The highest-elevation frog provides insights into mechanisms and evolution of defenses against high UV radiation.

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri. We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri. Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri. We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri. Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

From asymmetrical to balanced genomic diversification during rediploidization: Subgenomic evolution in allotetraploid fish.

A persistent enigma is the rarity of polyploidy in animals, compared to its prevalence in plants. Although animal polyploids are thought to experience deleterious genomic chaos during initial polyploidization and subsequent rediploidization processes, this hypothesis has not been tested. We provide an improved reference-quality de novo genome for allotetraploid goldfish whose origin dates to ~15 million years ago. Comprehensive analyses identify changes in subgenomic evolution from asymmetrical oscillation in goldfish and common carp to diverse stabilization and balanced gene expression during continuous rediploidization. The homoeologs are coexpressed in most pathways, and their expression dominance shifts temporally during embryogenesis. Homoeolog expression correlates negatively with alternation of DNA methylation. The results show that allotetraploid cyprinids have a unique strategy for balancing subgenomic stabilization and diversification. Rediploidization process in these fishes provides intriguing insights into genome evolution and function in allopolyploid vertebrates.

New country records for three species of frog from Myanmar including two genera (Nasutixalus and Oreolalax).

Myanmar, a biodiversity hotspot, harbors a striking diversity and endemism of species. Despite this, its herpetofauna remains one of the least explored in continental Asia due to restrictions of crossing political boundaries and infrastructure in remote regions. Many species in adjacent China and India are hypothesized to occur in Myanmar but records are wanting. Recent fieldwork found the frogs Polypedates braueri, Nasutixalus jerdonii and Oreolalax jingdongensis there, and the latter two species represent new generic records for Myanmar. All major morphological characters of these populations match the original descriptions. In addition, our matrilineal genealogy based on DNA barcoding confirms their identities. Overall, these findings confirm that the amphibian diversity is underestimated and this has important implications for conservation. Analyses indicate that northern Myanmar is a biogeographic corridor for the Himalayas, southern China, and northeastern India.

Multilocus phylogeny and cryptic diversity of white-toothed shrews (Mammalia, Eulipotyphla, Crocidura) in China.

BACKGROUND: Crocidura, the most speciose mammalian genus, occurs across much of Asia, Europe and Africa. The taxonomy of Chinese representatives has been studied primarily based on cursory morphological comparisons and their molecular phylogenetic analyses remain unexplored. In order to understand the phylogeny of this group in China, we estimated the first multilocus phylogeny and conducted species delimitation, including taxon sampling throughout their distribution range. RESULTS: We obtained one mitochondrial gene (cytb) (~ 1, 134 bp) and three nuclear genes (ApoB, BRCA1, RAG1) (~ 2, 170 bp) for 132 samples from 57 localities. Molecular analyses identified at least 14 putative species that occur within two major well-supported groups in China. Polyphyletic C. wuchihensis appears to be composed of two putative species. Two subspecies, C. rapax rapax and C. rapax kurodai should be elevated to full species status. A phylogenetic tree based on mitochondrial gene from Asian Crocidura species showed that the C. rapax rapax is embedded within C. attenuata, making the latter a paraphyletic group. Three strongly supported undescribed species (C. sp.1, C. sp.2 and C. sp.3) are revealed from Zada County of Tibet (Western China), Hongjiang County of Hunan Province (Central China) and Dongyang County of Zhejiang Province (Eastern China), Motuo County of Tibet, respectively. The divergence time estimation suggested that China's Crocidura species began to diversify during the late Pliocene (3.66 Ma) and the Early Pleistocene (2.29 Ma), followed by a series of diversifications through the Pleistocene. CONCLUSIONS: The cryptic diversity found in this study indicated that the number of species is strongly underestimated under the current taxonomy. We propose that the three undescribed species should be evaluated using extensive taxon sampling and comprehensive morphological and morphometric approaches. Climate change since the late Pliocene and the uplift of the Qinghai-Tibet Plateau may result in the diversification and speciation of China's Crocidura species. In short, the underestimated diversity underlines the need for a taxonomic revision of Chinese Crocidura species.

