4839).

Supplementary material

10336_2019_1653_MOESM1_ESM.docx (180 kb)
Supplementary material 1 (DOCX 179 kb)

References

  1. Alerstam T, Hedenström A, Åkesson S (2003) Long-distance migration: evolution and determinants. Oikos 103:247–260CrossRefGoogle Scholar
  2. Allen JA (1884) Zoölogical nomenclature. Auk 1:338–353CrossRefGoogle Scholar
  3. Alström P, Rheindt FE, Zhang R, Zhao M, Wang J, Zhu X, Gwee CY, Hao Y, Ohlson J, Jia C, Prawiradilaga DM, Ericson PGP, Lei F, Olsson U (2018) Complete species-level phylogeny of the leaf warbler (Aves: Phylloscopidae) radiation. Mol Phylogenet Evol 126:141–152CrossRefGoogle Scholar
  4. Bairlein F, Norris DR, Nagel R, Bulte M, Voigt CC, Fox J, Hussell DJT, Schmaljohann H (2012) Cross-hemisphere migration of a 25 g songbird. Biol Lett 8:505–507CrossRefGoogle Scholar
  5. Bandelt H-J, Forster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol 16:37–48CrossRefGoogle Scholar
  6. Bazzi G, Cecere JG, Caprioli M, Gatti E, Gianfranceschi L, Podofillini S, Possenti CD, Ambrosini R, Saino N, Spina F, Rubolini D (2016) Clock gene polymorphism, migratory behaviour and geographic distribution: a comparative study of trans-Saharan migratory birds. Mol Ecol 25:6077–6091CrossRefGoogle Scholar
  7. Bensch S, Åkesson S, Irwin DE (2002) The use of AFLP to find an informative SNP genetic differences across a migratory divide in willow warbler. Mol Ecol 11:2359–2366CrossRefGoogle Scholar
  8. Bensch S, Bengtsson G, Åkesson S (2006a) Patterns of stable isotope signatures in willow warbler Phylloscopus trochilus feathers collected in Africa. J Avian Biol 37:323–330CrossRefGoogle Scholar
  9. Bensch S, Irwin DE, Irwin JH, Kvist L, Åkesson S (2006b) Conflicting patterns of mitochondrial and nuclear DNA diversity in Phylloscopus warblers. Mol Ecol 15:161–171CrossRefGoogle Scholar
  10. Bensch S, Grahn M, Muller N, Gay L, Åkesson S (2009) Genetic, morphological, and feather isotope variation of migratory willow warblers show gradual divergence in a ring. Mol Ecol 18:3087–3096CrossRefGoogle Scholar
  11. Brock SE (1910) The Willow-Wrens of Loathian Wood. Zoologist 14:401–417Google Scholar
  12. Caprioli M, Ambrosini R, Boncoraglio G, Gatti E, Romano A, Romano M, Rubolini D, Gianfranceschi L, Saino N (2012) Clock gene variation is associated with breeding phenology and maybe under directional selection in the migratory barn swallow. PLoS One 7:e35140CrossRefGoogle Scholar
  13. Carson RJ, Spicer GS (2003) A phylogenetic analysis of the emberizid sparrows based on three mitochondrial genes. Mol Phylogenet Evol 29:43–57CrossRefGoogle Scholar
  14. Clements JF, Schulenberg TS, Iliff MJ, Roberson D, Fredericks TA, Sullivan BL, Wood CL (2018) The eBird/Clements checklist of birds of the world: v2018. http://www.birds.cornell.edu/clementschecklist/download/
  15. Endler JA (1978) Geogrpahic variation, speciation and clines (monographs in populatin biology 10). Princetone University Press, PrincetoneGoogle Scholar
  16. Fitzpatrick JW (2010) Subspecies are for convenience. Ornithol Monogr 67:54–61CrossRefGoogle Scholar
  17. Glutz von Blotzheim UN, Bauer KM (1991) Handbuch der Vögel Mitteleuropas Bd. 12 Passeriformes. Teil 2. Sylviidae. Verlag, WiesbadenGoogle Scholar
  18. Goudet J (1995) FSTAT (version 1.2): a computer program to calculate F-statistics. J Hered 86:486CrossRefGoogle Scholar
