Mitochondrial DNA of pre‐last glacial maximum red deer from NW Spain suggests a more complex phylogeographical history for the species

The major climatic oscillations that characterized the Quaternary had a great influence on the evolution and distribution of several species. During cold periods, the distribution of temperate‐adapted species became fragmented with many surviving in southern refugia (Iberian, Italian, and Balkan Peninsulas). Red deer was one of the species that contracted its original range to southern refugia. Currently, two main lineages have been described for the species: western and eastern. We have analyzed fossils pre‐dating the last glacial maximum (LGM) from Liñares cave (NW Spain) that belongs to the peripheral range of the western clade, and fossils from the Danish Holocene belonging to the central part of the same clade. Phylogenetic analyses place our samples in the western clade. However, some specimens from Liñares represent an early split in the tree along with other pre‐LGM western samples from previous studies. Despite low bootstrap values in the Bayesian phylogenies, haplotype networks connect these foreign haplotypes to the eastern clade. We suggest a mixed phylogeographical model to explain this pattern with range expansions from the east during the expansion phase after the cold periods in marine isotope stage 3. We find slight isolation by distance in post‐LGM populations that could be a consequence of the recolonization from southern refugia after the LGM.     

Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionize phylogeography.     

Pleistocene climate change and phylogeographic structure of the Gymnocarpos przewalskii (Caryophyllaceae) in the northwest China: Evidence from plastid DNA,ITS sequences,and Microsatellite

Northwestern China has a wealth of endemic species, which has been hypothesized to be affected by the complex paleoclimatic and paleogeographic history during Quaternary. In this paper, we used Gymnocarpos przewalskii as a model to address the evolutionary history and current population genetic structure of species in northwestern China. We employed two chloroplast DNA fragments (rps16 and psbB‐psbI), one nuclear DNA fragment (ITS), and simple sequence repeat (SSRs) to investigate the spatial genetic pattern of G. przewalskii. High genetic diversity (cpDNA: h_S = 0.330, h_T = 0.866; ITS: h_S = 0.458, h_T = 0.872) was identified in almost all populations, and most of the population have private haplotypes. Moreover, multimodal mismatch distributions were observed and estimates of Tajima's D and Fu's FS tests did not identify significantly departures from neutrality, indicating that recent expansion of G. przewalskii was rejected. Thus, we inferred that G. przewalskii survived generally in northwestern China during the Pleistocene. All data together support the genotypes of G. przewalskii into three groups, consistent with their respective geographical distributions in the western regions—Tarim Basin, the central regions—Hami Basin and Hexi Corridor, and the eastern regions—Alxa Desert and Wulate Prairie. Divergence among most lineages of G. przewalskii occurred in the Pleistocene, and the range of potential distributions is associated with glacial cycles. We concluded that climate oscillation during Pleistocene significantly affected the distribution of the species.     

Biogeography and evolution of European cave salamanders, Hydromantes (Urodela: Plethodontidae), inferred from mtDNA sequences

Aim To infer the phylogenetic relationships and biogeography of Hydromantes, with special emphasis on the European taxa. In particular, we aimed to test: (1) the monophyly of the European species and current views on their interrelationships; and (2) previously proposed timings of the separation of European and American Hydromantes, and of biogeographically important events within Europe. Location California and the Western Mediterranean Basin, specifically south‐east France, Italy, and the island of Sardinia. Methods Partial sequences of mitochondrial genes (cytochrome b and 12S rRNA) were obtained from 45 specimens of Hydromantes, including all European extant species and subspecies, and two species from California. In addition, a fragment of the mitochondrial 16S rRNA gene was amplified for 16 specimens. Data sets were aligned using Clustal X, and well‐supported phylogenetic trees were produced using maximum‐likelihood, Bayesian and maximum‐parsimony methods. Estimates of divergence times were obtained with the program r8s , the molecular clock being calibrated using the opening of the Strait of Gibraltar, the final event in the Messinian Salinity Crisis of 5.3 Ma. Results Separation between the American and European clades occurred approximately 13.5 Ma, most probably before or after westward dispersal across the Bering Land Bridge. In Europe, divergence started in the late Miocene, when Hydromantes (A.) genei separated from other members of the genus 9 Ma and colonized south‐west Sardinia. Movement between the European mainland and Sardinia, by a member of the subgenus Speleomantes, occurred in the Messinian Salinity Crisis, after the Mediterranean Basin desiccated almost completely 5.96 Ma. Subsequent widespread aridification fragmented the geographical ranges of Hydromantes, which live in cool and humid conditions, resulting in the origin of the six species in the subgenus Speleomantes. In contrast, a second period of diversification, in continental Europe 2–1.3 Ma, was probably caused by very cold interludes during the climatic oscillations that characterized the Pleistocene. Main conclusions The molecular clock used here indicates that the separation of Californian and European Hydromantes occurred more recently than previously believed, and the same is true of some subsequent phylogenetic divergences within Europe. Estimated dates for these divergence events are consistent with known geophysical and climatic events that could have caused or facilitated them.     

