Claudio Verdugo

A genetic signature of the evolution of loss of flight in the Galapagos cormorant

Loss of flight in the Galapagos cormorant Although rare among existing birds, the loss of flight appears to have occurred multiple times in evolutionary history. However, the genetic changes that ground avian species are not well understood. Burga et al. sequenced genomes from three cormorant species and compared them with that of the flightless Galapagos cormorant (see the Perspective by Cooper). They identified variants in genes involved in primary ciliogenesis. Functional analyses of these variants suggest that the impaired function of the genes may be responsible for skeletal changes associated with the Galapagos cormorant’s loss of flight. Science, this issue p. eaal3345; see also p. 904 Genomic analysis points to CUX1 and cilia-related genes likely underlying a loss of flight in the Galapagos cormorant. INTRODUCTION Changes in the size and proportion of limbs and other structures have played a key role in the evolution of species. One common class of limb modification is recurrent wing reduction and loss of flight in birds. Indeed, Darwin used the occurrence of flightless birds as an argument in favor of his theory of natural selection. Loss of flight has evolved repeatedly and is found among 26 families of birds in 17 different orders. Despite the frequency of these modifications, we have a limited understanding of their underpinnings at the genetic and molecular levels. RATIONALE To better understand the evolution of changes in limb size, we studied a classic case of recent loss of flight in the Galapagos cormorant (Phalacrocorax harrisi). Cormorants are large water birds that live in coastal areas or near lakes, and P. harrisi is the only flightless cormorant among approximately 40 extant species. The entire population is distributed along the coastlines of Isabela and Fernandina islands in the Galapagos archipelago. P. harrisi has a pair of short wings, which are smaller than those of any other cormorant. The extreme reduction of the wings and pectoral skeleton observed in P. harrisi is an attractive model for studying the evolution of loss of flight because it occurred very recently; phylogenetic evidence suggests that P. harrisi diverged from its flighted relatives within the past 2 million years. We developed a comparative and predictive genomics approach that uses the genome sequences of P. harrisi and its flighted relatives to find candidate genetic variants that likely contributed to the evolution of loss of flight. RESULTS We sequenced and de novo assembled the whole genomes of P. harrisi and three closely related flighted cormorant species. We identified thousands of coding variants exclusive to P. harrisi and classified them according to their probability of altering protein function based on conservation. Variants most likely to alter protein function were significantly enriched in genes mutated in human skeletal ciliopathies, including Ofd1, Evc, Wdr34, and Ift122. We carried out experiments in Caenorhabditis elegans to confirm that a missense variant present in the Galapagos cormorant IFT122 protein is sufficient to affect ciliary function. The primary cilium is essential for Hedgehog (Hh) signaling in vertebrates, and individuals affected by ciliopathies have small limbs and ribcages, mirroring the phenotype of P. harrisi. We also identified a 4–amino acid deletion in the regulatory domain of Cux1, a highly conserved transcription factor that has been experimentally shown to regulate limb growth in chicken. The four missing amino acids are perfectly conserved in all birds and mammals sequenced to date. We tested the consequences of this deletion in a chondrogenic cell line and showed that it impairs the ability of CUX1 to transcriptionally up-regulate cilia-related genes (some of which contain function-altering variants in P. harrisi) and to promote chondrogenic differentiation. Finally, we show that positive selection may have played a role in the fixation of the variants associated with loss of flight in P. harrisi. CONCLUSION Our results indicate that the combined effect of variants in genes necessary for the correct transcriptional regulation and function of the primary cilium likely contributed to the evolution of highly reduced wings and other skeletal adaptations associated with loss of flight in P. harrisi. Our approach may be generally useful for identification of variants underlying evolutionary novelty from genomes of closely related species. Comparative and predictive genomics of loss of flight. Comparison of the genomes of four closely related cormorant species allowed us to predict function-altering variants exclusively affecting the Galapagos cormorant and to test their functional consequences. Our results implicate ciliary dysfunction as a likely contributor to the evolution of loss of flight. We have a limited understanding of the genetic and molecular basis of evolutionary changes in the size and proportion of limbs. We studied wing and pectoral skeleton reduction leading to flightlessness in the Galapagos cormorant (Phalacrocorax harrisi). We sequenced and de novo assembled the genomes of four cormorant species and applied a predictive and comparative genomics approach to find candidate variants that may have contributed to the evolution of flightlessness. These analyses and cross-species experiments in Caenorhabditis elegans and in chondrogenic cell lines implicated variants in genes necessary for transcriptional regulation and function of the primary cilium. Cilia are essential for Hedgehog signaling, and humans affected by skeletal ciliopathies suffer from premature bone growth arrest, mirroring skeletal features associated with loss of flight.

