Report Evolutionary History, Genomic Adaptation to Toxic Diet, and Extinction of the Carolina Parakeet Highlights Authors d First whole genome from the extinct Carolina parakeet and Pere Gelabert, the sun parakeet Marcela Sandoval-Velasco, Aitor Serres, ..., Agostinho Antunes, d Divergence time between Conuropsis and Aratinga around 3 M. Thomas P. Gilbert, mya Carles Lalueza-Fox d Evidence for potential adaptation to toxic diet in two extremely conserved proteins Correspondence tgilbert@bio.ku.dk (M.T.P.G.), d No signs of inbreeding in the Carolina parakeet suggest the carles.lalueza@upf.edu (C.L.-F.) extinction was abrupt In Brief Gelabert et al. report the whole genome of the extinct Carolina parakeet and provide evidence of its phylogeny, adaptation to a toxic cocklebur diet, and demographic history. The lack of signs of recent inbreeding typically found in endangered species suggests its abrupt extinction was human mediated. Gelabert et al., 2020, Current Biology 30, 108–114 January 6, 2020 ª 2019 The Author(s). Published by Elsevier Ltd. https://doi.org/10.1016/j.cub.2019.10.066 Current Biology Report Evolutionary History, Genomic Adaptation to Toxic Diet, and Extinction of the Carolina Parakeet Pere Gelabert,1,2 Marcela Sandoval-Velasco,3 Aitor Serres,1 Marc de Manuel,1 Pere Renom,1 Ashot Margaryan,3 Josefin Stiller,4 Toni de-Dios,1 Qi Fang,5 Shaohong Feng,5 Santi Mañosa,6 George Pacheco,3 Manuel Ferrando-Bernal,1 Guolin Shi,7 Fei Hao,7 Xianqing Chen,8 Bent Petersen,3,9 Remi-Andre  Olsen,10 Arcadi Navarro,1,11,12 Yuan Deng,5 Love Dalen,13 Tomàs Marquès-Bonet,1,11,12,14 Guojie Zhang,4,15,16,17 Agostinho Antunes,18,19 M. Thomas P. Gilbert,3,20,* and Carles Lalueza-Fox1,21,* 1Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Dr. Aiguader 88, 08003 Barcelona, Spain 2Department of Evolutionary Anthropology, University of Vienna, Althanstraße 14, 1090 Vienna, Austria 3Section for Evolutionary Genomics, The GLOBE Institute, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen, Denmark 4Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark 5BGI-Shenzhen, Beishan Industrial Zone, Building 11, Shenzhen 518083, China 6Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Facultat de Biologia, Avinguda Diagonal 643, 08028 Barcelona, Spain 7Center of Special Environmental Biomechanics & Biomedical Engineering, School of Life Sciences, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China 8Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China 9Centre of Excellence for Omics-Driven Computational Biodiscovery (COMBio), Faculty of Applied Sciences, AIMST University, Semeling Road, 08100 Kedah, Malaysia 10ScieLifeLab, Department of Biochemistry and Biophysics, Stockholm University, Frescativa €gen 40, SE-17121 Solna, Sweden 11Catalan Institution of Research and Advanced Studies (ICREA), Passeig de Lluı́s Companys 23, 08010 Barcelona, Spain 12CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Baldiri i Reixac 4, 08036 Barcelona, Spain 13Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Frescativa €gen 40, 10405 Stockholm, Sweden 14Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, c. de les Columnes s/n, 08193 Cerdanyola del Vallès, Barcelona, Spain 15China National GeneBank, BGI-Shenzhen, Jinsha Road, Shenzhen 518120, China 16State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming 650223, China 17Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming 650223, China 18CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto, Portugal 19Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal 20NTNU University Museum, Erling Skakkes gate 47c, 7012 Trondheim, Norway 21Lead Contact *Correspondence: tgilbert@bio.ku.dk (M.T.P.G.), carles.lalueza@upf.edu (C.L.-F.) https://doi.org/10.1016/j.cub.2019.10.066 SUMMARY specimen held in a private collection in Espinelves (Gi- rona, Spain), as well as of a close extant relative, Ara- As the only endemic neotropical parrot to have tinga solstitialis. We identified two non-synonymous recently lived in the northern hemisphere, the Carolina genetic changes in two highly conserved proteins parakeet (Conuropsis carolinensis) was an iconic known to interact with CAT that could underlie a spe- North American bird. The last surviving specimen cific dietary adaptation to this toxin. Our genomic ana- died in the Cincinnati Zoo in 1918 [1]. The cause of its lyses did not reveal evidence of a dramatic past demo- extinction remains contentious: besides excessive graphic decline in the Carolina parakeet; also, its mortality associated to habitat destruction and active genome did not exhibit the long runs of homozygosity hunting, their survival could have been negatively that are signals of recent inbreeding and are typically affected by its range having become increasingly found in endangered species. As such, our results patchy [2] or by the exposure to poultry pathogens suggest its extinction was an abrupt process and [3, 4]. In addition, the Carolina parakeet showed a pre- thus likely solely attributable to human causes. dilection for cockleburs, an herbaceous plant that con- tains a powerful toxin, carboxyatractyloside, or CAT RESULTS [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To The Carolina Parakeet and the Sun Parakeet Genomes explore the demographic history of this bird, we gener- Given that de novo genome assembly is impractical with the typi- ated the complete genomic sequence of a preserved cally short and degraded DNA found in historic and ancient 108 Current Biology 30, 108–114, January 6, 2020 ª 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Figure 1. Phylogenetic Relationships of the Carolina Parakeet Calibrated phylogeny built with BEAST2 based on 50 nuclear UCE loci from 18 species of parrots and a passerine (common names in bold). The analysis was constrained to a topology obtained from maximum likelihood analysis of 4,988 nuclear loci (9,864,148 bp), in which all nodes had 100 bootstrap support. Node ages were