Molecular analysis of an 11,700-year-old rodent midden from the Atacama Desert, Chile

DNA was extracted from an 11,700-year-old rodent midden from the Atacama Desert, Chile and the chloroplast and animal mitochondrial DNA (mtDNA) gene sequences were analysed to investigate the floral environment surrounding the midden, and the identity of the midden agent. The plant sequences, together with the macroscopic identifications, suggest the presence of 13 plant families and three orders that no longer exist today at the midden locality, and thus point to a much more diverse and humid climate 11,700 years ago. The mtDNA sequences suggest the presence of at least four different vertebrates, which have been putatively identified as a camelid (vicuna), two rodents (Phyllotis and Abrocoma), and a cardinal bird (Passeriformes). To identify the midden agent, DNA was extracted from pooled faecal pellets, three small overlapping fragments of the mitochondrial cytochrome b gene were amplified and multiple clones were sequenced. These results were analysed along with complete cytochrome b sequences for several modern Phyllotis species to place the midden sequence phylogenetically. The results identified the midden agent as belonging to an ancestral P. limatus. Today, P. limatus is not found at the midden locality but it can be found 100 km to the north, indicating at least a small range shift. The more extensive sampling of modern Phyllotis reinforces the suggestion that P. limatus is recently derived from a peripheral isolate.     

Diversification and phylogeographic structure in widespread Azteca plant-ants from the northern Neotropics

The Neotropical myrmecophytic tree Cordia alliodora hosts symbiotic Azteca ants in most of its widespread range. The taxonomy of the genus Azteca is notoriously difficult, which has frequently obscured species identity in ecological studies. We used sequence data from one mitochondrial and four nuclear loci to infer phylogenetic relationships, patterns of geographic distribution, and timing of diversification for 182 colonies of five C. alliodora-dwelling Azteca species from Mexico to Colombia. All morphological species were recovered as monophyletic, but we identified at least five distinct genetic lineages within the most abundant and specialized species, Azteca pittieri. Mitochondrial and nuclear data were concordant at the species level, but not within species. Divergence time analyses estimated that C. alliodora-dwelling Azteca shared a common ancestor approximately 10-22million years ago, prior to the proposed arrival of the host tree in Middle America. Diversification in A. pittieri occurred in the Pleistocene and was not correlated with geographic distance, which suggests limited historical gene flow among geographically restricted populations. This contrasts with the previously reported lack of phylogeographic structure at this spatial scale in the host tree. Climatic niches, and particularly precipitation-related variables, did not overlap between the sites occupied by northern and southern lineages of A. pittieri. Together, these results suggest that restricted gene flow among ant populations may facilitate local adaptation to environmental heterogeneity. Differences in population structure between the ants and their host trees may profoundly affect the evolutionary dynamics of this widespread ant-plant mutualism.     

Mitochondrial DNA sequence variation and phylogeographic patterns in harbour porpoises (Phocoena phocoena) from the North Atlantic

The harbour porpoise (Phocoena phocoena)experiences high rates of incidental mortalityin commercial fisheries, and in some areasthese rates are sufficiently high to justifyconcern over population sustainability. Giventhe high incidental mortality, the resolutionof population structure will be important toconservation and management, but in the NorthAtlantic the relationships among many of theputative populations remain unclear. Aprevious genetic study demonstrated substantialgenetic differences between eastern and westernNorth Atlantic populations, however thelocation of this break remained unresolved. Inthe present study, we addressed this issue byincluding new samples from Iceland. Toinvestigate population structure, variation inthe mitochondrial DNA of 370 porpoises wascompared among six locations corresponding toseveral of the putative populations (Gulf ofMaine, Gulf of St. Lawrence, Newfoundland, WestGreenland, Iceland, Norway). The first 342base pairs of the control region were sequencedand genetic variation investigated by analysisof molecular variance (F _ST and _ST ) and ² withpermutation. Although some fine scalepopulation structure was detected, porpoisesfrom Iceland were found to be more similar tothe western populations (W. Greenland, Gulf ofSt. Lawrence, Newfoundland, Gulf of Maine) thanto Norway. Furthermore, porpoises from Norwaywere different from all other regions. Thesepatterns suggest the existence of adiscontinuity between Iceland and Norway,possibly the result of isolating events causedby repeated range contractions and expansionsthroughout Quaternary glaciation events withinthe North Atlantic. These results suggest thatharbour porpoise populations within the NorthAtlantic are distinguishable, but patterns mustbe interpreted in light of their historicalbiogeography.     

