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Keywords:

  • cryptic species;
  • DNA barcoding;
  • Japanese birds;
  • Palearctic region;
  • taxonomy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese-breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour-joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan-specific cryptic species from trans-Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans-Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

DNA barcoding is a useful and proven tool for species identification. A partial region (648 bp) of the cytochrome c oxidase I (COI) gene in the mitochondrial genome is used as a standard DNA barcoding region for most animals (Hebert et al. 2003). In vertebrates, the DNA barcoding region has been very useful in identifying species due to its high interspecific and low intraspecific variation (Ward et al. 2005; Hernández-Dávila et al. 2012), and moreover, for identifying cryptic species (Clare et al. 2007; Lara et al. 2010).

The DNA barcoding library of bird DNA includes several geographical regions, such as the Nearctic (Hebert et al. 2004), South Korea (Yoo et al. 2006), the Neotropics (Kerr et al. 2009a; Tavares et al. 2011), the eastern Palearctic (Kerr et al. 2009b) and Scandinavia (Johnsen et al. 2010), and is continually expanding (Milá et al. 2012). DNA barcoding of distinct conspecific genetic divergences has revealed lineages of many cryptic species within continents (Hebert et al. 2004; Kerr et al. 2009b; Milá et al. 2012) and among trans-continents (Kerr et al. 2009a; Johnsen et al. 2010; Lijtmaer et al. 2011). Lohman et al. (2010) investigated the barcoding region of six resident birds in South-East Asia, which included many small islands, and found deep genetic divergence among the islands, suggesting that traditional taxonomy may have overlooked endemic species in the area.

Geographical barriers, such as mountain ranges and seas, prevent gene flow and form definite distribution boundaries for species and subspecies. The Japanese Archipelago is located off the eastern coast of the Eurasian continent, across the Sea of Japan and the East China Sea (Fig. 1). The avifauna of Japan consists of 633 bird species, including nonbreeding birds that largely share the eastern Eurasian continent despite the sea barrier; however, the Japanese Archipelago has 11 endemic resident bird species and six migratory bird species breeding only in that area (The Ornithological Society of Japan 2012). The archipelago is treated as a biodiversity hot spot (Conservation International 2012) and a new zoogeographic region based on the phylogenetic relationship of the birds and other vertebrates (Holt et al. 2013). The high biodiversity in Japan is due to a wide variety of climates and ecosystems, ranging from the humid subtropics in the Ryukyu and Ogasawara islands to the boreal zones in northern Japan, the alpine zone at over 3000 m above sea level, and more than 3000 islands (Conservation International 2012). The islands and the straits between them have separated avian species over geographical time. Traditional taxonomists have repeatedly disputed whether the populations isolated on islands and/or by the straits should be recognized as subspecies or as separate species (Ornithological Society of Japan 2012), as the birds form a morphologically well-defined species group. However, molecular analysis has determined that the straits and seas have split the Japanese birds into several distinct lineages, but these have currently only been revealed for a limited number of species such as the Arctic Warbler Phylloscopus borealis (Saitoh et al. 2010), the Eurasian Jay Garrulus glandarius (Akimova et al. 2007), the Varied Tit Sittiparus varius (Nishiumi et al. 2006) and the Ryukyu Robin Erithacus kamodori (Seki 2006; Seki et al. 2007).

image

Figure 1. Map of the Japanese Archipelago and surrounding regions, showing the location and the names of islands and areas. The so-called Blakiston's Line is a biogeographical boundary that separates the Japanese bird and mammal fauna between Hokkaido and the other areas.

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The aim of this barcoding study was to examine whether the diversification and phylogeographic structure of Japanese birds is related to the complex arrangement of islands, which extend northeast and southwest of Japan. We focused on current Japanese-breeding bird species. We generated a DNA barcoding library of bird species to test whether DNA barcoding was a suitable method for species identification and to identify the prevalence of cryptic species. However, the region covered by our survey was not sufficient to fully establish a relationship between genetic diversification and the Japanese Archipelago because many Japanese birds are common throughout Eurasia. A wider-scale investigation was needed, and accordingly, we conducted a comprehensive survey to assess the genetic divergence of bird species breeding in the east Eurasian Continent and the Japanese Archipelago using published barcoding libraries.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