estimated using two fossil calibrations (highlighted in blue HPD intervals); gray bars indicate 95% HPD in- tervals of unconstrained nodes. Clade names follow a recent nomenclature revision [23]. See also Figure S2. substitutions in all genes (dN/dS) is 0.48, and values similar to these have been reported in other bird genomes [15–17]. Sex determination of the specimens is difficult using morphological observa- tions alone, as they have been described as being alike in coloration [4]. Females specimens [6, 7], we chose to generate a de novo assembly are the heterogametic sex (ZW) in birds; using genetic data, we genome of the species’ closest extant relative (Aratinga solstitia- were able to conclude that our specimen was a female because lis; the sun parakeet), against which we could subsequently map it showed about half the average depth coverage on the sex and call full-genome variants from the sequenced Carolina par- chromosomes (e.g., 13.43 genome-wide versus 7.113 at the akeet (STAR Methods). Previous analyses of the phylogenetic DMRT1 gene that is located in the Z chromosome). relationship of the Conuropsis genus to extant parrots have been assessed based on morphology [8] and a short (876-nucle- Phylogenetic Relationships otide) fragment of the mtDNA genome retrieved from the toes of In order to investigate the phylogenetic placement of Conuropsis six specimens [9]. Both studies concluded that Conuropsis within Psittaciformes and estimate its divergence time, we used falls in a clade as a sister group to three Aratinga species [9]. 4,988 nuclear loci (ultraconserved elements [UCEs], comprising Guided by this information, we generated a de novo reference 9,864,148 bp) extracted from the genomes of C. carolinensis, 17 genome of Aratinga solstitialis from a bird’s breeder specimen. extant parrots, and the rifleman Acanthisitta chloris (Passeri- This genome was assembled and annotated using the B10K formes) as an outgroup. Individual gene trees summarized into consortium pipelines [10] to render it consistent with previously a coalescent species tree were congruent with concatenated published avian nuclear genomes during subsequent analyses. datasets and supported by maximum local posterior probability The genome was based on Illumina reads from three long-range in all but one node. Gene trees suffered from the few and unusu- Nextera libraries of different insert sizes and assembled with ally short loci of one of the samples (Strigops habroptila; 77% of ALLPATH to a final N50 scaffold measure of 19.5 Mbp. loci missing and 95.81% gaps across the concatenated align- Following subsequent whole-genome shotgun sequencing ment), which resulted in a low supported relationship with of the Carolina parakeet DNA extract using the BGISeq-500 Psittacoidea (support = 0.23). This sample was also problematic platform, which has been demonstrated to be effective for in coalescent-based analyses in the original study that gener- ancient DNA [11], we were able to map 209,887,920 unique ated the data [18–21]. Concatenated analyses of all loci and of reads from C. carolinensis against the 1,168,990,576 bp of 95% and 100% completeness were congruent and had A. solstitialis genome, covering 93% of the genomic positions, maximum bootstrap support for Strigops+Nestor as the sister with a mean depth of 13.43 (STAR Methods). We also recovered to all other parrots, as found before [18–21]. All other relation- the entire mtDNA genome to 1503 depth of coverage. The reads ships and the placement of Conuropsis were entirely congruent exhibit characteristic ancient DNA deamination pattern at their between analyses suppress unambiguous and highly supported. ends [12], with a value close to 5% (Figure S1) that is consistent Conuropsis was consistently placed as the sister group of with our sample being just 100 years old [13]. We determined Aratinga, which in turn is sister group to the macaw Ara within which positions were derived in C. carolinensis or A. solstitialis Arinae (macaws, conures, and allies) (Figure 1). We also used using the chicken G. gallus as outgroup. A total of 28,348 the complete coding region of the mitochondrial (mtDNA) missense and 152 nonsense mutations were identified between genome sequence to investigate the placement of Conuropsis Conuropsis and Aratinga. Of the former, 502 mutations were against a greater sampling within the Arini and found the same predicted to be deleterious mutations using SIFT software [14]. placement as with nuclear data (Figure S2). Molecular clock The Carolina parakeet transition/transversion (Ts/Tv) value is analysis employing two fossil calibrations [21] suggests that 2.309, the ratio of the non-synonymous to synonymous the Aratinga-Conuropsis split occurred around 2.8 mya (1.6– Current Biology 30, 108–114, January 6, 2020 109 Figure 2. Demographic History of the Carolina Parakeet Pairwise sequentially Markovian coalescent (PSMC) plot shows Conuropsis carolinensis population history. We performed 100 bootstrap repetitions. The PSMC plot shows demographic fluctuations of the parakeet population size starting with the beginning of the Last Glacial Period. See also Figure S3. 