Phylogeography and evolution of a fungal–insect association on the Tibetan Plateau

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high‐frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis–ghost moth parasitoidism on the Tibetan Plateau.     

Two hypotheses of the evolution of endemic sponges in Lake Baikal (Lubomirskiidae)

Complete and nearly complete mtDNA genome sequences were used to resolve differences between two palaeontology‐based hypotheses on timing of the origin of the Baikal endemic sponge family Lubomirskiidae (Haplosclerida, Demospongiae). Bayesian ratio test, when coupled with estimates of substitution rates based on known palaeontological findings, provided strong evidence for the Miocene origin over the Late Oligocene one. The common ancestor of the present day sponges in Lake Baikal diverged about 2,3 million years ago (Ma), while Lubomirskia baicalensis, Rezinkovia echinata, Baikalospongia intermedia profundalis and B. bacillifera split in the Pleistocene about 0,7 Ma. A phylogenetic analysis within the family suggested that speciation may have coincided with the occurrence of cold climatic conditions. We argue that the cause of speciation may be niche splitting due to temperature, depth and possibly feeding preferences.     

Resequencing and comparison of whole mitochondrial genome to gain insight into the evolutionary status of the Shennongjia golden snub‐nosed monkey (SNJ R. roxellana)

Shennongjia Rhinopithecus roxellana (SNJ R. roxellana) is the smallest geographical population of R. roxellana. The phylogenetic relationships among its genera and species and the biogeographic processes leading to their current distribution are largely unclear. To address these issues, we resequenced and obtained a new, complete mitochondrial genome of SNJ R. roxellana by next‐generation sequencing and standard Sanger sequencing. We analyzed the gene composition, constructed a phylogenetic tree, inferred the divergence ages based on complete mitochondrial genome sequences, and analyzed the genetic divergence of 13 functional mtDNA genes. The phylogenetic tree and divergence ages showed that R. avunculus (the Tonkin snub‐nosed monkey) was the first to diverge from the Rhinopithecus genus ca. 2.47 million years ago (Ma). Rhinopithecus bieti and Rhinopithecus strykeri formed sister groups, and the second divergence from the Rhinopithecus genus occurred ca. 1.90 Ma. R. roxellana and R. brelichi diverged from the Rhinopithecus genus third, ca. 1.57 Ma. SNJ R. roxellana was the last to diverge within R. roxellana species in 0.08 Ma, and the most recent common ancestor of R. roxellana is 0.10 Ma. The analyses on gene composition showed SNJ R. roxellana was the newest geographic population of R. roxellana. The work will help to develop a more accurate protection policy for SNJ R. roxellana and facilitate further research on selection and adaptation of R. roxellana.     

Diploid hybrid origin of Ostryopsis intermedia (Betulaceae) in the Qinghai‐Tibet Plateau triggered by Quaternary climate change

Despite the well‐known effects that Quaternary climate oscillations had on shaping intraspecific diversity, their role in driving homoploid hybrid speciation is less clear. Here, we examine their importance in the putative homoploid hybrid origin and evolution of Ostryopsis intermedia, a diploid species occurring in the Qinghai‐Tibet Plateau (QTP), a biodiversity hotspot. We investigated interspecific relationships between this species and its only other congeners, O. davidiana and O. nobilis, based on four sets of nuclear and chloroplast population genetic data and tested alternative speciation hypotheses. All nuclear data distinguished the three species clearly and supported a close relationship between O. intermedia and the disjunctly distributed O. davidiana. Chloroplast DNA sequence variation identified two tentative lineages, which distinguished O. intermedia from O. davidiana; however, both were present in O. nobilis. Admixture analyses of genetic polymorphisms at 20 SSR loci and sequence variation at 11 nuclear loci and approximate Bayesian computation (ABC) tests supported the hypothesis that O. intermedia originated by homoploid hybrid speciation from O. davidiana and O. nobilis. We further estimated that O. davidiana and O. nobilis diverged 6–11 Ma, while O. intermedia originated 0.5–1.2 Ma when O. davidiana is believed to have migrated southward, contacted and hybridized with O. nobilis possibly during the largest Quaternary glaciation that occurred in this region. Our findings highlight the importance of Quaternary climate change in the QTP in causing hybrid speciation in this important biodiversity hotspot.     