Novel Avulaviruses in Penguins, Antarctica

We identified 3 novel and distinct avulaviruses from Gentoo penguins sampled in Antarctica. We isolated these viruses and sequenced their complete genomes; serologic assays demonstrated that the viruses do not have cross-reactivity between them. Our findings suggest that these 3 new viruses represent members of 3 novel avulavirus species.

Molecular divergence between insular and continental Pudu deer (Pudu puda) populations in the Chilean Patagonia

Island–continent isolation scenarios have played major roles in the understanding and development of evolutionary theories. During the last glacial maximum (LGM) in southern Chile, ice sheet advances and retreats formed the Patagonian archipelago along the southern Pacific Ocean. Chiloé Island is the largest island from the archipelago isolated from the continent by a narrow and shallow stretch of ocean. Pudu puda is a species endemic to Chile and part of Argentina, distributed along the Valdivian Temperate rainforest. We used mtDNA control region (654 bp) and cytochrome b (734 bp) sequences to understand the consequences in the evolutionary history and population structure of Pudu puda island–continent isolation. Bayesian and Maximum Likelihood phylogenetic analysis revealed two divergent clades corresponding to the continent and Chiloé Island. The Median Joining Network also supports these findings with an isolation of 10 mutational steps between Chiloé and the continent. We also found a significant high genetic structure (Φst = 0.75) and a sequence divergence percentage of 2.3% between the two clades. On the other hand, on the continent we found high haplotype genetic diversity (H = 0.9790 ± 0.0103) but no clear geographical population structure or phylogenetic clades. Our results suggest that the southern Pudu deer populations were isolated since the interglacial period (less than 0.5 million years ago) from the continent leading to two reciprocally monophyletic clades. We propose two subspecies to be considered in the development of future conservation programs for the species.

Postmortem findings in four south American sea lions (Otaria byronia) from an urban colony in Valdivia, Chile.

Abstract We performed postmortem examination on four South American sea lions (Otaria byronia) from an urban colony in Valdivia, Chile. Chronic leptospirosis and suspected morbillivirus-like infection were diagnosed in one individual. Antibodies against Toxoplasma gondii and the zoonotic helminthes Contracaecum sp., Pseudoterranova sp., and Diphyllobothrium sp. were also detected.

Infection with Borrelia chilensis in Ixodes stilesi ticks collected from Pudu puda deer

Lyme borreliosis is a vector-borne zoonosis caused by Borrelia burgdorferi sensu lato species complex spirochetes, which are maintained in transmission cycles among vertebrates and Ixodes ticks. Recently, a new genospecies within this complex, Borrelia chilensis, was described in Ixodes stilesi collected from the environment and from rodents in Chile. This tick also infests the native Southern pudu deer (Pudu puda). The objectives of this study were to investigate the prevalence, intensity of infestation, and aggregation of hard ticks on this deer species, and to determine the presence of borrelial pathogens in the ticks. Sixty-six deer were examined over a two-year period. A total of 179 ticks of two species, I. stilesi and Ixodes taglei, were collected. Of those, 100 were adults, 78 were nymphs, and one was a larva. Ixodes stilesi was the most prevalent tick (47%) and was highly aggregated (D=0.77) on the deer. Deer body weight was positively associated with tick burden. Borrelia spirochetes were detected in two (6.45%) of the examined I. stilesi ticks. Phylogenetic analyses of 16S and flaB gene sequences positioned these samples in the same clade with Borrelia chilensis VA1 previously described from Chile. These findings suggest that I. stilesi may play a role in the local persistence of B. chilensis. Further studies are required to fully understand the mechanisms of natural transmission of B. chilensis and the risk of infection in humans.

Sarcoptic Mange in a South American Gray Fox (Chilla Fox; Lycalopex griseus), Chile

Abstract Mange, a prevalent disease of dogs in Chile, is also a serious threat to wildlife. We report a case of sarcoptic mange in a South American gray fox or chilla fox (Lycalopex griseus). Further research is needed to understand the impact of mange in wildlife populations.