Historical museum collections clarify the evolutionary history of cryptic species radiation in the world's largest amphibians.

Inaccurate taxonomic assessment of threatened populations can hinder conservation prioritization and management, with human-mediated population movements obscuring biogeographic patterns and confounding reconstructions of evolutionary history. Giant salamanders were formerly distributed widely across China, and are interpreted as a single species, Andrias davidianus. Previous phylogenetic studies have identified distinct Chinese giant salamander lineages but were unable to associate these consistently with different landscapes, probably because population structure has been modified by human-mediated translocations for recent commercial farming. We investigated the evolutionary history and relationships of allopatric Chinese giant salamander populations with Next-Generation Sequencing methods, using historical museum specimens and late 20th-century samples, and retrieved partial or near-complete mitogenomes for 17 individuals. Samples from populations unlikely to have been affected by translocations form three clades from separate regions of China, spatially congruent with isolation by either major river drainages or mountain ranges. Pliocene-Pleistocene divergences for these clades are consistent with topographic modification of southern China associated with uplift of the Qinghai-Tibet Plateau. General Mixed Yule Coalescent model analysis indicates that these clades represent separate species: Andrias davidianus (Blanchard, 1871) (northern Yangtze/Sichuan), Andrias sligoi (Boulenger, 1924) (Pearl/Nanling), and an undescribed species (Huangshan). Andrias sligoi is possibly the world's largest amphibian. Inclusion of additional reportedly wild samples from areas of known giant salamander exploitation and movement leads to increasing loss of biogeographic signal. Wild Chinese giant salamander populations are now critically depleted or extirpated, and conservation actions should be updated to recognize the existence of multiple species.

CRISPR/Cas9 Application for Gene Copy Fate Survey of Polyploid Vertebrates.

Polyploidization occurs widely in eukaryotes, and especially in plants. Polyploid plants and some fishes have been commercialized. Typically, severe genomic perturbations immediately follow polyploidization and little is known about how polyploid offspring survives the genetic and epigenetic changes. Investigations into this require the identification of genes related to polyploidization and the discrimination of dosage-balance from paternal and maternal copies, and regardless of the mechanism being either autopolyploidization or allopolyploidization. New approaches and technologies may discern the mosaic of novel gene functions gained through the recombination of paternal and maternal genes in allopolyploidization. Modifications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR-associated system (Cas) protein 9 (CRISPR/Cas9) have been employed in studies of polyploidization of plants. However, the approach has seldom been applied to polyploidization in vertebrates. Herein, we use CRISPR/Cas9 to trace gene-fate in tetraploid goldfish, and specifically to identify the functional differentiation of two divergent copies of fgf20a, which are expressed differently throughout embryonic development. We expect this gene editing system will be applicable to studies of polyploids and the genetic improvement of polyploid livestock.

Phylogeny of Riama (Squamata: Gymnophthalmidae), impact of phenotypic evidence on molecular datasets, and the origin of the Sierra Nevada de Santa Marta endemic fauna.

Riama is the most speciose genus of the Neotropical lizard family Gymnophthalmidae. Its more than 30 montane species occur throughout the northern Andes, the Cordillera de la Costa (CC) in Venezuela, and Trinidad. We present the most comprehensive phylogenetic analysis of Riama to date based on a total evidence (TE) approach and direct optimization of molecular and morphological evidence. Analyses use DNA sequences from four loci and 35 phenotypic characters. The dataset consists of 55 ingroup terminals representing 25 of the 30 currently recognized species of Riama plus five undescribed taxa, including an endemic species from the Sierra Nevada de Santa Marta (SNSM) in Colombia, and 66 outgroup terminals of 47 species. Analysis results in a well-supported hypothesis in which Riama is polyphyletic, with its species falling into three clades. The Tepuian Anadia mcdiarmidi nests within one clade of Riama, and the recently resurrected Pantodactylus nests within Cercosaura. Accordingly, we propose a monophyletic taxonomy that reflects historical relationships. Analysis of character evolution indicates that the presence/absence of prefrontals-a cornerstone of the early genus-level taxonomy of cercosaurines-is optimally explained as having been plesiomorphically present in the most recent common ancestor of Cercosaurinae and lost in that of the immediately less inclusive clade. Multiple independent reversals to present and subsequent returns to absent occur within this clade. To evaluate the impact of phenotypic evidence on our results, we compare our TE results with results obtained from analyses using only molecular data. Although phenotypic evidence comprises only 1.2% of the TE matrix, its inclusion alters both the topology and support values of the clades that do not differ. Finally, current phylogenetic evidence reveals a SNSM-CC-Trinidad-tepuis biogeographical link. We hypothesize that an ancient connection facilitated the exchange of species between the SNSM and the CC.