A high frequency of allopolyploid speciation in the gymnospermous genus Ephedra and its possible association with some biological and ecological features

The origin and evolution of polyploids have been studied extensively in angiosperms and ferns but very rarely in gymnosperms. With the exception of three species of conifers, all natural polyploid species of gymnosperms belong to Ephedra, in which more than half of the species show polyploid cytotypes. Here, we investigated the origin and evolution of polyploids of Ephedra distributed in the Qinghai–Tibetan Plateau (QTP) and neighbouring areas. Flow cytometry (FCM) was used to measure the ploidy levels of the sampled species that are represented by multiple individuals from different populations, and then, two single‐copy nuclear genes (LFY and DDB2) and two chloroplast DNA fragments were used to unravel the possible origins and maternal donors of the polyploids. The results indicate that the studied polyploid species are allopolyploids, and suggest that allotetraploidy is a dominant mode of speciation in Ephedra. The high percentage of polyploids in the genus could be related to some of its biological attributes such as vegetative propagation, a relatively high rate of unreduced gamete formation, and a small genome size relative to most other gymnosperms. Significant ecological divergences between allotetraploids and their putative progenitors were detected by PCAs and anova and Tukey's tests, with the exception of E. saxatilis. The overlap of geographical distributions and ecological niches of some diploid species could have provided opportunities for interspecific hybridization and allopolyploid speciation.     

Inferring the mode of origin of polyploid species from next‐generation sequence data

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next‐generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa‐pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole‐genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels.     

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs

Subdivided Pleistocene glacial refugia, best known as “refugia within refugia”, provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and “ghost” mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD‐sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply‐diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation‐by‐distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.     

Assessing the monophyly of polyploid Gossypium species

The origin and monophyly of the polyploid cotton (Gossypium) species has been largely accepted, despite the lack of explicit phylogenetic evidence. Recent studies in other polyploid systems have demonstrated that multiple origins for polyploid species are much more common than once thought, raising the possibility that Gossypium polyploids also had multiple origins, as postulated by some authors. To test the monophyly of polyploid cotton, we sequenced a 2.8-kb intergenic region from all diploid species belonging to the genome groups from which the polyploid originates. The resulting phylogenetic analyses strongly support a single origin of polyploid cotton involving a D-genome ancestor related to Gossypium raimondii and an A-genome ancestor that was sister to both extant A-genome species.     

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B. nigra mitochondrial main circle genome with 232,407 bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B. rapa and B. oleracea had a better syntenic relationship than B. nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B. napus, in which napus‐type B. napus was derived from B. oleracea, while polima‐type B. napus was inherited from B. rapa. In addition, the mitochondrial genome of napus‐type B. napus was closer to botrytis‐type than capitata‐type B. oleracea. The sub‐stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.     

Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate?

Summary. The cytology of leaf cells from five different high-alpine plants was studied and compared with structures in chloroplasts from the typical high-alpine plant Ranunculus glacialis previously described as having frequent envelope plus stroma protrusions. The plants under investigation ranged from subalpine/alpine Geum montanum through alpine Geum reptans, Poa alpina var. vivipara, and Oxyria digyna to nival Cerastium uniflorum and R. glacialis. The general leaf structure (by light microscopy) and leaf mesophyll cell ultrastructure (by transmission electron microscopy [TEM]) did not show any specialized structures unique to these mountain species. However, chloroplast protrusion formation could be found in G. reptans and, to a greater extent, in O. digyna. The other species exhibited only a low percentage of such chloroplast structural changes. Occurrence of protrusions in samples of G. montanum and O. digyna growing in a mild climate at about 50 m above sea level was drastically reduced. Serial TEM sections of O. digyna cells showed that the protrusions can appear as rather broad and long appendices of plastids, often forming pocketlike structures where mitochondria and microbodies are in close vicinity to the plastid and to each other. It is suggested that some high-alpine plants may form such protrusions to facilitate fast exchange of molecules between cytoplasm and plastid as an adaptation to the short, often unfavorable vegetation period in the Alps, while other species may have developed different types of adaptation that are not expressed in ultrastructural changes of the plastids. Correspondence and reprints: Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Postglacial climate changes and rise of three ecotypes of harbour porpoises,Phocoena phocoena,in western Palearctic waters

Despite no obvious barriers to gene flow in the marine realm, environmental variation and ecological specializations can lead to genetic differentiation in highly mobile predators. Here, we investigated the genetic structure of the harbour porpoise over the entire species distribution range in western Palearctic waters. Combined analyses of 10 microsatellite loci and a 5085 base‐pair portion of the mitochondrial genome revealed the existence of three ecotypes, equally divergent at the mitochondrial genome, distributed in the Black Sea (BS), the European continental shelf waters, and a previously overlooked ecotype in the upwelling zones of Iberia and Mauritania. Historical demographic inferences using approximate Bayesian computation (ABC) suggest that these ecotypes diverged during the last glacial maximum (c. 23–19 kilo‐years ago, kyrbp ). ABC supports the hypothesis that the BS and upwelling ecotypes share a more recent common ancestor (c. 14 kyrbp ) than either does with the European continental shelf ecotype (c. 28 kyrbp ), suggesting they probably descended from the extinct populations that once inhabited the Mediterranean during the glacial and post‐glacial period. We showed that the two Atlantic ecotypes established a narrow admixture zone in the Bay of Biscay during the last millennium, with highly asymmetric gene flow. This study highlights the impacts that climate change may have on the distribution and speciation process in pelagic predators and shows that allopatric divergence can occur in these highly mobile species and be a source of genetic diversity.     