  19. Harris RB, Alstr P, Leach AD, Museum B, History N, Ecology A, Centre EB, Species S, Centre I, Sciences A, Se- U, Systematics Z, Academy C (2017) Discordance between genomic divergence and phenotypic variation in a rapidly evolving avian genus (Motacilla). Mol Phylogenet Evol 120:1–26Google Scholar
  20. Hedenström A, Pettersson J (1987) Migration routes and wintering areas of Willow Warblers Phylloscopus trochilus (L.) ringed in Fennoscandia. Ornis Fennica 64:137–143Google Scholar
  21. Hobson K, Van Wilgenburg SL, Wassenaar LI, Powell RL, Still CJ, Craine JM (2012) A multi-isotope (d13C, d15N, d2H) feather isoscape to assign Afrotropical migrant birds to origins. Ecosphere 3:1–20CrossRefGoogle Scholar
  22. Jakobsson S (1988) Territorial fidelity of willow warbler (Phylloscopus trochilus) males and success in competition over territories. Behav Ecol Sociobiol 22:79–84CrossRefGoogle Scholar
  23. Johnsen A, Fidler AE, Kuhn S, Carter KL, Hoffmann A, Barr IR, Biard C, Charmantier A, Eens M, Korsten P, Siitari H, Tomiuk J, Kempenaers B (2007) Avian Clock gene polymorphism: evidence for a latitudinal cline in allele frequencies. Mol Ecol 16:4867–4880CrossRefGoogle Scholar
  24. Kassambara A (2015) Factoextra: visualization of the outputs of a multivariate analysis. R Package version 1.0.1Google Scholar
  25. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefGoogle Scholar
  26. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  27. Larsson A (2014) AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30:3276–3278CrossRefGoogle Scholar
  28. Lawn MR (1984) Premigratory dispersal of juvenile willow warblers phylloscopus trochilus in southern england. Ring Migr 5:125–131CrossRefGoogle Scholar
  29. Lerche-Jørgensen M, Willemoes M, Tøttrup AP, Rachel K, Snell S (2017) No apparent gain from continuing migration for more than 3000 km: willow warblers breeding in Denmark winter across the entire northern Savannah as revealed by geolocators. Mov Ecol 5:1–7CrossRefGoogle Scholar
  30. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452CrossRefGoogle Scholar
  31. Liedvogel M, Larson KW, Lundberg M, Gursoy A, Wassenaar LI, Hobson KA, Bensch S, Åkesson S (2014) No evidence for assortative mating within a willow warbler migratory divide. Front Zool 11:1–9CrossRefGoogle Scholar
  32. Lundberg M, Boss J, Canbäck B, Liedvogel M, Larson KW, Grahn M, Åkesson S, Bensch S, Wright A (2013) Characterisation of a transcriptome to find sequence differences between two differentially migrating subspecies of the willow warbler Phylloscopus trochilus. BMC Genom 14:1–11CrossRefGoogle Scholar
  33. Lundberg M, Liedvogel M, Larson K, Sigeman H, Grahn M, Wright A, Akesson S, Bensch S (2017) Genetic differences between willow warbler migratory phenotypes are few and cluster in large haplotype blocks. Evol Lett 1:1–14CrossRefGoogle Scholar
  34. Martin PR, Tewksbury JJ (2008) Latitudinal variation in subspecific diversification of birds. Evolution 62:2775–2788CrossRefGoogle Scholar
  35. Mayr E (1982) Of what use are subspecies. Auk 99:593–595Google Scholar
  36. Milá B, Smith TB, Wayne RK (2006) Postglacial population expansion drives the evolution of long-distance migration in a songbird. Evolution 60:2403–2409CrossRefGoogle Scholar
  37. Pavlova A, Zink RM, Rohwer S (2005) Evolutionary history, population genetics, and gene flow in the common rosefinch (Carpodacus erythrinus). Mol Phylogenet Evol 36:669–681CrossRefGoogle Scholar