Respiratory Mites (Orthohalarachne diminuata) and β-hemolytic Streptococci-Associated Bronchopneumonia Outbreak in South American Fur Seal Pups (Arctocephalus australis)

Abstract:  Although mites of the Orthohalarachne genus are common parasites of otariids, their role as agents of disease and in causing population-level mortality is unknown. In the austral summer of 2016, there was an increase in mortality among South American fur seal (Arctocephalus australis) pups at Guafo Island, Northern Chilean Patagonia. Pups found dead or terminally ill had moderate to marked, multifocal, mucopurulent bronchopneumonia associated with large numbers of respiratory mites (Orthohalarachne diminuata) and rare Gram-positive cocci. In lung areas less affected by bronchopneumonia, acute interstitial pneumonia with marked congestion and scant hemorrhage was evident. Bacteria from pups dying of bronchopneumonia were isolated and identified as Streptococcus marimammalium and Streptococcus canis. Respiratory mites obstructed airflow, disrupted airway epithelial lining, and likely facilitated the proliferation of pathogenic β-hemolytic streptococci, leading to severe bronchopneumonia and death of fur seal pups. An abrupt increase in sea surface temperature in Guafo Island corresponded to the timing of the bronchopneumonia outbreak. The potential role of environmental factors in the fur seal pup mortality warrants further study.

MOLECULAR IDENTIFICATION OF AVIAN VIRUSES IN NEOTROPIC CORMORANTS (PHALACROCORAX BRASILIANUS) IN CHILE

Abstract We identified two RNA (paramyxovirus and coronavirus) and two DNA (adenovirus and herpesvirus) viruses in a common aquatic bird, the Neotropic Cormorant (Phalacrocorax brasilianus), and determined their phylogenetic relationships to other global circulating variants. We analyzed 104 cloacal swabs from individuals collected at locations in Central Chile. Sequences were obtained from amplicons using consensus primers targeting conserved genes of the virus families Paramyxoviridae, Coronaviridae, Adenoviridae, and Herpesviridae. A total of 20.2% of the samples was positive for coronavirus, 8.7% for adenovirus, and 3.8% for herpesvirus. No paramyxoviruses were detected. All coronaviruses were identified as viruses of the Gammacoronavirus genus, closely related to the infectious bronchitis virus clade (bootstrap clade support=75%). All adenovirus samples were identified as Aviadenovirus, related to a gull and falcon adenovirus (Bayesian posterior probability=0.86). The herpesviruses identified were related to the infectious laryngotracheitis virus (Gallid herpesvirus 1) of the genus Iltovirus (bootstrap clade support=99%). We provide information about the diversity of viruses circulating among apparently healthy Neotropic Cormorants.

Complete mitochondrial genome of the Neotropic cormorant (Phalacrocorax brasilianus)

Abstract The complete sequence of the Neotropic cormorant (Phalacrocorax brasilianus) mitochondrial DNA was obtained by the shotgun sequencing approach. The mitogenome is 19 042 bp in length and includes 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNA genes, a control region and a duplicated region of 2418 bp. The base composition is 32.1% for C, 31.8% for A, 22.6% for T and 13.4% for G, with an overall GC content of 45.5%. This is the first mitogenome of the P. brasilianus described and will be a useful tool for further phylogenetic and population genetic studies.

Avian tuberculosis in a Lady Amherst’s pheasant Chrysolophus amherstiae

espanolRESUMEN. Reportamos un caso de un faisan Lady Amherst (Chrysolophus amherstiae) con lesiones granulomatosas difusas en higado, bazo e intestino. Usando diagnostico histopatologico y molecular detectamos Mycobacterium avium subsp. avium, agente etiologico de tuberculosis aviar. Se requieren mas estudios para entender el rol de aves silvestres en la persistencia y transmision del patogeno. EnglishABSTRACT. We report a case of a Lady Amherst’s pheasant (Chrysolophus amherstiae) with diffuse granulomatous lesions in liver, spleen and intestine. Using histopathological and molecular diagnosis we detected Mycobacterium avium subsp. avium, etiological agent of avian tuberculosis. Further studies are needed to understand the role of wild birds in the persistence and transmission of this pathogen.