Nuclear DNA from a 180Yearold Study Skin Reveals the Phylogenetic Position of the Kinglet Calyptura Calyptura Cristata
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Ibis (2012), 154, 533–541 Nuclear DNA from a 180-year-old study skin reveals the phylogenetic position of the Kinglet Calyptura Calyptura cristata (Passeriformes: Tyrannides) JAN. I. OHLSON,1* MARTIN IRESTEDT,1 JON FJELDSÅ2 & PER G. P. ERICSON3 1Molecular Systematics Laboratory, Swedish Museum of Natural History, Box 50007, SE-104 05, Stockholm, Sweden 2Zoological Museum, University of Copenhagen, Universitetsparken 15, DK-2100, Copenhagen, Denmark 3Director of Science, Swedish Museum of Natural History, Box 50007, SE-104 05, Stockholm, Sweden The Kinglet Calyptura Calyptura cristata is one of the most enigmatic bird species in South America, known only from specimens collected in the 19th century and a few recent observations. Knowledge of its biology is scanty and its systematic position is obscure. Traditionally, Calyptura was placed in the Cotingidae, but associated with genera that are now known to fall outside the Cotingidae. In an attempt to clarify its phylogenetic position, sequence data from four nuclear markers were obtained from a 180-year-old museum study skin of Calyptura, and incorporated into a comprehensive dataset of tyrant flycatchers, cotingas, manakins and allies. Our analyses demonstrate that Calyptura is most closely related to Platyrinchus and Neopipo and that these three genera constitute a deep branch in the clade containing the Rhynchocyclidae (tody- tyrants and flatbills) and Tyrannidae (typical tyrant flycatchers). The Calyptura specimen is one of the oldest avian museum specimens from which a substantial amount of nuclear DNA sequence data have been obtained, and highlights the immense value of museum collections for DNA-based phylogenetic studies. Keywords: Platyrinchidae, Cotingidae, suboscines, nuclear DNA, museum specimen. The rare and enigmatic Kinglet Calyptura of Rio de Janeiro (Collar et al. 1992, Lambert & Calyptura cristata is one of the few members of Kirwan 2010). The species was rediscovered in Tyrannides (tyrant flycatchers, cotingas, manakins 1996 when a pair was observed near Teresópolis, and allies) whose relationships are still unknown. northeast of Rio de Janeiro (Pacheco & Fonseca Due to the almost complete lack of behavioural 2001). Subsequently, claims have been made that and anatomical data, its taxonomic history has the species was observed on three occasions near been rather uneventful, although speculative. It Ubatuba in the coastal part of São Paulo state, but was placed in Cotingidae by Sclater (1888), based these have been questioned (Lambert & Kirwan solely on its pycnaspidean tarsus, and this has 2010). remained its position until recently, when it was There are no anatomical specimens (skeletons given an incerta sedis position in some recent or whole birds stored in alcohol) and information systematic revisions (e.g. Remsen et al. 2012). on its ecology and behaviour is scanty and anec- All specimens of Calyptura were collected in dotal. P. W. Lund, who collected three specimens the 19th century, but for most of the c. 55 speci- (of which one was used for our molecular study) mens, precise locality data are either missing, or near Rosário in 1827–28, stated that they hopped are vague and potentially misleading. The only around in trees or shrubbery, one uttering a spar- specimens with reasonably precise and traceable row-like chirp, and that stomach contents locality data are from the Rosário area, northeast included small insects and probably also seeds (Krabbe 2007). Other information, summarized *Corresponding author. by Lambert and Kirwan (2010), indicates that the Email: [email protected] species inhabits the higher and wilder places in © 2012 The Authors Ibis © 2012 British Ornithologists’ Union 534 J. I. Ohlson et al. montane forest, sometimes at forest edge and in sequence data were incorporated into a compre- secondary growth, where it moves through both hensive dataset of Tyrannides, mostly consisting of canopy and lower shrubbery at edges, exploring data downloaded from GenBank. vine tangles and epiphytes in search of small ber- ries and insects, and that it has a surprisingly loud METHODS voice. Berries of a species of Solanacean scrub are mentioned as a potential food source and it has DNA was extracted from a c.29 2 9 1-mm also been suggested that the species may have a piece of tissue taken from the inside of an open predilection for mistletoe fruit (Snow 2004, Lam- study skin of Calyptura cristata. Extraction proce- bert & Kirwan 2010). dures largely followed those used in Irestedt et al. Habitat loss is likely to have caused a serious (2006), using the Qiagen DNeasy Tissue Micro decline in the population of Calyptura (Collar Kit, with some modification to their recom- et al. 1992, Lambert & Kirwan 2010). However, mended protocol; 20 lL of DTT (dithiothreitol) there may be additional explanations for the virtu- was added to break disulfide