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Bird identification: song two low, two high, five short


This morning I heard a bird that had a song which was two low notes, two high notes, and five quick notes in succession. Location is New England.

Is there any way, short of asking an expert, that I can find out what a bird is from a description of its song or call?

Update - Bird Song Uploaded

I have been able to host a sound clip on Vocaroo:

Unknown bird, Massachusetts, forested suburban environment


Best guess is that the bird is a Song Sparrow, Melospiza melodia. Cornell Lab of Ornithology has a good website for basic facts about birds. Here is the account for Song Sparrow. There is a sample of typical song, but there can occasionally be some variety.

The song is kind of complex and the recording you linked to seems to garble or not capture some of the song, but the changes in phrases throughout the duration seem to match as does the pitch of most of the phrases.


Common backyard birds of Ohio (lists, photos, ID)

I've put this resource together for you to answer the question: What birds are in my backyard in Ohio?

This article tells you what Ohio birds you can expect in your backyard and when they are most common. I also provide a photo and description section to help you with Ohio bird identification of the most common birds native to Ohio backyards.

The most common backyard birds throughout the year in the state of Ohio are these:

  1. Northern Cardinal (55% frequency)
  2. American Robin (50%)
  3. Blue Jay (45%)
  4. Mourning Dove (40%)
  5. Song Sparrow (40%)
  6. American Goldfinch (39%)
  7. Downy Woodpecker (38%)
  8. Red-bellied Woodpecker (36%)
  9. European Starling (34%)
  10. American Crow (32%)
  11. White-breasted Nuthatch (31%)
  12. Tufted Titmouse (30%)
  13. House Sparrow (29%)
  14. Common Grackle (25%)

These species occur on more than 25% of all eBird checklists for the state.

In this article:
Lists of the most common backyard birds of Ohio
Photos and ID of the most common backyard birds in Ohio
Other common birds you might see from your backyard in Ohio
Comparison of the most common backyard birds of Columbus, Ohio
Beyond your backyard

When we say "most common" when discussing eBird checklists, we're really talking about frequency, not absolute numbers. What birds do you see and hear the most frequently? Let's consider an example that explains the difference.

Downy Woodpeckers may occur widely over a large area in small numbers. But there may be far more Northern Pintails by number that only occur on one marsh in your area. The Downy Woodpecker would occur on checklists from many areas, the ducks from only checklists near the marsh, and not on other checklists except as occasional flyover sightings. So the woodpecker would be seen on a higher percentage of checklists from the larger area.

The percentage in parentheses, following the names on the above list, is the percentage of total bird checklists for the entire state that recorded each species. I created each of these lists from real bird watcher data on the eBird site.


Introduction

Millions of sequence variants have been identified through genome sequencing of cancer samples over the past decades [1]. The ability for us to interpret the functional impacts of these variants on cancer development remains poor. Pathogenic variants were mostly identified by the association of the variant with disease status, either in families or in a large cohort of people [2]. However, such information on the carriers of the variants is often difficult to collect. As a result, many of the genetic variants are classified as “variants of uncertain significance (VUS)” even though they are in well-known cancer genes. For example, for BRCA1 and BRCA2, two genes that are targets for prevention and therapy of several types of cancers [3], more than half of the 5095 and 8010 single nucleotide variants were classified as VUS or “conflicting interpretations” in the ClinVar database as of January 2020 [4].

In vitro functional assays have been used as important supporting evidence for determining the pathogenicity of variants [2], yet the method to introduce mutations into cells has limited their scalability. Most methods utilized exogenous vectors to carry cDNAs with mutations as transgenes while inhibiting or deleting the endogenous copy of the target gene [5,6,7]. These strategies could only assay mutations in coding regions and the mutations are not tested in their native genomic context. The newly developed CRISPR-Cas9 system is a way to directly introduce mutations in both coding and noncoding regions in the genome [8, 9]. Cas9-sgRNA complex could induce DNA double-strand breaks at target sites, which can be repaired to desired genotype in the presence of a repair template [10]. Saturated mutagenesis using CRISPR-Cas9 system with massive synthetic repair templates has been successfully applied for the functional assessment of all the nucleotide variations in 13 of the 23 exons of BRCA1 [11]. However, this method requires generation of DNA double-strand breaks, which could induce p53-mediated growth arrest in some cell types [12]. Also, the low ratio of base substitutions to insertion or deletion after the repair [13], and the requirement of synthesis of a large amount of templates make the saturated genome editing only feasible for relatively small loci.

Base editors, which are usually built by fusing a deoxynucleotide deaminase to a nuclease-deficient or nickase Cas protein [14, 15], is an efficient way to generate direct base substitutions throughout the genome without inducing DNA double-strand breaks [16,17,18,19,20]. Applying cytosine base editors in genetic screens has allowed functional assessment of C→T or C→G mutations in human cells and yeast [21, 22]. In these studies, the requirement of a protospacer adjacent motif (PAM) limited the targeting scope of base editing screens. For the canonical SpCas9-based base editors, a PAM sequence of “NGG” 13 to 17 nucleotides downstream the target site is required for efficient base editing. Recent development of the SpCas9 variants has relaxed the PAM requirement to “NGN”, making it possible to evaluate a much larger group of sequence variants in base editing screens.