Formal recognition of the species of Oreosaurus (Reptilia, Squamata, Gymnophthalmidae) from the Sierra Nevada de Santa Marta, Colombia.

Oreosaurus is one of the two genera extracted from the former Riamasensu lato, which was recently recognized as polyphyletic. Oreosaurus is a small clade (five named and two undescribed species) of montane gymnophthalmid lizards and exhibits an exceptional distributional pattern. Its nominal and undescribed species are discontinuously distributed on the Cordillera de la Costa of Venezuela, the tepuis from the Chimantá massif in Venezuela, the highlands of the island of Trinidad, and the Sierra Nevada de Santa Marta in Colombia (SNSM). Herein, we describe the species of Oreosaurus that is endemic to the SNSM. Historically, this species associates with two names that are currently nomina nuda: Proctoporus serranus and P. specularis. Formal nomenclatural recognition of Oreosaurus serranussp. n. renders specularis a permanently unavailable name for this taxon. Oreosaurus serranussp. n. is the sister of all remaining congeners, and differs primarily from them in having only one pair of genial scales, as well as a unique pattern of scutellation. We provide an identification key to the species of Oreosaurus.

ResumenOreosaurus es uno de los dos géneros que fueron extraídos de Riamasensu lato, el cual fue reconocido recientemente como polifilético. Oreosaurus es un clado pequeño (cinco especies nominales y dos indescritas) de gimnoftálmidos de montaña y presenta un patrón de distribución excepcional. Sus especies nominales e indescritas se distribuyen discontinuamente sobre la Cordillera de la Costa de Venezuela, los tepuyes del macizo de Chimantá en Venezuela, las tierras altas de la isla de Trinidad, y la Sierra Nevada de Santa Marta en Colombia (SNSM). Describimos la especie de Oreosaurus que es endémica de la SNSM. Históricamente, esta especie se asocia con dos nombres que son nomina nuda: Proctoporus serranus y P. specularis. El reconocimiento formal de Oreosaurus serranussp. n. hace que specularis sea un nombre permanentemente indisponible para este taxón. Oreosaurus serranussp. n. es la especie hermana de todos los congéneres restantes, y se diferencia de ellos principalmente por tener un único par de escamas geneiales, así como por presentar un patrón único de escamación. Proveemos una clave de identificación para las especies de Oreosaurus.

Genomic incompatibilities in the diploid and tetraploid offspring of the goldfish × common carp cross.

Polyploidy is much rarer in animals than in plants but it is not known why. The outcome of combining two genomes in vertebrates remains unpredictable, especially because polyploidization seldom shows positive effects and more often results in lethal consequences because viable gametes fail to form during meiosis. Fortunately, the goldfish (maternal) × common carp (paternal) hybrids have reproduced successfully up to generation 22, and this hybrid lineage permits an investigation into the genomics of hybridization and tetraploidization. The first two generations of these hybrids are diploids, and subsequent generations are tetraploids. Liver transcriptomes from four generations and their progenitors reveal chimeric genes (>9%) and mutations of orthologous genes. Characterizations of 18 randomly chosen genes from genomic DNA and cDNA confirm the chimera. Some of the chimeric and differentially expressed genes relate to mutagenesis, repair, and cancer-related pathways in 2nF1. Erroneous DNA excision between homologous parental genes may drive the high percentage of chimeric genes, or even more potential mechanisms may result in this phenomenon. Meanwhile, diploid offspring show paternal-biased expression, yet tetraploids show maternal-biased expression. These discoveries reveal that fast and unstable changes are mainly deleterious at the level of transcriptomes although some offspring still survive their genomic abnormalities. In addition, the synthetic effect of genome shock might have resulted in greatly reduced viability of 2nF2 hybrid offspring. The goldfish × common carp hybrids constitute an ideal system for unveiling the consequences of intergenomic interactions in hybrid vertebrate genomes and their fertility.