4.4; 95% highest posterior density [HPD] interval) from nuclear genome data and around 3.8 mya (2.73–5.05; 95% HPD interval) from mtDNA data. Both dates roughly coincide with the widely recognized date of 3 mya for the final closure of the Panama Isthmus [22]. It seems plausible, therefore, that the dispersal to North America occurred after the North and South American landmasses were continuous. Figure 3. Genetic Diversity among Birds Demographic History Logarithm of the average genome heterozygosity for most published avian We used the pairwise sequentially Markovian coalescent genomes. All species belong to different taxonomic orders except for Con- (PSMC) algorithm [24] to evaluate the past demographic evo- uropsis carolinensis and Aratinga solstitialis, which are both Psittaciformes. lution of Conuropsis and Aratinga species. We found that the Samples are colored by IUCN Conservation status. See also Figure S4. Carolina parakeet population experienced an increase in effective population size (Ne) during the Middle Pleistocene, followed by demographic fluctuations that started during the Mb) (Figure S4). Nevertheless, we report the presence of a sin- Last Glacial Period (~110 kya) and a subsequent population gle, long run of homozygosity of 7.15 Mb, which is suggestive decline that continued until recent times (Figure 2). In contrast, of recent inbreeding in the ancestors of the Espinelves spec- the PSMC of the endangered Aratinga solstitialis shows a imen. It cannot be discarded, in fact, that our specimen was orig- stronger and continuous population decline and a longer inally bred in captivity. Alternatively, having only a single long period of lower effective population size than Conuropsis RoH could be indicative of some selective sweep in that partic- (Figure S3). ular region. We then profiled both the overall heterozygosity across the genome and the distribution of long runs of homozygosity Adaptation to Toxic Diet (RoHs) (Figure S4). The former is a measure of overall genetic di- We leveraged our data to explore the genomic basis of a curious versity, whereas RoHs arise when identical chromosomal frag- behavior of this species, relating to its dietary habits. The Car- ments are inherited from a recent common ancestor. Thus, olina parakeet consumed a variety of fruits, seeds, and to a significantly reduced heterozygosity is typical of populations lesser extent, buds and flowers, but most remarkably, it showed that have been small and isolated for long periods, although a predilection for cockleburs (Xanthium strumarium). This is un- elevated levels of RoH are usually observed in inbred popula- usual, as cockleburs contain significant levels of a diterpenoid tions [25]. Both may therefore be typical of endangered species. glucoside, the carboxyatractyloside or CAT [5], a lethal toxin We found that Conuropsis had a heterozygosity slightly below that inhibits mitochondrial energy production [26]. In a collection the average across bird genomes [10] but clearly does not of 99 feeding observations of Conuropsis, the highest plant appear to be an outlier (Figure 3) (the low level of heterozygosity intake (n = 17) corresponded in fact to cockleburs [4]. CAT in- of our Aratinga specimen could be influenced by the fact that it hibits the function of four mitochondrial ATP transporters was an individual bred in captivity). In addition, 188 total RoHs (ANT1, ANT2, ANT3, and ANT4; encoded by SLC25A4, were detected for Conuropsis (9 of them >1 Mb), although for SLC25A5, SLC25A6, and SLC25A31, respectively), which is Aratinga, the number is much higher (611 total RoHs; 85 >1 lethal [26, 27]. We next explored these genes further by 110 Current Biology 30, 108–114, January 6, 2020 Figure 4. Adaptation to Toxic Diet (A) Mitochondria representation of the outer and inner membrane (zoom in to B). (B) Cartoon of the bovine ANT protein X-ray crys- tallographic structure (approximate location of the inhibition by CAT blocking the flux of ATP and ADP + Pi). (C) Three-dimensional modeled structure of the SLC25A4 in Conuropsis with variable positions of the SLC25A4 in yellow and SLC25A5 in light or- ange (both modeled protein structures are quite similar—so only one was represented for simplifi- cation). The red region of the protein corresponds to the pocket. (D) Inside view of the 3D modeled structure of the SLC25A4 in Conuropsis (positions labeled as in C). (E) Sequential depiction of the amino acids around the position A122V of SLC25A4 in Conuropsis (comparison bottom-down: human; cow; mouse; opossum; C. carolinensis; A. solstitialis; chicken; anoles; python; green turtle; and crocodile) and indication of the pocket sites (in red) in the protein segment represented. comparing them against their orthologs in other available of all Psittaciformes is at least ten times larger (about 34.4 avian genomes, including one recently generated dataset mya), we can conclude that the evolutionary history underlying representing 363 species spanning nearly all avian families the Carolina-parakeet-specific adaptations is a rather recent [28]. First, we found that SLC25A6 and SLC25A31 genes are process within this order of birds. not present in that dataset, presumably due to the annotation We also uncovered evidence of a past population history of pipeline used. We did, however, find that the Conuropsis expansions and contractions with low effective population size SLC25A4 and SLC25A5 genes carry two non-synonymous but no dramatic signals of widespread, recent inbreeding that amino acid changes with respect to the Aratinga annotation: interestingly were discernable in Aratinga. This suggests that, A122V in SLC25A4 (a C to T substitution covered by 14 DNA despite the perception of high parakeet abundance based on reads) and T126S in SLC25A5 (an A to T substitution covered observations of large and noisy flocks, this species had experi- by 13 DNA reads). An additional variant in this gene, V227A, is enced population contractions that were likely associated shared with 24 other species from different orders. The two to past climatic oscillations. However, scarce evidence of SLC25A4 and SLC25A5 substitutions found are conserved in inbreeding indicates that it suffered a very quick extinction a diverse dataset of vertebrates, in 37 previously published process that left no traces in the genomes of the last specimens. avian genomes [10], and in the newly available avian genome da- In fact, the bird’s final extinction was likely accelerated by col- taset (Figure 4). Among the large avian dataset, additional non- lectors and trappers when it became evident that it was synonymous substitutions in the four codons preceding and extremely rare [8]. opposite these two positions have only been found in one We found evidence