However, the variability in editing efficiencies poses a challenge for quantitative functional assessment of variants in base editing screens. In a pooled screen, the change of sgRNA abundance between two conditions is often used to evaluate the effects of sgRNAs [23]. Because the activities of sgRNAs to induce an editing event at a target site are highly variable [24, 25], the functional effect of a mutation could be masked by the low editing efficiency of its targeting sgRNA. In addition, several bases could be edited simultaneously in the editing window, but at different frequencies [24, 25], making the interpretation of sgRNA effects difficult.

In order to account for the variability in editing activities, we developed a framework to incorporate editing efficiency correction in base editing screens. We demonstrated that our efficiency correction framework improved the identification of loss-of-function variants from base editing screens. Applying base editing screens with efficiency correction, we assessed functional impacts of C·G→T·A or A·T→G·C conversions for about 9000 sites in BRCA1 and BRCA2 genes, and identified 910 variants that have negative effects on BRCA1/2 function. These variants include 185 variants that were marked as VUS or “benign/likely benign” in ClinVar, suggesting the power of the method for determining the functional significance for previously unknown variants.


2. Mark and Cut 2 pieces to attach the platform feeder to the post (6½ inches each).

3. Assemble the frame for the platform birdfeeder.

  • Drill holes for screws to prevent wood from splitting
  • Apply glue to surfaces to be attached
  • Attach with screws (two side pieces will be attached inside of the top and bottom pieces making the frame 24 inches in one direction and 25½ inches the other direction).
  • Cut the screen material and make sure to leave several inches overlap on each side.
  • Fold the extra screen material several times for strength to hold the staples and pull the material tight around a corner and attach with staples.

Click For Larger Photo

5. Mark the frame and screen supports for attachment of post attachment pieces.

  • The two frame and screen support pieces need to be set apart the width of the post (I used a 4X4, which was 3½ inches)
  • The two post attachment pieces also need to be set 3½ inches apart and need to be centered on the frame support pieces to help balance the platform on the post

6. Attach the frame and screen supports to post attachment pieces.

  • Attach with screws and glue or with bolts (bolts are much stronger for these small pieces of wood. Align them to the marks and drill holes for either the bolts or the screws (I used ¼ inch bolts where are larger than needed, but that's what I had on hand)

7. Attach support pieces to the Post and to the Platform Feeder

  • Drill holes and attach to post with screws as shown
  • Drill holes and attach the frame and screen support pieces to the platform frame with screws

Click For Larger Photo


The completed platform bird feeder can be seen in the photo below.

You may have noticed that the wooden 4x4 post is attached to one of the wire fence T-posts with "zip ties" or cable ties.

This is so we can test several places before deciding where to permanently fix the post and feeder.

Zip ties will last at least a year exposed to the sun before they deteriorate.

So if you use this idea, you will need to inspect and replace zip ties as needed or your feeder may fall over.

Click For Larger Photo


Materials and methods

Study area

The SNSM is an isolated mountain massif located in northern Colombia. Reaching elevations of nearly 5800 m and being only c. 40 km from the Caribbean coast, the SNSM is the highest coastal mountain range in the world. Owing to its isolation, the SNSM has high levels of endemism across multiple groups (Cleef et al., 1984 ).

Our study was conducted on the San Lorenzo slope, located in the north-western SNSM (Fig. 1). Mean annual precipitation in San Lorenzo is 2840 mm at 600 m and 2520 mm at 2250 m elevation (Cleef et al., 1984 ). We focused on lower- and upper-montane forests, which extend from c. 600 to 2500–2700 m and from c. 2500 to 2800 m, respectively. Lower-montane forests are characterized by trees with dense foliage and often with buttressed roots, and a 25–35 m-tall canopy arborescent ferns and palms are common in the understory, and vascular epiphytes are abundant (Cleef et al., 1984 ). Upper-montane forests are characterized by a high incidence of cloud cover. Trees are smaller (8–20 m), unbuttressed, and there are high densities of epiphytes (Bromeliaceae, Orchidaceae, pteridophytes, bryophytes and lichens). Owing to selective timber extraction and deforestation, some areas at upper elevations have been transformed into early secondary habitats, with Chusquea bamboo dominating the understory (Cleef et al., 1984 ).

Study system

The Grey-breasted Wood-wren is a highly vocal and territorial bird of the forest understory broadly distributed in montane areas of Central and South America (Kroodsma & Brewer, 2005 ). Recent studies focused on Ecuadorian populations (Dingle et al., 2008 , 2010 ) have demonstrated song divergence presumably driven by adaptation to different acoustic environments along an elevational gradient. Birds from lower elevations sang within more-restricted bandwidths and lower-frequency ranges than birds at higher elevations, possibly a result of cicadas calling at high frequencies having a dominant presence only at lower elevations. Vocal divergence also correlated well with genetic differentiation.

Two different forms of Grey-breasted Wood-wren referred to as H. l. bangsi and H. l. anachoreta replace each other along the elevational gradient in the SNSM (Bangs, 1899 Ridgway, 1903 ). