Phylogenetic Relationships of the Extinct Carolina Parakeet (Conuropsis Carolinensis) Inferred

Running Head: Carolina Parakeet Ancient DNA

Phylogenetic relationships of the extinct Carolina Parakeet (Conuropsis carolinensis) inferred from ancient-DNA sequences

Jeremy J. Kirchman1, 3, Erin E. Schirtzinger2, Timothy F. Wright2

1New York State Museum, 3140 Cultural Education Center, Albany New York 12230

2Department of Biology, New Mexico State University, Las Cruces, NM 88003

3Address correspondence to this author. E-mail:

ABSTRACT

We obtained the first DNA sequences from the extinct Carolina Parakeet (Conuropsis carolinensis) and used these data to infer the phylogenetic relationships of this iconic North American parrot. We compared our sequences of the mitochondrial cytochrome oxidase I gene obtained from two Carolina Parakeet museum specimens to homologous sequences from individuals representing 43 species in 26 genera of Neotropical parrots (Tribe Arini), and four species from more distantly related Old World tribes of Order Psittaciformes. Maximum likelihood and Bayesian analyses place C. carolinensis on a long branch, most closely related to a well-supported clade of Aratinga parakeets that includes the most northern extant species of Neotropical parrots and species endemic to Cuba, Hispaniola, and Socorro Island. Our data do not support a close relationship with the Monk Parakeet (Myiopsitta monachus) with which Carolina Parakeet shares fully feathered ceres, a putative adaptation for cold tolerance that appears to have evolved independently in both species. Based on the high level of sequence divergence from all sampled species (uncorrected P > 5.6%), we recommend continued recognition of the monotypic genus Conuropsis. Taxonomic revision of the highly polyphyletic genus Aratinga is needed.

Key Words: ancient DNA, Arini, Carolina Parakeet, Conuropsis carolinensis, parrot, phylogenetics, systematics

Prior to its decline and extinction the Carolina Parakeet (Conuropsis carolinensis) was distributed patchily throughout the eastern half of North American as far north as the southern shores of Lake Erie and Lake Ontario, making it by far the most northerly distributed parrot (Psittaciformes) in the Americas (Forshaw 1989). Once common and abundant, the species was in decline by the 1830s and was rare by the end of the 19th century, by which time its distribution was largely limited to the swamps of Florida (Snyder and Russell 2002). The last reliable sightings were in the late 1920s, but unconfirmed reports suggest it persisted in Florida, Georgia and South Carolina into the late 1930s (Snyder and Russell 2002). Although the exact timing and cause of its ultimate demise are unknown, it is generally acknowledged that Carolina Parakeets were shot for sport, food, and feathers, and to guard against crop depredations; destruction of bottomland forests likely also played a role in its extinction (Snyder 2004). Snyder (2004) has further hypothesized that a preference for eating weedy cockleburs may have brought it into contact with diseases carried by settlers and their livestock. The Carolina Parakeet now stands with the Passenger Pigeon (Ectopistes migratorius) as an iconic example of the ability of humans to exterminate even widespread and abundant continental bird species (Bucher 1992).

The extinction of the Carolina Parakeet occurred before any systematic study of its ecology and habits was undertaken, and what little is known regarding its life-history and historic distribution have been pieced together from the fragmentary accounts of 19th century naturalists. Daniel McKinley synthesized reports of its regional occurrence in a lengthy series of papers (e.g. McKinley 1960a, b, 1965), and summarized the available details of its nesting biology (McKinley 1978) and seasonal movements (McKinley 1977). Noel F. R. Snyder combined this work with interviews of the few remaining persons with first-hand knowledge of wild Carolina Parakeets and his own experience with parrot biology to provide the most complete picture to date of the natural history of this species (Snyder and Russell 2002; Snyder 2004). On the heels of Snyder’s work, additional insights concerning the natural history and extinction of the Carolina Parakeet will likely come from only two sources: reasoned inference from studies of related extant species, and additional study of museum specimens.

Inference of evolutionary patterns requires an informed hypothesis of the evolutionary history of a clade. The genus Conuropsis was erected as a monotypic genus by Linnaeus (Linnaeus 1758). Most workers have suggested that Conuropsis was most closely related to the genus Aratinga based on shared morphological features including a long, pointed tail and wings, feathered cheeks and lores, and comparatively broad and heavy bills (Forshaw 1989; Snyder 2004). Snyder (2004) further noted the shared presence of fully feathered ceres in Conuropsis and Myiopsitta monachus, the Monk Parakeet of temperate South America. Snyder (2004) concluded by expressing considerable uncertainty as to the true affinities of Conuropsis, and advocating broad sampling to include many of the genera in the Neotropical tribe Arini. In this study we used sequences of the mitochondrial gene, cytochrome oxidase I (COI), obtained from museum specimens of Conuropsis and representatives of 26 other genera of Neotropical parrots to produce the first study of the evolutionary relationships of the extinct Carolina Parakeet.

Methods

DNA extraction. - We used sterile scalpel blades and forceps to remove slivers of skin and connective tissue from the toes of four taxidermy-mount specimens at the New York State Museum (NYSM zo-9420, no data; NYSM zo-9421, female, Manatee County, Florida; NYSM zo-9422, male, Florida; NYSM zo-9436, female, Florida). All specimens have extensive yellow plumage on the head and are presumed to be adults. None of the specimens have reliable dates. Latex gloves, worn while handing specimens, were changed between specimens and instruments were sanitized with 10% bleach between specimens. Toe pad samples were removed to the dedicated ancient DNA lab at NYSM, where no previous work on parrots had been performed. All DNA extractions and polymerase chain reaction (PCR) set-ups from C. carolinensis were performed in this lab following procedures intended to minimize and highlight contamination from exogenous DNA sources, including negative extraction controls (containing no toe pad), negative PCR controls (containing no DNA extract), glove changes between handling each sample, ultraviolet irradiation of all plastics, exclusive use of aerosol-barrier pipette tips, and daily sanitation of all equipment and surfaces with 10% bleach solution.

DNA was extracted from toe-pads using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following two different modifications of the manufacturer’s protocol. In the first set of extractions, double volumes of Buffer ATL (380 ul) and proteinase K (40 ul) were used and samples were incubated overnight at 55ºC with agitation. Undigested chunks were ground with micro-pestles and an additional 20 ul of proteinase K was then added to each tube. Digestion was continued for a second night at 55ºC with agitation. Following complete digestion, double volumes of the Buffer AL and ethanol were added to the sample and the resulting mixture was applied to the silica-membrane spin columns in 2 or 3 batches, centrifuging each batch through the membrane to bind the DNA. Following binding, the manufacturer’s protocol was followed for two rounds of washing, and DNA was eluted in 75 ul of Fisher DNA-grade water (filtered, autoclaved, DNase free). Elution was repeated and both filtrates were combined for a total volume of 150 ul of DNA in water. Extracts from two specimens (NYSM zo-9421, NYSM zo-9436) yielded PCR products and DNA sequences in initial trials; these specimens were re-sampled as above and subjected to a second extraction that involved a two-day washing and hydration procedure (de Moraes-Barros and Morgante 2007) followed by the modified DNEasy protocol above. Extracts from this second round yielded additional PCR products and sequences, which confirmed the sequencing results from the first set of extracts.

Extractions, PCR, and sequencing for additional taxa were performed at New Mexico State University. At NMSU we extracted DNA from blood and frozen tissue samples from captive birds or museum specimens (Table 1) using the DNeasy Blood and Tisue Kit following the manufacturer’s protocol as described in Wright et al. (2008).

COI amplification and sequencing. - A portion of the mitochondrial protein-coding, cytochrome oxidase I (COI) gene was amplified from non-ancient samples using the primers LCOIp and HCOIp and reaction conditions described in Wright et al (2008). To obtain homologous sequences from C. carolinensis, we tested five PCR primer pairs that were used by Eberhard and Birmingham (2004) to amplify and sequence short, overlapping fragments of COI from the Yellow-crowned Amazon Amazona ochrocephala (L7506, H7523, L7628, H7642, L7773, H7813, L7804, and H7879) in combination with CO1a and CO1f from Palumbi (1996). We amplified these fragments in 50 ul PCRs that contained 5 ul of DNA extract (concentration unquantified), 1.0 unit of HotMaster™ DNA Polymerase (Eppendorf) in the manufacturer’s buffer with MgCl2, 0.10mM of each dNTP, 0.5mM of each primer, and 0.025mg of bovine serum using the following thermal program: 95ºC for 60 s followed by 50 cycles of 94ºC for 30 s, 50ºC for 30 s, 65ºC for 45 s, and a final extension step of 65ºC for 120 s.

Amplification success was determined by visualizing 5 ul of each PCR product on 1.5% agarose gels stained with ethidium bromide. We cleaned PCR products using a QiaQuick PCR Purification Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. We sequenced each PCR product in both directions using the PCR primers and Big Dye v3.1 Terminator Cycle Sequencing chemistry (Applied Biosystems Inc, Foster City, CA) on an ABI 3100 Genetic Analyzer. Raw sequences were edited and assembled into consensus sequences for each specimen in Sequencher 4.7 (Gene Codes, Ann Arbor MI). We used MacClade 4.0 (Maddison and Maddison 2003) to convert the sequence alignment from all specimens into Nexus format and to establish codon positions.

Phylogenetic Analysis and Taxon Sampling. In addition to the two specimens of C. carolinensis, we included single representatives from 43 species of Neotropical parrots from 26 genera of the Neotropical tribe Arini (taxonomy following Forshaw (2006)). We selected single representatives from 21 genera, 2 or 3 representatives from the speciose genera Amazona, Ara, Pionus and Pyrrhura, and 13 representatives from Aratinga, the hypothesized sister clade to Conuropsis (Table 1). In total, this sample represents 28% of the 162 species and all but four genera (Primolius, Guaruba, Triclaria, Ognorhynchus) in Tribe Arini. Outgroups included one representative each from four Old World tribes: Psittacus erithacus of the Afrotropical tribe Psittacini; Strigops habroptilus, of the New Zealand tribe Strigopini; Melopsittacus undulatus, of the Australian and Oceanian tribe Platycercini, and Calyptorhynchus funereus of the Australasian tribe Calyptorhynchini.