DNA barcoding and the identification of tree frogs (Amphibia: Anura: Rhacophoridae).

The DNA barcoding gene COI (cytochrome c oxidase subunit I) effectively identifies many species. Herein, we barcoded 172 individuals from 37 species belonging to nine genera in Rhacophoridae to test if the gene serves equally well to identify species of tree frogs. Phenetic neighbor joining and phylogenetic Bayesian inference were used to construct phylogenetic trees, which resolved all nine genera as monophyletic taxa except for Rhacophorus, two new matrilines for Liuixalus, and Polypedates leucomystax species complex. Intraspecific genetic distances ranged from 0.000 to 0.119 and interspecific genetic distances ranged from 0.015 to 0.334. Within Rhacophorus and Kurixalus, the intra- and interspecific genetic distances did not reveal an obvious barcode gap. Notwithstanding, we found that COI sequences unambiguously identified rhacophorid species and helped to discover likely new cryptic species via the synthesis of genealogical relationships and divergence patterns. Our results supported that COI is an effective DNA barcoding marker for Rhacophoridae.

Genetic assessments and parentage analysis of captive Bolson tortoises (Gopherus flavomarginatus) inform their "rewilding" in New Mexico.

The Bolson tortoise (Gopherus flavomarginatus) is the first species of extirpated megafauna to be repatriated into the United States. In September 2006, 30 individuals were translocated from Arizona to New Mexico with the long-term objective of restoring wild populations via captive propagation. We evaluated mtDNA sequences and allelic diversity among 11 microsatellite loci from the captive population and archived samples collected from wild individuals in Durango, Mexico (n = 28). Both populations exhibited very low genetic diversity and the captive population captured roughly 97.5% of the total wild diversity, making it a promising founder population. Genetic screening of other captive animals (n = 26) potentially suitable for reintroduction uncovered multiple hybrid G. flavomarginatus×G. polyphemus, which were ineligible for repatriation; only three of these individuals were verified as purebred G. flavomarginatus. We used these genetic data to inform mate pairing, reduce the potential for inbreeding and to monitor the maintenance of genetic diversity in the captive population. After six years of successful propagation, we analyzed the parentage of 241 hatchlings to assess the maintenance of genetic diversity. Not all adults contributed equally to successive generations. Most yearly cohorts of hatchlings failed to capture the diversity of the parental population. However, overlapping generations of tortoises helped to alleviate genetic loss because the entire six-year cohort of hatchlings contained the allelic diversity of the parental population. Polyandry and sperm storage occurred in the captives and future management strategies must consider such events.

Genealogy and Demographic History of a Widespread Amphibian throughout Indochina.

Relatively little is known about spatial patterns of cryptic diversity in tropical species and the processes that generate them. Few studies examine the geographic distribution of genetic lineages in Southeast Asia, an area hypothesized to harbor substantial cryptic diversity. We investigated the evolutionary history of Asian tree frogs of the Polypedates leucomystax complex (n = 172) based on 1800 bp of the mtDNA genes ND1 and cytochrome b and tested hypotheses pertaining to climate, geology, and dispersal patterns. Analyses revealed substantial genetic diversity and lineage divergence throughout the region with evidence for widespread sympatric lineages and a general north versus south clustering. Relaxed molecular clock analysis and tests for demographic expansion identified an initial cladogenesis during the Miocene with subsequent Plio-Pleistocene diversification, with the former corresponding to periods of increased aridity and the onset of monsoonal weather systems. Rates of diversification were relatively constant until the Early Pleistocene when rates increased exponentially. We found equivocal evidence for isolation-by-distance and a potential role of some landscape features as partial barriers to dispersal. Finally, our analyses showed that divergence between insular and mainland populations occurred before Homo sapiens colonized Southeast Asia, suggesting that historical human-mediated dispersal did not drive insular diversification. Our results suggested that demographic expansion in the Late Pleistocene resulted in widespread sympatric lineages in the P. leucomystax complex throughout southern China and Indochina and further clarified the evolutionary history of lineages within P. leucomystax.