In total, 1367 voucher specimens representing 234 Japanese bird species collected throughout the Japanese Archipelago (Appendix S1, Supporting information) were included in this research. Blood and frozen tissue samples (pectoral muscle) were taken from voucher specimens held at the National Museum of Nature and Science, Tokyo (NSMT) and the Yamashina Institute for Ornithology (YIO). All the samples were linked to voucher specimens from the NSMT (49.3%), the YIO (40.1%), the Higashi Taisetsu Museum of Natural History (6.8%), the Botanical Garden at the Field Science Centre for Northern Biosphere, Hokkaido University (1.9%), the Kushiro-Shitsugen Wildlife Centre (1%) and other institutions (0.8%). Detailed information about these samples is accessible via ‘Birds of Japan, NSMT’ and ‘Birds of Japan, YIO’ projects on the Barcode of Life Data Systems (BOLD) website (http://www.boldsystems.org/).

To estimate the full genetic divergence of birds in East Asia, including the insular region of the Japanese Archipelago, we also used 1737 sequences representing 142 bird species from the intersection of 437 Eurasian species (Yoo et al. 2006; Kerr et al. 2009b) and 234 Japanese species. The taxonomy followed Clements (2007) as recommended by the ‘All Birds Barcoding Initiative’ (http://www.barcodingbirds.org/).

DNA extraction, PCR amplification and DNA sequencing of the COI barcoding region was conducted at the NSMT and YIO laboratories. DNA was extracted from blood or tissue samples using a standard phenol–chloroform procedure at NSMT and using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) at YIO. Several pairs of primers were used as standard primers for amplification of the COI barcoding region; Bird F1 (5′-TTCTCCAACCACAAAGACATTGGCAC-3′), Bird R1 (5′-ACGTGGGAGATAATTCCAAATCCTG-3′) and Bird R2 (5′-ACTACATGTGAGATGATTCCGAATCCAG-3′), alongside newly designed primers, L6697Bird (5′-TCAACYAACCACAAAGAYATCGGYAC-3′) and H7390Thrush (5′-ACGTGGGARATRATTCCAAATCCTG-3′) for passerine birds. If this approach was unsuccessful, an alternative forward primer, FalcoFA (5′-TCAACAAACCACAAAGACATCGGCAC-3′), or a reverse primer, VertebrateR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′), was used (Kerr et al. 2007). The 25 μL polymerase chain reaction (PCR) reaction mix comprised 19.2 μL ultrapure water, 1.0 U Taq polymerase (Ex Taq, TaKaRa, Shiga, Japan), 2.5 μL PCR buffer (Mg2+ free), 0.3 μL of each primer (0.24 mM), 2.5 μL of each dNTP (2.5 mM) and 0.4–1.0 μL of DNA. The amplification protocol was as follows: 94 °C for 3 min followed by five cycles at 94 °C for 30 s, 48 °C for 30 s, 72 °C for 1 min, then 30 cycles at 94 °C for 30 s, 51 °C for 30 s, 72 °C for 1 min and a final 72 °C for 5 min. The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide and purified using ExoSAP-IT (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions. Sequencing reactions were carried out using BigDye Terminator v1.1 (ABI, Paisley, UK), and analysis was performed on an ABI 3130 Genetic Analyser (ABI) at NSMT, and BigDye Terminator v3.1 (ABI) and an ABI 3100 at YIO. COI sequences were re-covered for all 234 bird species and did not contain insertions, deletions, or nonsense- or stop codons, supporting the absence of nuclear pseudogene amplification (Song et al. 2008).

Genetic distances of breeding birds in Japan were ascertained using the Distance Summary and the Barcode Gap Analysis applications of the BOLD website, with the Kimura 2-parameter model (K2P) (Ratnasingham & Hebert 2007). Neighbour-joining (NJ) trees with bootstrap values (1000 replications) were constructed from the COI data using the K2P model and Mega 6.05 (Tamura et al. 2013). In cases of very low nearest NJ distances (<1%), the Diagnostic Character application on the BOLD website was used for a molecular diagnostic of species assignment. We applied >1.6% intraspecific variation as the criterion to find candidates of cryptic species (Kerr et al. 2009b). Similarly, genetic distances of 142 common birds from the Japanese Archipelago and the Eurasian continents were calculated using the applications on the BOLD website. The mean genetic distances among populations and bootstrap values of the NJ tree (1000 replications) were ascertained using MEGA 6.05 (Tamura et al. 2013). Bird species with a high bootstrap value (>95%) were grouped to estimate the phylogeographic structure.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