that the Carolina parakeet was adapted to single species (Pomatorhinus ruficollis). The two sites are the cocklebur’s toxin, but we caution that this feeding behavior is located in a helix of the protein and are flanking pocket sites, not exclusive of Conuropsis; parrots in general ingest fruits and likely influencing the functionality of both proteins. Therefore, it seeds known to be toxic to other vertebrates [29]. It has been pro- is possible that these mutations conferred the species with a posed that some species could neutralize them by consuming unique adaptive mechanism for dealing with the toxic CAT pre- clay from river banks, which would have a toxin-absorbing func- sent in its diet, although we do not know whether they could tion [30], although other physiological detoxification mechanisms be shared with other Aratinga species (besides A. solstitialis). cannot be discarded. Nevertheless, it would be interesting in the future to functionally test the two variants detected in the DISCUSSION SLC25A4 and SLC25A5 genes using avian cell lines. Other potential factors for Conuropsis extinction, such as the The extinct Carolina parakeet’s genome could provide evidence exposure to poultry pathogens, will likely require a metagenomic for specific adaptive peculiarities of this species and also help screening of at least several parakeet specimens; however, answer questions related to the population history and extinction preliminary results from our sample do not show a significant dynamics of this paradigmatic bird. presence of bird viruses. Taking advantage of having eighteen available parrot ge- The potential adaptation to the CAT toxin and the lack of evi- nomes, we have generated the first Psittaciformes genome- dence for a dramatic long-term decline and widespread wide phylogeny, which showed that the divergence time for inbreeding suggests that no additional factors contributed to Conuropsis evolutionary lineage and its subsequent colonization the extinction process. Therefore, the abrupt disappearance of of the North American subcontinent took place around 3 mya. the Carolina parakeet seems to be directly attributable to human Considering that the time to the most recent common ancestor pressures. Current Biology 30, 108–114, January 6, 2020 111 STAR+METHODS A.A. performed the in silico protein analysis. J.S. and A.A. worked in visualiza- tion. P.G., M.P.T.G., and C.L.-F. wrote the manuscript with input from all coauthors. Detailed methods are provided in the online version of this paper and include the following: DECLARATION OF INTERESTS d KEY RESOURCES TABLE The authors declare no competing interests. d LEAD CONTACT AND MATERIALS AVAILABILITY d EXPERIMENTAL MODEL AND SUBJECT DETAILS Received: July 5, 2019 B Conuropsis carolinensis specimen Revised: October 3, 2019 B Aratinga solstitialis specimen Accepted: October 30, 2019 d METHOD DETAILS Published: December 12, 2019 B Conuropsis DNA extraction and sequencing B Aratinga solstitialis DNA extraction REFERENCES B Aratinga solstitialis sequencing and assembly 1. Elphick, C.S., Roberts, D.L., and Reed, J.M. (2010). Estimated dates of B Aratinga solstitialis annotation recent extinctions for North American and Hawaiian birds. Biol. Conserv. B Conuropsis carolinensis mapping and variant calling 143, 617–624. B Sex determination 2. Burgio, K.R., Carlson, C.J., and Tingley, M.W. (2017). 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Genome Res. 26, 1211–1218. 114 Current Biology 30, 108–114, January 6, 2020 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Biological Samples Conuropsis carolinensis tissue samples Arboretum Masjoan private N/A collection at Espinelves (Girona, Spain) Aratinga solstitialis Blood sample from N/A Aratinga breeder Deposited Data Whole genome data of one historical Conuropsis This paper PRJEB33130 carolinensis sample Aratinga solstitialis assembly This paper PRJEB33135 Aratinga solstitialis annotation data This paper PRJEB33153 Taeniopygia guttata 3.2.4 annotated proteins from N/A Release 85 Ensembl Gallus gallus 5.0 annotated proteins from Ensembl N/A Release 85 Whole-genome sequencing data of Acanthisitta chloris [10] NCBI Project ID: PRJNA212877 Whole-genome sequencing data of Agapornis [31] NCBI Project ID: PRJNA355979 roseicollis Whole-genome sequencing data of Amazona aestiva [32] NCBI Project ID: PRJNA294082 Whole-genome sequencing data of Amazona collaria N/A NCBI Project ID: PRJNA490036 Whole-genome sequencing data of Amazona guildingii [28] Ultraconserved Elements (UCEs) extracted from whole genome data Whole-genome sequencing data of Amazona vittata [33] NCBI Project ID: PRJNA171587 Whole-genome sequencing data of Ara macao [34] NCBI Project ID: PRJNA175470 Whole-genome sequencing data of Eolophus [28] Ultraconserved Elements (UCEs) roseicapilla extracted from whole genome data Whole-genome sequencing data of Melopsittacus N/A NCBI Project ID: PRJNA72527 undulatus Whole-genome sequencing data of Nestor notabilis [10] NCBI Project ID: PRJNA212900 Whole-genome sequencing data of Probosciger [28] Ultraconserved Elements (UCEs) aterrimus extracted from whole genome data Whole-genome sequencing data of Psittacula krameri N/A NCBI Project ID: PRJNA377329 Targeted capture ultraconserved elements for This paper N/A Conuropsis carolinensis Targeted capture ultraconserved elements for Aratinga This paper N/A solstitialis Targeted capture ultraconserved elements for [21] https://doi.org/10.5061/dryad.2vd01grFile: Amazona autumnalis, Cacatua sulphurea, Psittacula passerines.unaligned.uce.contigs.tar.gz alexandri, Psittacus erithacus, Strigops habroptila 4,988 aligned UCE loci This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 4,988 gene trees from UCE loci and summary tree from This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 ASTRAL-III Concatenated dataset of 4,988 aligned UCE loci and This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 resulting IQTREE tree Concatenated dataset