Identification of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance.

Stress proteins such as metallothioneins (MTs) play a key role in cellular protection against environmental stressors. In nature, insects such as houseflies (Musca domestica) are commonly exposed to multiple stressors including heavy metals (e.g. Cadmium, Cd) and high temperatures. In this paper, we identify two novel MT genes from the cDNAs of M. domestica, MdMT1 and MdMT2, which putatively encode 40 and 42 amino acid residues respectively. Expression of the two MTs' mRNAs, which are examined in the fat body, gut, hemocyte, and the epidermis. From our study, we saw that the expression of MdMT1 and MdMT2 are enhanced by Cd and thermal stress. Levels of expression are highest at 10 mM Cd(2+) within a 24-h period, and expressions increase significantly with exposure to 10 mM Cd for 12h. Levels of the mRNAs are up-regulated after heat shock and that of MdMT2 reaches its maximum peak faster than MdMT1. Both of the MT genes might be involved in a transient systemic tolerance response to stressors and they may play important roles in heavy metal and high temperature tolerance in the housefly. To detect whether or not the MTs bind heavy metals, the target genes are cloned into the prokaryotic expression vector pET-DsbA to obtain fusion protein expressed in Escherichia coli BL21 (DE3). Recombinant DsbA-MdMT1 significantly increases tolerance of the host bacteria to Cd(2+), but DsbA-MdMT2 is absent. These differential characteristics will facilitate future investigations into the physiological functions of MTs.

A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae.

BACKGROUND: The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous. RESULTS: We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades: ((((Gallus, Bambusicola) Francolinus) (Coturnix, Alectoris)) Pavo) and (((((((Chrysolophus, Phasianus) Lophura) Syrmaticus) Perdix) Pucrasia) (Meleagris, Bonasa)) ((Lophophorus, Tetraophasis) Tragopan))). CONCLUSIONS: The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution.

New insights to the molecular phylogenetics and generic assessment in the Rhacophoridae (Amphibia: Anura) based on five nuclear and three mitochondrial genes, with comments on the evolution of reproduction.

The phylogenetic relationships among 12 genera of treefrogs (Family, Rhacophoridae), were investigated based on a large sequence data set, including five nuclear (brain-derived neurotrophic factor, proopiomelanocortin, recombination activating gene 1, tyrosinase, rhodopsin) and three mitochondrial (partial 12S and 16S ribosomal RNA and the complete valine t-RNA) genes. Phylogenetic analysis of the nuclear gene sequences resolved three major clades. The first group included Philautus, Pseudophilautus, Kurixalus, Gracixalus, and Theloderma moloch; Pseudophilautus and Kurixalus were sister taxa. The second group consisted of Nyctixalus and Theloderma. The third group contained Feihyla, Polypedates, Rhacophorus, and Chiromantis vittatus; Polypedates and Feihyla were sister taxa. Analyses of the nuclear and mitochondrial genes supported the following results: (1) Genus Liuixalus formed the sister group of all other rhacophorines. (2) Philautus, Theloderma, and Chiromantis were not resolved as monophyletic genera. Four groups, including Philautus ocellatus and P. hainanus, P. longchuanensis and P. gryllus, P. banaensis, and P. quyeti nested well within the genera Liuixalus, Pseudophilautus, Kurixalus, and Gracixalus, respectively. (3) Theloderma moloch and Chiromantis vittatus did not cluster with other species of Theloderma and Chiromantis, respectively. Foam nesting evolved only once, as did laying eggs in a jelly-like matrix containing some bubbles. Terrestrial direct development evolved twice in the Rhacophoridae.