Species identification in Japanese birds

Sequence data from the COI barcoding region were obtained for 1367 specimens of 234 bird species from the Japanese Archipelago. Of these species, 200 were represented by multiple specimens. The average K2P genetic distances within species and genus were 0.49% (range 0–6.13) and 8.82% (0–16.39), respectively (Fig. 2). Average K2P intraspecific distance was 0.21% based on 200 species from multiple specimens (0–6.13%). Of the 234 species, 226 (96.6%) had unique DNA sequences that did not overlap with any other species. Ninety-six percentage of the species were >1.0% divergent from their nearest neighbour, and 92.7% were >1.5% divergent. The NJ tree of K2P genetic distances formed 192 monophyletic clusters (96% of the species represented by multiple specimens) that were well supported by bootstrap values (>95%). The bootstrap value of Erithacus (94%) was slightly lower than the reliable bootstrap value (95%) for phylogenetic structure.

image

Figure 2. Frequency histogram of COI among Japanese-breeding birds showing (a) the distribution of the average distance within species (200 species) and (b) the average congeneric distance (47 genus).

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Of the 234 bird species, five pairs of sister species showed relatively low interspecific genetic variation (<1.0%) (Table 1). Five pairs were exhibited on the NJ tree with unreliable bootstrap values (30–72) or on nonmonophyletic clades. For example, the Pleske's Warbler Locustella pleskei and the Middendorff's Warbler Locustela ochotensis had bootstrap values of 55% and 65%, respectively, and were unable to be identified by diagnostic subsitutions due to small sample sizes (n = 2 and 6, respectively). Only one diagnostic substitution was observed between the Izu Thrush Turdus celaenops and the Brown-headed Thrush T. chrysolaus. Two Guillemot species (Cepphus carbo and C. columba) could not be discriminated based on the diagnostic sequences due to small sample sizes (n = 1 and 1, respectively). The Common and Oriental cuckoos were paraphyletic and could not be perfectly discriminated. Some Spot-billed Ducks Anas poecilorhyncha and Mallards A. platyrhynchos shared the same sequences, although the majority of A. poecilrhyncha had diverged with a cluster supported by 96% bootstrap values (0.79% genetic distance). Thus, 226 Japanese bird species (96.6%) were identified by their COI barcoding regions using either a distance-based criterion or a NJ tree with bootstrap values and diagnostic sequences in cases of low interspecific genetic distances (<1.0%).

Table 1. Japanese bird species with small (<1.0%) interspecific genetic COI sequence distances
 OrderCommon nameScientific name n Genetic distance (%)
1AnseriformesMallard Anas platyrhynchos 40
Spot-billed Duck Anas poecilorhyncha 7 
2PasseriformesIzu Thrush Turdus celaenops 40.15
Brown-headed Thrush Turdus chrysolaus 12 
3CuculiformesCommon Cuckoo Cuculus canorus 50.3
Oriental Cuckoo Cuculus optatus 6 
4PasseriformesMiddendorff's Warbler Locustella ochotensis 60.63
Pleske's Warbler Locustella pleskei 2 
5CharadriiformesSpectacled Guillemot Cepphus carbo 10.85
Pigeon Guillemot Cepphus columba 1 

Deep genetic divergence within the Japanese Archipelago

Eleven bird species showed deep intraspecific divergence (>1.6% K2P) and had two or more clusters supported by high bootstrap values (>95%) (Table 2). Nine of 11 birds had three distinct geographical splits, separating the lineages within the Japanese Archipelago. The Tsugaru Strait splits Hokkaido and Honshu in the south and phylogeographically separates Garrulus glandarius, Phylloscopus borealis and the Ural Owl Strix uralensis. The Narcissus Flycatcher Ficedula narcissina and several Brown-eared Bulbul Ixos amaurotis populations collected from the Ryukyu Islands had different lineages from those collected at Kyushu in the north. Erithacus komadori split into two distinct lineages between the Okinawa Islands and the Amami Islands in the north. Certain populations of the Oriental Greenfinch Carduelis sinica, Sittiparus varius and the Scaly Thrush Zoothera dauma, which occupy a specific island and its neighbouring small islands, had sequences that had deeply diverged from sequences from other areas, although the three species are widely distributed throughout the Japanese Archipelago. Distinct lineages were found for C. sinica on the Ogasawara Islands, S. varius on the Iriomote Island and Z. dauma on the Amami-Oshima island. No evidence of a geographical split was detected between the two distinct lineages of both the Oriental Turtle-Dove Streptopelia orientalis and the Ryukyu Scops-Owl Otus elegans. The Japanese Scops-Owl O. semitorques formed two clusters, one of which could reliably be discerned as originating from the Ryukyu Islands (>95% bootstrap value), and the other, with a moderate cluster value (87%), from Honshu, despite having relatively low maximum intraspecific distances (0.9%).