bonds; and an addi- ally complete absence of records from the entire tional 10–20 lL of Proteinase K was added 20th century. Lambert and Kirwan (2010) con- roughly half-way through the lysis process, as sider several possible explanations pertaining to its Proteinase K may become inactive or depleted ecology, such as nomadism or altitudinal migra- during long lysis periods (in this case c. 24 h). In tion, microhabitat specialization or a highly cryp- the elution stage, elution buffer volume was tic behaviour. They also suggest that part of the decreased to yield a higher DNA concentration problem may be purely logistic: large portions of and the sample was incubated for 5 min. The the remaining potential habitat are never visited extraction from the Calyptura specimen was car- and it is generally difficult to obtain good views of ried out in separate facilities dedicated to extract- the forest canopy at many places that receive ing old DNA. All equipment and surfaces were regular visits from ornithologists and birdwatchers. sterilized before the extraction, by irradiation with The original reason for placing Calyptura in ultraviolet light or sanitized with 10% bleach. Cotingidae was its type of tarsal scutellation PCR-amplification and sequencing of frag- (Sclater 1888) but molecular systematic studies mented DNA from old study skins require careful have demonstrated that tarsal scutellation is of lit- primer design, as target regions need to be broken tle phylogenetic value in Tyrannides. In his review down into short overlapping fragments. By using of Cotingidae systematics, Snow (1973) grouped primer combinations that amplify short DNA Calyptura with the three species of purpletuft fragments, the risk of amplifying contaminations is (Iodopleura), although he noted that the two were minimized, as study skins generally contain large not obviously related. In light of the molecular sys- numbers of short fragments, whereas longer frag- tematics of Tyrannides (Johansson et al. 2002, ments are absent or only exist in small numbers Chesser 2004, Ericson et al. 2006, Ohlson et al. (Irestedt et al. 2006). For our Calyptura specimen, 2008, Tello et al. 2009), there is little support for we used primers that amplified fragments of 200– a close relationship between Calyptura and Cotin- 250 bp, as this fragment length has been found to gidae. Iodopleura has now been transferred to the work for the majority of museum study skins Tityridae (Ericson et al. 2006, Ohlson et al. 2008, (Irestedt et al. 2006, and personal experience). Tello et al. 2009) and Calyptura is now often trea- However, for a few fragments we were able to ted as incerta sedis (Remsen et al. 2012). With the amplify almost 350 bp from our Calyptura speci- recent advances in laboratory procedures for work- men. A list of primers used in this study can be ing with old museum study skins (Irestedt et al. found in the supporting information (Table S1). 2006, Kirchman et al. 2010), it is now feasible to Thermocycling programmes varied depending on obtain high-quality DNA sequence data from spec- estimated annealing temperatures of the primers, imens well over 100 years of age. Here we present but typically involved a 5-min denaturation at an attempt to assess the systematic position of 95 °C followed by three phases of denaturation Calyptura cristata, using DNA sequence data from (95 °C) for 40 s, annealing (typically 55–65 °C) four different nuclear markers. The DNA was for 1 min and extension (72 °C) for 1 min, with a extracted from a tissue sample of one of the speci- decrease in annealing temperature of 2–3 °C mens collected by P. W. Lund in 1827–28 and the between each phase. The first two phases were © 2012 The Authors Ibis © 2012 British Ornithologists’ Union Relationships of Calyptura cristata 535 run for four cycles and the third for 32–36 cycles. temperature of 0.2. All BI analyses were per- A final extension phase at 72 °C was run for formed with four chains, one cold chain and three 8 min. incrementally heated chains and were run for 25 All the PCR products were loaded on an aga- million generations, with trees sampled every rose gel for electrophoresis and the target bands 1000 generations. Trees saved before the target were excised and cleaned from gel remains using distribution had been reached (the burnin phase) the Sigma GelClean Kit. Strong bands were were discarded and the final phylogenetic tree was sequenced directly, whereas weaker bands were estimated from 15 000 trees from each run. reamplified using the same settings and primer combinations as in the initial PCR. Sequencing RESULTS was carried out on an ABI 3130xl capillary array, using the same primers that had been used during Sequence characteristics PCR-amplification. Each segment of the target sequences was checked separately against the cor- We obtained sequence data from RAG-1, myoglo- responding sequence segments in a number of bin intron 2, ODC introns 6–7 and G3PDH other Tyrannides species to control for the intron 11 for Calyptura cristata.