  38. Poelstra JW, Vijay N, Bossu CM, Lantz H, Ryll B, Müller I, Baglione V, Unneberg P, Wikelski M, Grabherr MG, Wolf JBW (2014) The genomic landscape underlying phenotypic integrity in the face of gene flow in crows. Science 344:1410–1414 (New York, NY) CrossRefGoogle Scholar
  39. Price T (2008) Speciation in birds. Roberts and Co, ColoradoGoogle Scholar
  40. Richardson DS, Jury FL, Blaakmeer K et al (2001) Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler (Acrocephalus sechellensis). Mol Ecol 10:2263–2273.  https://doi.org/10.1046/j.0962-1083.2001.01355.x CrossRefGoogle Scholar
  41. Rolshausen G, Segelbacher G, Hobson KA, Schaefer HM (2009) Contemporary evolution of reproductive isolation and phenotypic divergence in sympatry along a migratory divide. Curr Biol 19:2097–2101CrossRefGoogle Scholar
  42. Saino N, Romano M, Caprioli M, Fasola M, Lardelli R, Micheloni P, Scandolara C, Rubolini D, Gianfranceschi L (2013) Timing of molt of barn swallows is delayed in a rare Clock genotype. Peer J 1:e17CrossRefGoogle Scholar
  43. Saino N, Bazzi G, Gatti E, Caprioli M, Cecere JG, Possenti CD, Galimberti A, Orioli V, Bani L, Rubolini D, Gianfranceschi L, Spina F (2015) Polymorphism at the Clock gene predicts phenology of long-distance migration in birds. Mol Ecol 24:1758–1773CrossRefGoogle Scholar
  44. Salomonsen F (1928) Die geographische variation des Phylioscopns trochilus (L.). J für Ornithol 3:451–461CrossRefGoogle Scholar
  45. Schweizer M, Warmuth V, Kakhki NA, Aliabadian M, Förschler M, Shirihai H, Suh A, Burri R (2018) Parallel plumage color evolution and introgressive hybridization in wheatears. J Evol Bio 32(1):100–110CrossRefGoogle Scholar
  46. Seebohm H (1901) The birds of Siberia. Ballantyne Press, LondonGoogle Scholar
  47. Shirihai H, Svensson L (2018) Handbook of Western Palearctic Birds. Helm, LondonGoogle Scholar
  48. Sokolovskis K, Bianco G, Willemoes M, Solovyeva D, Bensch S, Åkesson S (2018) Ten grams and 13, 000 km on the wing—route choice in willow warblers Phylloscopus trochilus yakutensis migrating from Far East Russia to East Africa. Mov Ecol 6:1–10CrossRefGoogle Scholar
  49. Stach R, Kullberg C, Jakobsson S, Ström K, Fransson T (2016) Migration routes and timing in a bird wintering in South Asia, the Common Rosefinch Carpodacus erythrinus. J Ornithol 157:671–679CrossRefGoogle Scholar
  50. Svensson L (1992) Identification guide to European Passerines, 4th edn. Page Bros, NorwichGoogle Scholar
  51. Tiainen J (1983) Dynamics of a local population of the Willow Warbler Phylloscopus trochilus in Southern Finland. Ornis Scand 14:1–15CrossRefGoogle Scholar
  52. Tukey JW (1949) comparing individual means in the analysis of variance. Biometrics 5:99–114CrossRefGoogle Scholar
  53. Wesołowski T (2012) Faulty practice in field biology—what should be done? For Biol 1:1–7Google Scholar
  54. Williamson K (1976) Identification for ringers the genus phylloscopus. Maund and Irvine Limited, TringGoogle Scholar
  55. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214CrossRefGoogle Scholar
  56. Zink RM (2004) The role of subspecies in obscuring avian biological diversity and misleading conservation policy. Proc R Soc B Biol Sci 271:561–564CrossRefGoogle Scholar

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of BiologyLund UniversityLundSweden
  2. 2.Behavioural Genomics Research GroupMax Plank Institute for Evolutionary BiologyPlönGermany
  3. 3.Laboratory of OrnithologyInstitute of Biological Problems of the NorthMagadanRussia

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