Table 2. Japanese bird species with large (>1.6%) intraspecific COI divergence, treated as candidates of cryptic species
 Common nameScientific name n BootstrapMax intraspecific distance (%)Collection areaaGenetic break
  1. a

    A = Amami Is., H = Hokkaido, Hn = Honshu (including Kyushu and Shikoku), I = Izu Is., M = Miyakojima I., Mi = Minami-Daitojima I., O = Okinawa Is., Og = Ogasawara Is., T = Tokara Is., Y = Yaeyama Is., and Yk = Yakushima I.

1Ryukyu Robin Erithacus komadori 3/4100/1006.13O/A, YkRyukyu Is.
2Arctic Warbler Phylloscopus borealis 1/4/3–/89/1005.06Hn/H/HnTsugaru Strit
3Eurasian Jay Garrulus glandarius 4/3100/1004.43H/HnTsugaru Strit
4Scaly Thrush Zoothera dauma 2/18100/993.73A/Y, Og, I, Hn, HIsolated Islands
5Narcissus Flycatcher Ficedula narcissina 7/1099/1003.71Y, O, A/Hn, HRyukyu Is.
6Brown-eared Bulbul Ixos amaurotis 1/10/331/99/993.57Mi/M, O, A/Y, Og, I, Hn, HRyukyu Is.
7Oriental Greenfinch Carduelis sinica 9/199/–3.37Tk, I, Hn, H/OgIsolated Islands
8Ryukyu Scops-Owl Otus elegans 7/6100/1002.90Y, O/O, A, TSympatric
9Varied Tit Sittiparus varius 4/18100/1002.82Y/O, A, T, Yk, I, Hn, HIsolated Islands
10Ural Owl Strix uralensis 8/13100/992.81H/HnTsugaru Strit
11Oriental Turtle-Dove Streptopelia orientalis 3/5100/992.42Hn, H/I, Hn, YSympatric

Comparison of the Eurasian and Japanese birds

COI barcoding sequences of 1437 individuals were collected from 142 species that represented multiple specimens from the Japanese Archipelago and the eastern Eurasian continent (Appendix S2, Supporting information). The average intraspecific genetic distance (K2P) was 0.46% (0–6.13%). The NJ tree of K2P genetic distances described 139 monophyletically distinct species supported by high bootstrap values (>95%). Cuculus optatus and C. canorus were not reciprocally monophyletic. Motacilla flava was part of a paraphyletic relationship between a sample from western Eurasia and samples from Japan and eastern Eurasia.

Forty-one of the 142 species had two or more distinct clusters supported by high bootstrap values (>95%). Of these 41 species, 23 displayed phylogeographic patterns that suggested genetic splits between the Eurasian continent and the Japanese Archipelago (Table 3). Of these 23 species, 19 had two or more clusters differing by at least 1.6% K2P. Some species formed clades that included multiple samples derived from Japanese birds, and one sample from Eurasia (the Eurasian Skylark Alauda arvensis, the Carrion Crow Corvus corone, the Large-billed Crow C. macrorhynchos, the Grey-faced Woodpeeker Picus canus and the Eurasian Magpie Pica pica), for which it was not possible to ascertain any geographical splits between Eurasia and the Japanese Archipelago. In addition to those mentioned above, three species formed Japanese-specific monophyletic clusters with moderate to relatively high bootstrap support (77–94%). A clade for the Siberian Blue Robin Luscinia cyane, which included specimens form Hokkaido and the South Kuril Islands (77% bootstrap value), was separated from another clade from Eurasia and Honshu (91%), differing by 1.06%. A clade for the Asian Brown Flycatcher Muscicapa dauurica from the Japanese Archipelago (94%) differed by 0.88% from a Eurasian continent clade (88%). The Rock Ptarmigan Lagopus muta formed a Japan-specific monophyletic clade (82%) that differed by 0.3% from another monophyletic clade formed of Eurasian samples (68%).

