Phylogeographic and Demographic Analysis of the Asian Black Bear (<i>Ursus thibetanus</i>) Based on Mitochondrial DNA

<div><p>The Asian black bear <i>Ursus thibetanus</i> is widely distributed in Asia and is adapted to broad-leaved deciduous forests, playing an important ecological role in the natural environment. Several subspecies of <i>U</i>. <i>thibetanus</i> have been recognized, one of which, the Japanese black bear, is distributed in the Japanese archipelago. Recent molecular phylogeographic studies clarified that this subspecies is genetically distantly related to continental subspecies, suggesting an earlier origin. However, the evolutionary relationship between the Japanese and continental subspecies remained unclear. To understand the evolution of the Asian black bear in relation to geological events such as climatic and transgression-regression cycles, a reliable time estimation is also essential. To address these issues, we determined and analyzed the mt-genome of the Japanese subspecies. This indicates that the Japanese subspecies initially diverged from other Asian black bears in around 1.46Ma. The Northern continental population (northeast China, Russia, Korean peninsula) subsequently evolved, relatively recently, from the Southern continental population (southern China and Southeast Asia). While the Japanese black bear has an early origin, the tMRCAs and the dynamics of population sizes suggest that it dispersed relatively recently in the main Japanese islands: during the late Middle and Late Pleistocene, probably during or soon after the extinction of the brown bear in Honshu in the same period. Our estimation that the population size of the Japanese subspecies increased rapidly during the Late Pleistocene is the first evidential signal of a niche exchange between brown bears and black bears in the Japanese main islands.</p><p>This interpretation seems plausible but was not corroborated by paleontological evidence that fossil record of the Japanese subspecies limited after the Late Pleistocene. We also report here a new fossil record of the oldest Japanese black bear from the Middle Pleistocene, and it supports our new evolutionary hypothesis of the Japanese black bear.</p></div

Divergence times of the family Ursidae.

<p>Nodal numbers indicate the estimated divergence time (Ma). Horizontal gray bars spanning the nodes mark the 95% confidence interval for the divergence time. The phylogenetic positions of extinct lineages, which are indicated by the dashed lines, follow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref018" target="_blank">18</a>]. Horizontal black bars indicate temporal range based on fossil evidence. The temporal range of the common ancestor of Ursidae based on fossil evidence is indicated by the shading. Extinct species are marked by a dagger (†).</p

Summary Statistics for the demography.

<p>N: numbers of the samples</p><p>Θπ: Theta based on the nucleotide diversity</p><p>Θw: Waterson's theta</p><p>*All of them were not significant in this study (p-values> 0.1)</p><p>Summary Statistics for the demography.</p

Dynamics of the population sizes and tMRCAs of all Asian black bears, the Japanese population and the continental population.

<p>(a) Dynamics of the population sizes estimated by Bayesian Skyline Plot analysis. The y-axes indicates the effective population size × generation intervals, and the x-axes indicate the time in years before present. (b) Posterior distributions of the tMRCAs. The times of formation of land bridges before the oldest record of the Japanese black bear (337–330 Kilo annum) are indicated by shaded bars, following Dobson and Kawamura [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref060" target="_blank">60</a>] and Rohling et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref063" target="_blank">63</a>] with recalibrations by Lisiecki and Raymo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref067" target="_blank">67</a>]. The shading around the lines indicates 95% confidence interval of effective population size for each time point.</p

Middle Pleistocene black bear from Japan with a comparison of the left upper second molars (M2) among bears.

<p>1, Middle Pleistocene (ca. 337–330 Kilo annum, MIS 9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref068" target="_blank">68</a>]) black bear, <i>Ursus thibetanus</i> subsp. indet., from Aomori Prefecture, northern Japan (NMNS-PV 22666). 2, Extant Japanese black bear, <i>Ursus thibetanus japonicus</i>, from Nagano Prefecture, central Japan (NMNS-PO 52). 3, Extant Tibetan black bear, <i>Ursus thibetanus thibetanus</i>, from Thailand (NMNS-PO 207). 4, Extant Hokkaido brown bear, <i>Ursus arctos yesoensis</i>, from Hokkaido, northern Japan (NMNS-PO 208). The black bears share a combination of M2 characters such as a relatively large metacone (as large as the paracone), a distinct constriction between the paracone and the metacone, and a less developed posterior talon. In contrast, the brown bear has a smaller metacone relative to the paracone, a less distinct constriction between the two cusps, and a well developed posterior talon in M2. These comparisons suggest that the tooth of Middle Pleistocene Japanese black bear is closer in size and shape to the teeth of continental black bears than it is to extant Japanese black bears, but it is far from the brown bears.</p

Coalescent times of the Asian black bear.