of 2,755 aligned UCE loci which This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 are present in 95% of taxa and resulting IQTREE tree Concatenated dataset of 893 aligned UCE loci which This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 are present in 100% of taxa and resulting IQTREE tree (Continued on next page) Current Biology 30, 108–114.e1–e5, January 6, 2020 e1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER BEAST input files for 2 sets of 50 loci with calibration This paper https://doi.org/10.17632/ p4wt7jc9 dw.1 constraints and resulting trees Commands used for bioinformatic processing and N/A https://doi.org/10.17632/ p4wt7jc9 dw.1 phylogenetic analysis SOFTWARE AND ALGORITHMS ALLPATHS-LG [35] v.52485 AbySS [36] v.1.3.5 SOAPdenovo [37] v.2 TblastN [38] v.2.2.2 genBlastA [39] v.1.0.4 GeneWise [40] v.2.4.1 MUSCLE [41] v.3.8.31 cutadapt [42] v1.9.1 BWA [43] v.0.7.1 Picard [44] v2.0.1 Samtools [45] v.1.6 mapdamage2 [46] 2.7.12 GATK [47] 3.7 ClustalOmega [48] 1.2.1 BEAST [49] v.1.8.4 (v.2) JmodelTest [50] v.2.1.10 BamUtil [51] v1.0.13 PSMC [52] v1.0 SNPeff [53] v4.3 PLINK [54] v1.9b R [55] v3.5.1 SIFT [14] v6.2.1 Schmutzi [56] v1.5.4 Pomegranate [57] python v3 bcftools N/A v.1.9 PHYLUCE [58] v.1.6.6 IQTREE (incl. ModelFinder and UFBoot) [59–61] v.1.6.10 ASTRAL-III [62] v.5.6.3 BEAST2 [63] v.2.6.0 Tracer [64] v.1.7 LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Pere Gelabert (peregelabertx@gmail.com). This study did not generate new unique reagents. EXPERIMENTAL MODEL AND SUBJECT DETAILS Conuropsis carolinensis specimen At least 720 skins and 16 Carolina parakeet skeletons are preserved in museum collections globally [65, 66]. We sampled one such specimen with the intention of generating the first near-complete whole genome information of the species. The specimen is pre- served in a private collection in the village of Espinelves (Girona, Spain), and was collected at the beginning of the 20th century by the Catalan naturalist Marià Masferrer i Rierola (1856-1923). The Carolina parakeet is believed to have consisted of two subspecies: Conuropsis carolinensis carolinensis, that was principally distributed in Florida and along the Southeast coast of United States, and Conuropsis carolinensis ludovicianus that was distributed e2 Current Biology 30, 108–114.e1–e5, January 6, 2020 across the central states of the country [2]. Both subspecies could be differentiated by morphological features such as coloration and body size. The wing, bill, and tail lengths of all adult C. c. ludovicianus significantly averaged more than in all adult C. c. carolinensis [4]. The wing and tarsal lengths (Figure S1), as well as the general color pattern of the Espinelves specimen, indicate it belongs to C. c. carolinensis. Aratinga solstitialis specimen A sample of blood was obtained in vivo from a female specimen from an official Aratinga breeder. METHOD DETAILS Conuropsis DNA extraction and sequencing Two different samples of about 100 mg were obtained, one from the femur (leg bones were preserved inside the naturalized spec- imen) and one from toepads, with the help of a Dremel machine. The two samples were digested using 2mL of extraction buffer containing 10mM Tris-HCL (pH 8), 10mM NaCl, 5mM CaCl2, 2.5mM EDTA, 1% SDS, 1% Proteinase K and 40mM DTT. The solution was resuspended by vortexing and was incubated in a rotating plate overnight at 55 C. Digested samples were purified, and DNA was isolated following a combination of Phenol/Chloroform and column purification, as outlined below. After incubation, digested samples were centrifuged for 3 min at 3000 3 g and the supernatant was collected and mixed with 1X volume of Phenol. The sample solution was incubated on a rotor for 5min at RT. After, it was centrifuged for 3 min at 5000 3 g and the upper aqueous layer was collected in new low-bind Eppendorf tube. The collected aqueous layer was mixed with 1X volume of Chloroform and the process was repeated. Again, the upper aqueous layer was collected in new tube and mixed with 10X volume of binding buffer prepared as previously described [67].The sample solution mixed with the binding buffer was poured into a binding apparatus constructed by fitting an ZymoV extension reservoir in a MinElute column and set inside a 50mL Falcon tube (as in [68]. Samples were centrifuged at 300 3 g until all the liquid had passed through. The MinElute column was then separated from the reservoir and set into a new 2ml low-bind collection tube. The column was washed with 730mL of QIAGEN buffer PE, centrifuged at 3,300 3 g, flow-though was discarded and the MinElute column was dry-spun for 1 min at 6000 3 g in a bench-top centrifuge. DNA was eluted in a final volume of 50mL by adding twice 25mL of QIAGEN EB buffer and incubating for 5 min at 37 C between each elution. Samples were centrifuged at 6000 3 g and extracted DNA was quantified using a Qubit instrument. Following extraction, 15 mL of DNA extract was built into blunt-end libraries using both the NEBNext DNA Sample Prep Master Mix Set 2 (Cat No. E6070) and the BEST protocol using BGI adapters (as in [11]. Two libraries were built for each method. For the NEB protocol, the libraries were prepared according to manufacturer’s instructions, only skipping the initial nebulization step. The resulting DNA library for each method was then amplified and indexed in 4 PCR reactions of 50mL each with 16mL of DNA template on each, mixed with 25 mL 2X KAPA U+ Buffer, 1.5 mL of BGI amplification primer (10 mM)(sequences described in [11]. Ther- mocycling conditions were 3 min at 98 C, followed by 22 cycles of 20 s at 98 C, 30 s at 60 C and 30 s at 72 C, and a final 7 min elongation step at 72 C. The amplified library was purified with PB buffer on QIAGEN MinElute columns, before being eluted in 30 mL EB. Negative library controls, constructed with H2O, were included, as well as libraries constructed on the negative extraction controls; both subsequently yielded no