Table 3. Mean genetic distance and distinguishable lineages for birds that breed in the Palearctic region including the Japanese Archipelago. We listed the bird species with robust divergent clusters with high bootstrap values (>95%). Superscripts show groups arranged in the NJ tree
 Species n BootstrapMean K2P (%)aMax K2P (%) Eurasia vs. JapanCollection areabJapan-specific lineagescGenetic split within JapanCyptic species in relation to Japan
  1. a

    Mean population genetic distance (K2P) between clades.

  2. b

    E = Eurasian continent, S = Sakhalin, Ss = South Kril Is., Y = Yaeyama Is., O = Okinawa Is., M = Miyakojima I., Mi = Minami-Daitojima, A = Amami Is., Yk = Yakushima I., Og = Ogasawara Is., I = Izu Is., H = Hokkaido, and Hn = Honshu (including Kyushu and Shikoku).

  3. c

    Including the area of Sakhalin and S Kuril Is.

  4. d

    Duplication of cryptic species candidates in Table 2.

1 Alauda arvensis 1a/6b/10ca/99b/99c7.95a-b, 4.80a-c8.38Ea/E, Sb/S, H, Hnc  Yes
2 Delichon dasypus 1a/1b/9ca/–b/99c6.40a-b, 3.85a-c6.4Ea/Sb/Hn, HcYes Yes
3 Caprimulgus indicus 4/599/995.96.04E/H, HnYes Yes
4 Phylloscopus borealis 8a/9b/4c/1d99a/99b/90c/–d3.60a-b, 1.78a-c, 3.57a-d5.15Ea/S, Hb/Hnc/HndYesYesYesd
5 Pica pica 9/599/993.84.73E/E, H  Yes
6 Garrulus glandarius 3a/12b/3c99a/99b/99c2.72a-b, 3.59a-c, 4.32b-c4.53Ea/E, Hb/HncYesYesYesd
7 Emberiza spodocephala 8/1799/993.553.95E/Hn, H, S, SsYes Yes
8 Zoothera dauma 2/2599/993.633.73A/E, Y, Og, I, Hn, HYesYesYesd
9 Ficedula narcissina 7/1299/992.883.71Y, O, A/Hn, H, SYesYesYesd
10 Ixos amaurotis 10a/37b/1c99a/99b/–c2.53a-b, 3.19a-c3.57E, Y, Mi, Og, I, Hn, Ha/O, Ab/MicYesYesYesd
11 Cettia diphone 2/3596/992.933.43E/Y, O, A, Og, I, Hn, H, S, SsYes Yes
12 Muscicapa sibirica 6/399/992.662.92E/Y, Hn, SYes Yes
13 Dendrocopos major 4/1199/992.692.82E/Hn, H, SYes Yes
14 Strix uralensis 10/1399/992.812.81E, H/HnYesYesYesd
15 Corvus corone 7/785/982.122.45E/E, Hn, H  Yes
16 Urosphena squameiceps 4/599/992.12.21E/Hn, H, SYes Yes
17 Corvus macrorhynchos 2/2399/781.552.13E/E, Y, O, A, Yk, Hn, H, S  Yes
18 Lanius cristatus 3/199/–1.912.01E/YYes Yes
19 Uragus sibiricus 5/1297/991.491.82E/Hn, H, S, SsYes Yes
20 Picus canus 7/1499/881.251.44E/E, H   
21 Luscinia calliope 6/998/681.031.34E/Hn, HYes  
22 Tachybaptus ruficollis 1/4–/961.051.05E/Y, M, A, HnYes  
23 Mergus merganser 1/2–/980.590.59E/HnYes  

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

The COI barcoding region enabled identification of 226 species from 234 Japanese-breeding birds, with distance threshold criteria, a NJ tree with high bootstrap values and diagnostic substitution analyses. The average congeneric difference (8.8%) was 18-fold that of the average conspecific genetic difference (0.49%) (Fig. 2). The COI barcoding region has a high resolution for identification of Japanese bird species, making it an effective tool for the identification of bird species inhabiting insular regions, such as the Japanese Archipelago. DNA barcodes have identified 24 cryptic species candidates in the Japanese Archipelago, suggesting that the sea and the straits around the Japanese Archipelago act as effective genetic barriers, despite their relatively small area.