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice

The related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon‐like population, referred to as Taxon A, and O. meridionalis‐like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short‐ and long‐read next‐generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement.     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.     

Epidermal UV-screening of arctic and alpine plants along a latitudinal gradient in Europe

This study reports epidermal UV-transmittance in field-grown leaves of ecotypes of six species at three sites along a latitudinal UV-B gradient from Arctic Svalbard, via southern Norway to the French Alps for the years 1999–2001. Unexpectedly, Arctic populations had just as high epidermal UV-screening as alpine populations from lower latitudes. Dryas octopetala was the only species that significantly increased epidermal screening with increasing natural UV-B. Most species, however, showed clear differences in transmittance between years.Under controlled conditions in a growthroom, no ecotypic differences with respect to epidermal UV-B screening were found in Arctic and alpine ecotypes of Oxyria digyna, either in the absence or presence of UV-B radiation. Furthermore, UV-B transmittance in the absence of UV-B radiation in the growthroom was as low (5–6%) as in field-grown plants, indicating a high constitutive screening. Analysis of UV-B-absorbing phenolic compounds in O. digyna displayed no difference between the French Alps and Svalbard ecotypes, while the S. Norway ecotype contained significantly higher amounts of screening compounds. The qualitative analysis showed that the French Alps ecotype had a different composition of flavonoids compared with the two others, and that the ratio between di- and monohydroxylated flavonoids increased from south to north.     

Pleistocene Chinese cave hyenas and the recent Eurasian history of the spotted hyena,Crocuta crocuta

The living hyena species (spotted, brown, striped and aardwolf) are remnants of a formerly diverse group of more than 80 fossil species, which peaked in diversity in the Late Miocene (about 7–8 Ma). The fossil history indicates an African origin, and morphological and ancient DNA data have confirmed that living spotted hyenas (Crocuta crocuta) of Africa were closely related to extinct Late Pleistocene cave hyenas from Europe and Asia. The current model used to explain the origins of Eurasian cave hyena populations invokes multiple migrations out of Africa between 3.5–0.35 Ma. We used mitochondrial DNA sequences from radiocarbon‐dated Chinese Pleistocene hyena specimens to examine the origin of Asian populations, and temporally calibrate the evolutionary history of spotted hyenas. Our results support a far more recent evolutionary timescale (430–163 kya) and suggest that extinct and living spotted hyena populations originated from a widespread Eurasian population in the Late Pleistocene, which was only subsequently restricted to Africa. We developed statistical tests of the contrasting population models and their fit to the fossil record. Coalescent simulations and Bayes Factor analysis support the new radiocarbon‐calibrated timescale and Eurasian origins model. The new Eurasian biogeographic scenario proposed for the hyena emphasizes the role of the vast steppe grasslands of Eurasia in contrast to models only involving Africa. The new methodology for combining genetic and geological data to test contrasting models of population history will be useful for a wide range of taxa where ancient and historic genetic data are available.     

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha‐proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early‐diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein‐coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.     

Colonization of islands in the Mona Passage by endemic dwarf geckoes (genus Sphaerodactylus) reconstructed with mitochondrial phylogeny

Little is known about the natural history of the Sphaerodactylus species endemic to the three islands located in the Mona Passage separating the Greater Antillean islands of Hispaniola and Puerto Rico. In this study, parts of two mitochondrial genes, 16S rRNA and 12S rRNA, were sequenced to determine the relationships between the sphaerodactylids that live in the Mona Passage and other Caribbean species from the same genus. While the main goal was to identify the biogeographical origin of these species, we also identified a genetically distinct type of dwarf gecko that warrants future evaluation as a possible new species. According to the reconstructed phylogenies, we propose a stepwise model of colonization wherein S. nicholsi from southwestern Puerto Rico or a very close ancestor gave rise through a founder event to Sphaerodactylus monensis on Mona Island. In a similar fashion, S. monensis or a very close ancestor on Mona Island gave rise to S. levinsi on Desecheo Island. This study also suggests that the most recent common ancestor between the species from the islands in the Mona Passage and Puerto Rico existed approximately 3 MYA.