DNA sequences. Amplified libraries were quantified using a TapeStation instrument (Agilent) and two sequencing pools were created by merging the amplified libraries for each method and sequenced on 2 lanes of a BGISEQ-500 sequencing instrument using 100SR chemistry. Libraries prepared from tibia bone powder exhibited longer DNA reads in comparison with the toe tissue (x = 83bp versus x = 61 bp, p < 0.001). NEB libraries yielded longer DNA reads than BEST libraries (x = 84.86 and x = 63.45 versus x = 75.39 and x = 60.31 in tibia and toe respectively, p < 0.001). NEB libraries were also the ones that yielded higher endogenous DNA content as well as lower clonality. Aratinga solstitialis DNA extraction Parallel genomic DNA extractions were performed on blood from a single Aratinga solstitialis female individual using the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) following manufacturer’s instructions. The resulting DNA extracts were quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) with no modification of its standard protocol. To check for mo- lecular integrity, each DNA extract was run on the 2200 TapeStation (Agilent Technologies, Santa Clara, CA) following manufacturer’s protocol. Aratinga solstitialis sequencing and assembly Using the high molecular weight (HMW) DNA extracts, a short PCR-free insert library with 180 bp inserts was prepared using TruSeq DNA kit (Illumina, CA, USA) according to the manufacturer’s instructions. In addition, three different mate-pair libraries were built us- ing the Nextera protocol (Illumina, CA, USA). These comprised one 3 kb mate-pair library, one 5 kb mate-pair library, and one 20kb mate-pair library. All libraries were indexed to enable de-multiplexing after sequencing. The libraries were subsequently sequenced on the Illumina HiSeq X platform (using 2 3 150 bp reads), where the first lane was used for the 180 bp insert library. For the second lane, the three mate-pair libraries were pooled in equimolar ratios prior to sequencing. Current Biology 30, 108–114.e1–e5, January 6, 2020 e3 In order to generate a de novo assembly, three different assemblers were used: ALLPATHS-LG v.52485, ABySS v.1.3.5 and SOAPdenovo. Out of the three assemblers, ALLPATHS-LG gave the best result, with an N50 scaffold measure of 19.5Mbp. Aratinga solstitialis annotation We used a homology-based method to annotate the protein-coding genes in the Aratinga genomes by using Ensembl gene sets (release 85) of chicken (Gallus gallus), zebra finch (Taeniopygia guttata) and human (Homo sapiens), and genes derived from pub- lished avian transcriptomes. The protein sequences of the reference gene set compiled above were used as references for homol- ogy-based gene prediction. We aligned reference protein sequences to the genome by TBLASTN with an E-value cut-off of 1e-5. We linked the hits into candi- date gene loci with genBlastA and removed candidate loci with a homologous block length shorter than 30% of length of query pro- tein. We extracted genomic sequences of candidate gene loci and 2,000bp upstream/downstream sequences as input for GeneWise to predict gene models in the genome. Then we translated the predicted coding regions into protein sequences, and ran MUSCLE for each pair of predicted protein and reference protein. We filtered out the predicted proteins with length of < 30aa or percent identity < 40%, as well as the pseudogenes (genes containing > 2 frameshifts or pre-mature stop codons) and retrogenes. The output of GeneWise could include redundant gene models, which overlap at the same genome regions. Hierarchical clustering was applied to the output of GeneWise to build a non-redundant gene set. Genes that overlapped in > 40% of their coding sequence were clus- tered and kept the sequence with the highest identity to the reference genes. We removed the highly duplicated genes (frequency of duplications > 10) in two conditions: 1) with a single exon; 2) with > 70% repeat sequences in coding region. Conuropsis carolinensis mapping and variant calling The ancient DNA reads were clipped using cutadapt; sequencing adapters were removed. Only reads longer than 30bp were kept. Filtered reads were mapped against the A. solstitialis assembly with Burrows-Wheeler Aligner (BWA) [69], setting no trimming, disabling seed, increasing stringency for edit distance, and allowing opening of 2 gaps. Duplicated reads were removed using Pic- ard-tools MarkDuplicates. Mapped reads with mapping quality below 30 were removed using Samtools. The resulting reads were examined with mapdamage2 to assess the degradation rate of the data, which is a sign of authenticity. We detected the presence of typical aDNA-damaged bases at the end of reads. To avoid problems in the next steps, we trimmed 2 nt from each read end using BamUtil trimbam. Genotypes were estimated using GATK UnifiedGenotyper. We removed calls with base quality below 30 (-mbq), and we set the rest of parameters as default. The average depth of coverage of the sample was 13.4X. To prevent variant calling errors in repetitive or complex regions, we used GATK SelectVariants to exclude the calls with depths of coverage below 10x and above 35X. Afterward we also used GATK SelectVariants and GATK FilterVariants to exclude from the call-set InDels and heterozygous calls in allele fre- quencies below 0.2 and above 0.8. We subsequently used the A. solstitialis assembly annotations to build a SNPeff database and used Gallus annotations to determine derived alleles. Sex determination The Aratinga genome -which we knew was a female- showed, as expected, half of coverage in the ZW chromosomes [70]. We plotted the depth of coverage distribution for each scaffold of the Carolina parakeet using Samtools and found identical coverage distribu- tion. We subsequently searched for the DMRT1 gene [71] to confirm the Aratinga Z chromosome scaffold. Ultraconserved Elements (UCE) phylogenetic tree For phylogenetic analysis, we targeted 5,060 UCE