There were five pairs of nearest-neighbour species that had small interspecific genetic differences (<1.0%) in the COI barcoding region (Table 1). Identical sequences were shared between Anas platyrhynchos and Anas poecilorhyncha, which has also been found for other geographical regions in previous studies (Yoo et al. 2006; Park et al. 2011). Anas platyrhynchos is closest to A. poecilorhyncha based on cytochrome b and ND2 (Johnson & Sorenson 1999), as well as on COI; thus, further taxonomic re-evaluation of these species should be performed. Two scenarios were proposed for the sequence similarities of these species. An ancestral duck species could have split relatively recently into the two species of duck. Alternatively, the two species of duck may have hybridized in the Russian Far East, resulting in their sharing of the barcoding region (Kulikova et al. 2004). Cuculus canorus and C. optatus also had a very low interspecific distance (0.3%) and formed paraphyletic clades; this pair was previously grouped together as a single clade (Kerr et al. 2009b), but hybrids have not been documented to date (Sorenson & Payne 2005). The interspecific genetic difference between Cepphus carbo and C. columba was 0.85%, which is similar to results based on the cytochrome b gene (1.4%) (Friesen et al. 1996). Only a very small genetic distance (0.15%) separated T. chrysolaus, a migrant breeder in northern Japan, and Turdus celaenops, a resident bird living in several islands off the main islands of Japan (Ornithological Society of Japan 2012), with only a single substitution enabling the species to be individually identified. In addition to those mentioned above, the sequence of the Slaty-backed Gull Larus schistisagus that breeds in Japan completely matched those of other gulls recorded in previous studies (Yoo et al. 2006; Kerr et al. 2009b; Lijtmaer et al. 2011) that do not breed in Japan, but migrate to Japan as winter visitors (Ornithological Society of Japan 2012). The COI barcoding region could not distinguish between L. schistisagus breeding in Japan and the winter visitor gulls.

We found 11 species with deep intraspecific divergences (>1.6%) (Table 2) that were supported by high bootstrap values (>95%). The patterns of these deep intraspecific divergences were consistent with some of the biogeographical boundaries across the Japanese Archipelago, and these were classified into three groups according to the sea barrier: (i) the Tsugaru Strait, (ii) the Ryukyu Islands and (iii) other specific islands. Streptopelia orientalis and O. elegans, however, exhibited an intricate pattern of haplotypic distribution. Although haplotypes of the dove were clearly split into two clades with high bootstrap values, no geographical structure was observed across the Japanese Archipelago, including the Ryukyu Islands. Otus elegans only partially separated into two clades between Okinawa and Amami-Oshima because two Okinawa birds belonged to the Amami-Oshima clade. Takagi (2013) reported that the owl had a vocal divergence among Amami-Oshima, Okinawa Islands and Yaeyama Islands. The Okinawa population could be an interbred population between the two clades. Sympatric divergence of O. elegans and S. orientalis would reflect the admixture of multiple lineages isolated over a geographical timescale (Webb et al. 2011; Hogner et al. 2012).

The Tsugaru Strait lies between Hokkaido and Honshu and divides the lineages of S. uralensis, Garrulus glandarius and Phylloscopus borealis into two clades (Table 2). In recent studies, P. borealis has been split into two species, the Kamchatka Leaf Warbler Phylloscopus examinandus on Hokkaido and the Japanese Leaf Warbler Phylloscopus xanthodryas on Honshu, based on morphological, behavioural and genetic assessments (Saitoh et al. 2010; Alström et al. 2011). Strix uralensis and G. glandarius have also been split in different subspecies in the Tsugaru Strait (Ornithological Society of Japan 2012). The Tsugaru Strait (so-called Blakiston's Line) represents one of the biogeographical boundaries that separate Japanese avifauna south of Honshu from typical Eurasian and Hokkaido avifauna (Blakiston 1883).