Lineage diversification and historical demography of a sky island salamander,Plethodon ouachitae,from the Interior Highlands

Sky islands provide ideal opportunities for understanding how climatic changes associated with Pleistocene glacial cycles influenced species distributions, genetic diversification, and demography. The salamander Plethodon ouachitae is largely restricted to high‐elevation, mesic forest on six major mountains in the Ouachita Mountains. Because these mountains are separated by more xeric, low‐elevation valleys, the salamanders appear to be isolated on sky islands where gene flow among populations on different mountains may be restricted. We used DNA sequence data along with ecological niche modelling and coalescent simulations to test several hypotheses related to diversifications in sky island habitats. Our results revealed that P. ouachitae is composed of seven well‐supported lineages structured across six major mountains. The species originated during the Late Pliocene, and lineage diversification occurred during the Middle Pleistocene in a stepping stone fashion with a cyclical pattern of dispersal to a new mountain followed by isolation and divergence. Diversification occurred primarily on an east–west axis, which is likely related to the east–west orientation of the Ouachita Mountains and the more favourable cooler and wetter environmental conditions on north slopes compared to south‐facing slopes and valleys. All non‐genealogical coalescent methods failed to detect significant population expansion in any lineages. Bayesian skyline plots showed relatively stable population sizes over time, but indicated a slight to moderate amount of population growth in all lineages starting approximately 10 000–12 000 years ago. Our results provide new insight into sky island diversifications from a previously unstudied region, and further demonstrate that climatic changes during the Pleistocene had profound effects on lineage diversification and demography, especially in species from environmentally sensitive habitats in montane regions.     

Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.     

Nuclear gene sequences and DNA variation of Cryptomeria japonica samples from the postglacial period

Genomic DNA was extracted from heartwood blocks of six Cryptomeria japonica individuals that had been buried (in an area now covered by rice fields) for about 3600 years. Attempts were made to determine the sequences of five nuclear genes following polymerase chain reaction amplification, using previously obtained C. japonica expressed sequence tag (EST) information. We detected 15 nucleotide substitutions and four insertion/deletions (indels) in a partial GapC gene sequence among 13 individuals of the buried and an extant population, which allowed us to estimate the extent of DNA variation within the buried populations, and the level of genetic differentiation between the buried population and the extant population growing in a neighbouring area. For the entire haplotypes of the GapC region, pi and theta nucleotide diversity estimates were 0.0063 and 0.0010, respectively, when both populations were included, while corresponding figures for the buried population alone were 0.0009 and 0.0017. Estimates of DNA divergence statistics (dXY = 0.0062, dA = 0.0005, FST = 0.0832 and KST = 0.0935) suggest that differentiation between the two populations was not great. However, permutation tests gave FST and KST values rejecting the null hypothesis (that populations were not differentiated) at the 5% and 1% probability levels, respectively. The significant genetic differentiation between the two populations was mainly caused by differences in haplotype diversity. The significant level of haplotype diversity in the extant population compared to the buried population might be the result of gene flow from neighbouring artificial forests. Alternatively, it is possible that we failed to detect all the DNA variation in the buried population because of clonal growth in the buried population.     

Phylogeographic inferences from chloroplast DNA: quantifying the effects of mutations in repetitive and non-repetitive sequences

Phylogeographic inference can be a powerful tool in reconstructing species’ evolutionary histories; however, although inferred phylogeographic patterns should depend in part on the underlying types and rates of mutations, the effects of different types of mutations have seldom been quantified. In this study we identified two chloroplast minisatellites in the common reed Phragmites australis, and showed that these are more variable than chloroplast microsatellites. We then recreated parsimony networks of the global phylogeography of P. australis based on data that either included or excluded repetitive sequences (minisatellites and microsatellites), thereby illustrating the influence that these repetitive sequences can have on large‐scale phylogeographic inference. The resulting networks differed in the numbers of mutational steps, degrees of uncertainty, and total numbers of haplotypes. In addition, the suggested ancestor‐descendant relationships among lineages changed substantially depending on whether repetitive sequences were included. We therefore caution against the inclusion of repetitive sequences in large‐scale networks because of their high potential for homoplasy. Nevertheless, we advocate the inclusion of repetitive sequences in other analyses: specifically, we show that the ratio of mutations in repetitive vs. non‐repetitive regions can provide insight into the relative ages of lineages.     