loci from 14 species with whole genome sequences (including the two new ge- nomes presented here) and from 5 parrots that were included in a previous UCE capture study [21]. The Tetrapods-UCE-5Kv1 bait set [72] was applied to 18 parrots and the outgroup Acanthisitta chloris. A total of 4,988 UCE sequences were identified and ex- tracted with the flanking 1000bp to both sides, aligned and trimmed using PHYLUCE (commands in 10.17632/p4wt7jc9dw.1). Stri- gops habroptila from the targeted capture study had significantly fewer and shorter loci than all other samples (1,648 loci, 269bp length on average compared to 757bp on average across samples from [21]) but we kept the sample because of its significance for fossil calibration. We used coalescent and concatenation approaches to infer phylogenetic relationships. First, we constructed maximum likelihood gene trees for all 4988 alignments using IQTREE with 1000 ultrafast bootstrap replicates after determining the most appropriate nucleotide substitution model with ModelFinder. The resulting gene trees were summarized into a coalescence-based species tree using ASTRAL-III. Second, we concatenated all loci (9,864,148bp), the 2,755 loci that were present in 95% of all species (5,561,275bp) and the 893 loci that were present in 100% species (1,840,245bp) and analyzed them as above. For calibration analyses, we drew two random samples of 50 loci that had all taxa and had the same substitution model (HKY+ F+G4, the most common model across all loci). For both random samples, we executed two MCMC chains (100 million generations, sampled every 5,000 generations) in BEAST2 on the CIPRES Science Gateway [73]. Each analysis was performed on the topology from concatenation, employing a birth-death model, a relaxed clock model with lognormal distribution on the rate prior and HKY+ F+G4 as the substitution model. The age of two nodes was constrained with lognormal distributions following the thorough published fossil justifications [21]. First, a lognormal prior was placed on the root of the tree (Passeriformes+Psittaciformes, Eozygodactylus americanus) with an offset of 51.81 Mya and a 97.5% quantile encompassing 66.5 Mya. Second, a lognormal prior was placed on e4 Current Biology 30, 108–114.e1–e5, January 6, 2020 the MRCA of Strigops+Nestor (Nelepsittacus minimus) with an offset of 15.9 Mya and a 97.5% quantile at 66.5 Mya. Replicate runs were checked for convergence in Tracer, combined and annotated after a burning of 30% with LogCombiner and TreeAnnotator. Mitochondrial phylogenetic tree Trimmed DNA reads (209,887,920) were mapped against A. solstitialis mtDNA genome (JX441869). The mtDNA consensus sequence of Conuropsis was obtained by using schmutzi endoCaller and aligned with Clustal Omega to 11 other Arini mtDNA genomes and Amazona ventralis as outgroup. The obtained alignment of 13 sequences of 18,731bp in total length was dated using BEAST based on a fixed clock rate of 0.0042 substitutions/site/MY for all coding regions, which was previously determined for the brown-throated Parakeet Eupsittula (formerly Aratinga) pertinax [74]. The number of polymorphic sites of Conuropsis mtDNA genome in the alignment was 4,369. We used the GTR+I+G nucleotide substitution model selected by jModelTest with the Akaike Information Criterion. Conuropsis population history We used the Pairwise Sequentially Markovian Coalescent (PSMC) model to explore the demographic history of C. carolinensis. We obtained a fastq sequence of C. carolinensis for autosomal regions in scaffolds longer than 100Kbps. Only positions with a depth of coverage above 8X and below 50X were kept. Posteriorly a PSMC was built using the following parameters: -N25, -t15, -r5, -p ‘‘4+25*2+4+6.’’ We used age of sexual maturity (1 year) [3], multiplied by a factor of two as a proxy for generation time, following the same approach as in a previous study of PSMC in 38 avian species [52] and a mutation rate of 2.3x10 9, estimated from bird pedigree information [75]. Conuropsis average genome heterozygosity To identify regions of the C. carolinensis genome that shows signs of homozygosity we plotted the distribution of heterozygous positions across the genome sequence. We examined the scaffolds counting the number of heterozygous positions in windows of 50Kb with 10Kb of overlap. We define the average genome heterozygosity as the proportion of heterozygous sites genome- wide divided by the total number of callable bases. We kept only SNV sites applying the following filtering criteria: Read Depth > 10, Genotype Quality > 20, Allele Balance 0.2 < AB < 0.8 (hypergeometric distribution 0.95 CI [0.233-0.766]). All variable repeats, indels and multiallelic sites were removed. Non-variable sites were considered callable if their read depth was larger than 10. Additional heterozygosity values for other bird species were extracted from published avian genomes [10]. Conuropsis Runs of Homozygosity (RoHs) RoHs were called based on the density of heterozygous sites in the genome using a Hidden Markov Model (HMM) for segmentation: First, the Aratinga reference genome was partitioned into 50Kb windows guided by the Conuropsis callability mask, namely, uncal- lable Conuropsis sites were omitted in the window tally. Heterozygosity values were calculated for each window as described above. Next, an HMM (python3 pomegrenate package) was fitted to the data. Emissions were modeled based on the empirical window het- erozygosity distribution with a two/three component Gaussian Mixture Model (GMM). The first component of the GMM was reserved to extremely small heterozygosity values in order to capture the RoH variability, while the second component was allowed to vary freely. If necessary, a third mixture component was added to capture outliers. The transition probabilities were trained using the Baum-Welch algorithm. QUANTIFICATION AND STATISTICAL ANALYSIS All statistical details of experiments can be found at the STAR