The Ryukyu Islands have also formed boundaries that separate some bird species and have led to deep genetic divergences. Ficedula narcissina is classified into two subspecies according to the breeding populations on the main islands of Japan and the Ryukyu Islands (Ornithological Society of Japan 2012). In the Narcissus Flycatcher, the narcissina and owstoni subspecies are sometimes treated as separate species, with F. narcissina present on the main islands of Japan and F. owstoni on the Ryukyu Islands (Brazil 2009). The I. amaurotis population from middle Ryukyu (Okinawa and Amami Islands) was formed as a single unique clade, supported by high bootstrap values, but individuals on the south Ryukyu (the Yaeyama and Miyakojima islands) belong to the same clade as those on the Japanese main islands. Hamao et al. (2013) also reported the complex genetic structure of the bulbuls among the Ryukyu Islands. Climate change has caused repeated emergences and disappearances of land-bridges between the Ryukyu Islands, temporarily connecting some of the islands, and even the continent. These islands biogeographically form the boundary between the Palearctic and the Oriental regions.

Four species showed large genetic differences on other islands (Table 2). For example, the sequence of C. sinica on the Ogasawara Islands was clearly separated from a clade clustered by other subspecies of C. sinica on the main islands of Japan and the continental regions. The Ogasawara Islands are oceanic islands located 1000 km south of the main islands of Japan. They contain some endemic bird species and subspecies, including some species which are now extinct (Ornithological Society of Japan 2012). Sittiparus varius shows deep genetic divergence between Iriomote Island and the other islands. The Iriomote population belongs to the same clade as the Taiwanese population (McKay et al. 2014). On Amami-Oshima, Z. dauma forms a separable clade to that on the Japanese islands. The Amami-Oshima Z. dauma is often treated as a separate species of Zoothera major, although this was not supported by genetic evidence (Dickinson 2003; Brazil 2009). Erithacus komadori is split into two clades with deep intraspecific divergence (6.13%): one clade, E. k. namiyei, originates from Okinawa, and the other clade, E. k. komadori, comes from Amami-Oshima and Yakushima Islands. Our findings closely resemble those of Seki (2006) and Seki et al. (2007). These deep divergences between the middle/southern Ryukyu Islands and the main islands of Japan may have occurred during the Pliocene, when the middle Ryukyu Islands finally separated from the main island (Kizaki & Oshiro 1980).

In total, 142 of 398 bird species that breed in the Japanese Archipelago and the eastern Eurasian Continent were analysed to reveal the genetic structure of trans-Japanese and Eastern Eurasian birds (Appendix S2, Supporting information). The average intraspecific distance was 0.46% (0–3.32%). Ten percentage of 234 species exhibit deep intraspecific divergence in relation to the Japanese Archipelago. This percentage is higher than for species in the Nearctic (2%) (Hebert et al. 2004; Kerr et al. 2007), Korea (1%) (Yoo et al. 2006), Scandinavia (1%) (Johnsen et al. 2010), and approximately equal to the Palearctic (10%) (Kerr et al. 2009b). Geographical barriers, such as mountains and oceans, have divided populations and prevented gene flow. For example, the birds inhabiting both regions of Scandinavia and the Nearctic had deep intraspecific divergence in 24% of species (Kerr et al. 2007) and the birds of the Nearctic and Argentina in 24% (Kerr et al. 2009a). Relatively high levels of divergence were reported between Palearctic and Nearctic populations of some Holarctic birds (e.g. Drovetski et al. 2004; Koopman et al. 2005; Zink et al. 2006). The avifauna of the Japanese Archipelago includes many possible cryptic species, despite the differences in size and geographical history of the region.

Japan has a relatively high proportion of cryptic bird species compared with South Korea and Scandinavia, which are similar in size to Japan, and a similar proportion as Eurasia, which is geographically much larger. Sea barriers would have led to the isolation of bird populations around Japan. The repeated southward spread of the ice sheets to 40°N in North America during the quaternary (Hewitt 2004) would have caused shallow genetic divergence (Ball & Avise 1992; Seutin et al. 1995; Zink 1996; Weir & Schluter 2004) and low genetic diversity (Hewitt 1996; Soltis et al. 1997; Conroy & Cook 2000) among the various taxa in the northern regions, while the ice sheets that were limited to 50°N in the east Palearctic region allowed bird populations to speciate. The southward spread of ice did not affect the Japanese Archipelago equally, and some areas would have been linked to the Eurasian Continental by land-bridges. The fluctuating sea levels during the quaternary would have led to repeated periods of connection, isolation and submergence of the islands of the Japanese Archipelago, which could have isolated bird populations for lengthy periods. Such populations would have retained geographical and temporal isolation without admixture of other populations and extinction for glacial and interglacial periods. These isolated populations may lead to the deep intraspecific genetic divergence observed in 11 Japanese bird species. The distinct intraspecific divergence observed between 23 trans-Palearctic species may reflect the history of isolation and gene flow of these bird populations with respect to the changes in the land-bridges over the straits between the Japanese Archipelago and the Eurasian continent.