<p>Nodal numbers indicate the estimated coalescent time (Ma). Horizontal gray bars show the 95% confidence interval for the coalescent times. (a-c) Geographical distribution of fossils of Asian black bear in (a) the Calabrian (Early Pleistocene) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref058" target="_blank">58</a>], (b) the lonian (Middle Pleistocene) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref058" target="_blank">58</a>, Kohno, unpublished] and (c) the Tarantian (Late Pleistocene) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref058" target="_blank">58</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136398#pone.0136398.ref060" target="_blank">60</a>]. The illustration is of a Japanese black bear (<i>Ursus thibetanus japonicus</i>), kindly provided by Utako Kikutani. Circles on maps indicate the fossil record of Asian black bears (Blue circles indicate the fossil records reported by previous studies and red circle indicates the fossil records newly reported by this study.).</p

Genealogy of eight Asian black bears as inferred from the 3rd codon positions of the complete mitochondrial protein genes using the GENETREE program.

<p>Since the program did not work on the whole data set, probably due to excessive numbers of mutation sites, the whole data set was separated into six fragments. The dots on the branches indicate the number of mutation sites. The values of ρ_ML (= 2×Nef×μ; μ is the mutation rate per sequence per generation) and tMRCAs were also estimated by GENETREE. The substitution rate of the 3rd codon positions of mitochondrial protein genes of Asian black bear was estimated to be 3.03×10⁻⁸/site/year (data not shown), and the average mutation rate of each of the six fragments is 5.99×10⁻⁵/sequence/generation.</p

Additional file 2: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

Figure S1. Performance of two-way active avoidance response of double transgenic (Gal4FF;UAS:zBoTxBLC:GFP) fish. The following Gal4FF transgenic fish lines were crossed with UAS:zBoTxBLC:GFP effector fish, and analyzed for two-way active avoidance fear conditioning. a hspGGFF10C (n = 6), b hspGGFF20A (n = 10), c hspGFF38B (n = 10), d hspGFF55B (n = 6), e SAGFF81B (n = 12), f SAGFF226F(n = 9), g SAGFF233A (n = 9), h SAGFF234A (n = 11), i hspGFFDMC12A (n = 12), j hspGFFDMC56B (n = 10), k hspGGFF19B (n = 9), l hspGGFF19C (n = 9), m hspGFF62A (n = 5), n gata6SAGFF94A (n = 6), o SAGFF27C (n = 6), p SAGFF38A (n = 8), q SAGFF87C (n = 5), r SAGFF92A (n = 8), s SAGFF183A (n = 5), t SAGFF195A (n = 6), u SAGFF212C (n = 5), v SAIGFF170B (n = 10), w hspGFFDMC76A (n = 10), x hspGFFDMC85C (n = 11). Mean ± SEM and avoidance (%) for individual fish are plotted. Performance of wild type fish (n = 28) described in Fig. 2 is shown in dotted lines. Two-way ANOVA, fish groups (wild type fish treated by CS-US, wild type fish treated by CS only and double transgenic fish including fish described in Figs. 2 and 3 × training session days (day 1, day 5), was performed (F = 7.236, P < 0.0001). Dunnett’s multiple comparison post-hoc tests were performed between avoidance percentage of wild type fish and double transgenic fish on session day 1 and day 5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant (P > 0.05). a–j Reduced performance of the active avoidance response was observed. k–x Performance of the active avoidance response was not significantly different between wild type and the double transgenic fish. (PPTX 8767 kb

Additional file 3: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

Figure S2. GFP expression patterns of 16 Gal4FF;UAS:GFP fish that showed reduced performance of the active avoidance response. A dorsal view, a ventral view, and a schematic side view with positions of coronal sections are shown on the top. Serial coronal sections with position numbers are shown in the bottom. a hspGGFF10C, b hspGGFF20A, c hspGFF38B, d hspGFF55B, e SAGFF36B, f SAGFF70A, g SAGFF81B, h SAGFF120A, i SAGFF226F, j SAGFF228A, k SAGFF231A, l SAGFF233A, m SAGFF234A, n SAGFF234D, o hspGFFDMC12A, p hspGFFDMC56B. Scale bars in whole brain images: 500 Îźm. Scale bars in coronal section images: 200 Îźm. (PDF 3264 kb

Additional file 8: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

Figure S4. GFP expression patterns in SAGFF120A;UAS:GFP and SAGFF120A;UAS:GFP;UAS:zBoTxBLC:GFP fish. a Dorsal views of the brains from eight SAGFF120A;UAS:GFP (~10 months old) fish and eight SAGFF120A;UAS:GFP;UAS:zBoTxBLC:GFP (~10 months old) fish are shown. Scale bars: 1 mm. b Areas having more intensity than background (the maximum intensity measured in the posterior part of the telencephalon) were identified by using ImageJ [57] and shown in red. Scale bars: 500 Îźm. c Immunohistochemistry using anti-GFP (green) and anti-NeuN (a neuronal marker, magenta) of coronal sections of the telencephalon and hypothalamus of brain samples from these transgenic fish. The fish numbers correspond to the numbers of the dorsal view images. Scale bars, 200 Îźm. (PPTX 6544 kb