Methods. The phylogenetic tree in Figure 1 was performed with BEAST 2 (https://www.beast2.org). The pattern of post-mortem damage in Figure S1 was generated with mapdamage2 and the contami- nation estimates at the mtDNA was done with Schmutzi program. Adaptors from the DNA reads were removed with cutadapt. Ge- netic differences between Conuropsis and Aratinga were explored with SIFT software and the prediction of effects of some polymor- phisms was done with SNPeff software. DATA AND CODE AVAILABILITY The accession numbers for the Conuropsis and Aratinga genomes reported in this paper are in the European Nucleotide Archive (ENA): PRJEB33130 and PRJEB33153, respectively. Current Biology 30, 108–114.e1–e5, January 6, 2020 e5 Current Biology, Volume 30 Supplemental Information Evolutionary History, Genomic Adaptation to Toxic Diet, and Extinction of the Carolina Parakeet Pere Gelabert, Marcela Sandoval-Velasco, Aitor Serres, Marc de Manuel, Pere Renom, Ashot Margaryan, Josefin Stiller, Toni de-Dios, Qi Fang, Shaohong Feng, Santi Mañosa, George Pacheco, Manuel Ferrando-Bernal, Guolin Shi, Fei Hao, Xianqing Chen, Bent Petersen, Remi-André Olsen, Arcadi Navarro, Yuan Deng, Love Dalén, Tomàs Marquès-Bonet, Guojie Zhang, Agostinho Antunes, M. Thomas P. Gilbert, and Carles Lalueza-Fox Figure  S1:  Morphological  and  post-­‐mortem  DNA  damage  analysis.  Related  to  the   STAR   Methods.   Above   left:   tarsal   measurements   of   the   Espinelves   female   studied   compared  with  average  and  range  measurements  for  adult  females  from  the  literature   [S1];  the  values  indicate  the  specimen  belongs  to  Conuropsis  carolinensis  carolinensis   subspecies.   Above   right:   X-­‐ray   image   used   to   measure   the   tarsal   bones.   Below:   nucleotide   deamination   pattern   at   the   end   of   the   Carolina   parakeet   reads,   deriving   from   cytosine   deamination   in   ancient   sequences   [S2].   The   ratio   (about   4-­‐5%   of   the   reads)  is  consistent  with  the  age  of  the  sample.   Figure S2: Calibrated   phylogeny   built   with   BEAST   based   on   mitochondrial   genomes   of   13   species   of   Neotropical   parrots.   Related   to   Figure   1.   A   0.0042   substitutions/site/MY  molecular  rate  was  used  to  calibrate  the  phylogeny.  95%  HPD   intervals   are   shown   in   the   nodes   in   addition   to   bootstrap   values   below   1.   The   Aratinga-­‐Conuropsis  split  occurred  around  3.8  Mya.   Figure S3: Pairwise Sequentially Markovian Coalescent (PSMC) plot of Aratinga solstitialis population history. Related to Figure 2. We performed 100 bootstrap repetitions.     ● Conuropsis carolinensis ● ● 0.007 500 ● ● ● ● 0.006 ● ● ● ● 400 ● ● ● 0.005 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 300 ● ● ● 0.004 ● ● ● ● ● ● ● ● ● Heterozygosity ● ● ● ● Occurrence ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.003 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● 200 ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 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● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● 0.002 ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ●● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● 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● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● 0.000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ●● ●● ●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ●● ● ●● ● ●● ● ●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ● ●●● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ●●● ●●● ●●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ●● ● ● ●● ●● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ● 0 ● ● ● ● ● ● ● ●●● ●● ●● ● ● ● ●● ● ●● ● ●● ●● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ●●●●● ●●● ● ●● ●●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●● ● ●●● ● ● ●●● ● ● ●● ●● ● ●●● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● 0e+00 2e+08 4e+08 6e+08 8e+08 1e+09 0.0e+00 1.0e+07 2.0e+07 Position in Scaffold (Conuropsis carolinensis) RoH Length (bps) Aratinga solstitialis 500 0.007 ● ● ● 0.006 400 0.005 ● 300 0.004 ● Heterozygosity Occurrence ● ● ● ● ● ● ● 0.003 ● ● ● 200 ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.002 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 100 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● 0.001 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● 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● ● ●●●●● ●●●● ●● ● ● ● ● ● ●● ● ●● ● ●● ● ●● ● ●●●●● ●● ● ●● ● ● ● ●● ●● ●● ● ●● ●● ●● ●● ●●●● ●● ●● ●● ● ●● ●● ● ●● ●● ● ●●●●● ● ●● ● ● ●● ●● ●● ● ●●● ●● ● ● ● ● ●● ●● ● ● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ● ●● ●● ● ●●●●● ● ●● ●● ● ● ●● ● ●●● ● ● ●● ● ●● ●●●●● ● ● ●●● ●●● ●● ● ● ● ● ● ● ● ● ●●●● ● ●● ● ●●● ● ●● ●● ● ●●● ● ●● ●● ● ●● ● ●●● ● ● ●● ●● ● ● ●●●● ● ●● ● ●● 0e+00 2e+08 4e+08 6e+08 8e+08 1e+09 0.0e+00 1.0e+07 2.0e+07 Position in Scaffold Aratinga (Aratinga solstitialis) RoH Length (bps) Figure   S4:   Heterozygosity   across   scaffolds   (vertical   doted   lines   represent   contigs  in  the  scaffold)  and  histogram  of  Runs  of  Homozygosity  (RoH).  Related   to  Figure  4.  Length  count  for  Conuropsis  carolinensis  (above)  and  Aratinga  solstitialis   (below),   at   the   same   scale.   Only   one   long   RoH   of   >7Mb   was   observed   in   the   former   species.   Supplemental  References       S1.   Jónsson,   H.,   Ginolhac,   A.,   Schubert,   M.,   Johnson,   P.,   Orlando,   L.   2013.   mapDamage2.0:   fast   approximate   Bayesian   estimates   of   ancient   DNA   damage   parameters.  Bionformatic  29  (13):  1682-­‐1684.     S2.   Snyder,   N.F.R.,   Russel,   K.   2002.   Carolina   Parakeet   (Conuropsis   carolinensis),version   2.0.   In:   Poole,   A.F.,   Gill,   F.B.   (Eds.),   The   Birds   of   North   America.   Cornell   Lab   of   Ornithology,  Ithaca.