In this study, we constructed a barcoding library comprising 93.2% of Japanese-breeding bird species and demonstrated an effective tool for the identification of these bird species. The relatively deep genetic divergence was related to the periodic occurrence and disappearance of sea barriers. We found 24 cryptic species candidates in this study, which suggests that traditional taxonomic methods for identification of East Asian birds do not fully reflect the divergences. For example, P. borealis has been split into three species in recent studies (Saitoh et al. 2010; Alström et al. 2011). Our study contributes additional Japanese bird data to the global DNA barcoding library and provides valuable data for future investigation of the taxonomy of East Asian birds.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

We thank the following institutions and people for providing specimens: the Higashi Taisetsu Museum of Natural History; the Botanical Garden, Field Science Centre for Northern Biosphere, Hokkaido University; the Hokkaido Seabird Centre, the Kushiro-Shitsugen Wildlife Centre, the Iriomote Wildlife Centre, the Amami Wildlife Centre, the Yambaru Wildlife Conservation Centre and Raptor Conservation Centre, the Ministry of the Environment, Japan; Miyakojima City Museum; the Hokkaido Research Centre, the Forestry and Forest Products Research Institute; Okhotsk Chapter and Sapporo Chapter, the Wild Bird Society of Japan; Shunkunitai Wild Bird Sanctuary; the Lake Utonai Wildlife Centre; the Lake Shikaribetsu Nature Centre; the Akkeshi Waterfowl Observation Centre; the Institute for Raptor Biomedicine Japan; the Ushiku Nature Sanctuary; the Ibaraki Nature Museum; the Gyotoku Wild Bird Observation Centre; the Hachijo visitor's centre; the Miyake Nature Centre; the Yonagunijima Ayamihabiru Museum; the Graduate School of Science, Osaka City University; the Graduate School of Fisheries Sciences, Hokkaido University; Hiroshima City Asa Zoological Park; the Ehime Prefectural Science Museum; Sanbonmatsu High School; Crane Park Izimi; Amami Ornithologists’ Club; the Ikeya Animal Hospital; and the Takizawa Animal Hospital. We also thank Dr. Takeshi Yamasaki and Dr. Sayaka Mori for helping us to obtain specimen records. This study was funded by the research project of the National Museum of Nature and Science, ‘DNA barcoding of birds from Japan and East Asia’, JSPS KAKENHI Grant Numbers 21370039 and 24657066, and a Grant-in-Aid for Scientific Research (Specially Designated Research Promotion), ‘Development and disclosure of the Yamashina Institute for Ornithology database system (2009–)’ from the Ministry of Education, Culture, Sports, Science & Technology in Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

T.S. collected the specimens, analysed the data and prepared the draft. N.S. provided the sequences, analysed the data and wrote the manuscript. S.S. performed the genetic analysis. Y.I. and S.K. collected the specimens. H.K., A.H., S.A. and Y.Y. provided many of the sequences and organized the sequence data. I.N. designed and coordinated the project.

Data accessibility

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information

DNA sequences: DDBJ accessions AB842491–AB843857. Sequence alignment and final neighbour-joining tree uploaded as Online Supporting Information. BOLD project BJNSM and YIO, accessions BJNSM001-08–BJNSM842-12 and YIO001-08–YIO558-12, respectively.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Data accessibility
  10. Supporting Information
FilenameFormatSizeDescription
men12282-sup-0001-AppendixS1-S3.pdfapplication/PDF474K

Appendix S1 List of Japanese-breeding bird species analysed in this study.

Appendix S2 List of the bird species analysed in this study (breeding in both Eurasia and the Japanese Archipelago).

Appendix S3 A K2P neighbour-joining tree of COI made with 1367 voucher specimens from 234 species breeding in the Japanese Archipelago.

men12282-sup-0002-SuppInfo.txtplain text document1514K 

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