Testing models of speciation from genome sequences: divergence and asymmetric admixture in Island South‐East Asian Sus species during the Plio‐Pleistocene climatic fluctuations

In many temperate regions, ice ages promoted range contractions into refugia resulting in divergence (and potentially speciation), while warmer periods led to range expansions and hybridization. However, the impact these climatic oscillations had in many parts of the tropics remains elusive. Here, we investigate this issue using genome sequences of three pig (Sus) species, two of which are found on islands of the Sunda‐shelf shallow seas in Island South‐East Asia (ISEA). A previous study revealed signatures of interspecific admixture between these Sus species (Genome biology, 14 , 2013, R107). However, the timing, directionality and extent of this admixture remain unknown. Here, we use a likelihood‐based model comparison to more finely resolve this admixture history and test whether it was mediated by humans or occurred naturally. Our analyses suggest that interspecific admixture between Sunda‐shelf species was most likely asymmetric and occurred long before the arrival of humans in the region. More precisely, we show that these species diverged during the late Pliocene but around 23% of their genomes have been affected by admixture during the later Pleistocene climatic transition. In addition, we show that our method provides a significant improvement over D‐statistics which are uninformative about the direction of admixture.     

Climate impacts on transocean dispersal and habitat in gray whales from the Pleistocene to 2100

Arctic animals face dramatic habitat alteration due to ongoing climate change. Understanding how such species have responded to past glacial cycles can help us forecast their response to today's changing climate. Gray whales are among those marine species likely to be strongly affected by Arctic climate change, but a thorough analysis of past climate impacts on this species has been complicated by lack of information about an extinct population in the Atlantic. While little is known about the history of Atlantic gray whales or their relationship to the extant Pacific population, the extirpation of the Atlantic population during historical times has been attributed to whaling. We used a combination of ancient and modern DNA, radiocarbon dating and predictive habitat modelling to better understand the distribution of gray whales during the Pleistocene and Holocene. Our results reveal that dispersal between the Pacific and Atlantic was climate dependent and occurred both during the Pleistocene prior to the last glacial period and the early Holocene immediately following the opening of the Bering Strait. Genetic diversity in the Atlantic declined over an extended interval that predates the period of intensive commercial whaling, indicating this decline may have been precipitated by Holocene climate or other ecological causes. These first genetic data for Atlantic gray whales, particularly when combined with predictive habitat models for the year 2100, suggest that two recent sightings of gray whales in the Atlantic may represent the beginning of the expansion of this species' habitat beyond its currently realized range.     

Historical stocking data and 19th century DNA reveal human‐induced changes to native diversity and distribution of cutthroat trout

Many species are threatened with extinction and efforts are underway worldwide to restore imperilled species to their native ranges. Restoration requires knowledge of species' historical diversity and distribution. For some species, many populations were extirpated or individuals moved beyond their native range before native diversity and distribution were documented, resulting in a lack of accurate information for establishing restoration goals. Moreover, traditional taxonomic assessments often failed to accurately capture phylogenetic diversity. We illustrate a general approach for estimating regional native diversity and distribution for cutthroat trout in the Southern Rocky Mountains. We assembled a large archive of historical records documenting human‐mediated change in the distribution of cutthroat trout (Oncorhynchus clarkii) and combined these data with phylogenetic analysis of 19th century samples from museums collected prior to trout stocking activities and contemporary DNA samples. Our study of the trout in the Southern Rocky Mountains uncovered six divergent lineages, two of which went extinct, probably in the early 20th century. A third lineage, previously declared extinct, was discovered surviving in a single stream outside of its native range. Comparison of the historical and modern distributions with stocking records revealed that the current distribution of trout largely reflects intensive stocking early in the late 19th and early 20th century from two phylogenetically and geographically distinct sources. Our documentation of recent extinctions, undescribed lineages, errors in taxonomy and dramatic range changes induced by human movement of fish underscores the importance of the historical record when developing and implementing conservation plans for threatened and endangered species.