The final alignment of sequences from 49 species was analyzed using Maximum Likelihood (ML) and Bayesian methods. The best nucleotide substitution model for the ML analysis of the complete alignment was selected using Modeltest v3.8 (Posada and Crandall 1998; Posada 2006) under the AIC criterion. ML tree searching was performed in PAUP* (Swofford 1999) using a Hasegawa-Kishino-Yano model (HKY+I+G) with a transition/transversion ration = 12.49, a proportion of invariant sites = 0.59 and a gamma distribution shape parameter = 1.46. Nodal support was evaluated with 100 ML bootstrap replicates in GARLI v0.951 using estimated model parameters, random starting trees and default parameters for the genetic search algorithm (Zwickl 2006). For Bayesian methods the dataset was divided into two partitions by codon position. The appropriate nucleotide substitution model for each partition was determined using MrModeltest (Nylander 2004) under the AIC criterion. The mixed model analysis of the partitioned dataset was undertaken in MrBayes v3.1 (Huelsenbeck and Ronquist 2001). The analysis consisted of 2 runs of 4 chains each (1 cold and 3 heated) under the default settings. The analysis was run for 10,000,000 generations, sampling every 1000 generations until the average deviation of split frequencies was less than or equal to 0.01 and the effective sample size (ESS) values for each parameter in Tracer v1.4 (Rambaut and Drummond 2007) were greater than 100. The posterior distribution of trees and model parameters were summarized using the “sumt” and “sump” commands in MrBayes discarding the first 25% (2500) of trees as burn-in.

Results

We obtained COI sequences totaling 251 nucleotides from two of the four specimens of C. carolinensis sampled. The sequences were obtained from two overlapping PCR products (using the primer pairs L7773 – H 7879 and L7804 – COIa) that were sequenced from replicated DNA extracts and replicated PCRs. Sequences from the two Carolina Parakeets differed by a single, synonymous nucleotide substitution at a third position site of a serine codon (a six-fold degenerate amino acid). Both sequences were unambiguously aligned with sequences totaling 570 nucleotides from 47 other parrot species (GenBank accession numbers in Table 1). Uncorrected pairwise genetic distances (p-distances) between C. carolinensis and other Arini species ranged from 5.3% (Aratinga mitrata) to 12.8% (Pionopsitta vulturina).

The ML search recovered two equally likely trees (ML scores = 5880.14), which did not differ in topology. More recent nodes in these trees were generally supported in the ML boostrap analysis and the Bayesian analysis, but more basal relationships differed among analyses and generally had low nodal support (Fig. 1). All analyses placed the two Conuropsis carolinensis samples together with strong support and sister to a well-supported clade of eight Aratinga species that includes the most northern extant species of the genus (A. holochlora) and species endemic to Cuba (A. euops), Hispaniola (A. chloroptera), and Socorro Island (A. brevipes). The Conuropsis plus northern Aratinga clade is nested within a well-supported (64% ML bootstrap, 1.0 Bayesian posterior probability) clade of long-tailed Neotropical parakeets and macaws. Members of the genus Aratinga were found in three distinct and well-supported clades that each contained members of other genera (Fig. 1). All analyses also recovered a well-supported (74% ML bootstrap, 1.0 Bayesian posterior probability) clade of short-tailed Neotropical parrots from the genera Amazona, Gradydidascalus, Pionus, Pionopsitta, and Haplosittaca.

Discussion

Systematics of Conuropsis carolinensis. - We obtained the first DNA sequence data from specimens of the extinct Carolina Parakeet. These data reveal a strongly-supported sister relationship with some species of Aratinga and help clarify the evolutionary relationships of this iconic North American parrot. Although maximum likelihood bootstrap and Bayesian support is low for many nodes that resolve generic relationships within the Arini, we find robust support for the placement of C. carolinensis within a diverse clade of long-tailed parakeets and macaws (Fig. 1). Within this clade C. carolinensis groups with northern and island-endemic species currently placed in the genus Aratinga, but it is clear that Aratinga itself is highly polyphyletic and in need of revision. Previous analyses have suggested that Aratinga is paraphyletic with regards to Nandayus (Ribas and Miyaki 2004; Tavares et al. 2006); ongoing work (by EES) on a much larger dataset of DNA sequences from a comprehensive taxonomic sample of Arini should help resolve relationships in this diverse group. We find no evidence for the previously hypothesized close relationship with Monk Parakeet, Myiopsitta monachus (Snyder 2004). Monk Parakeets are native to temperate southern regions of South America and have established year-round breeding colonies in several large metropolitan areas of North America that experience long harsh winters (Spreyer and Bucher 1998). Our data suggest that the shared presence of feathered ceres and other cold tolerances in Conuropsis and Myopsitta represent convergent traits in these temperate-dwelling taxa.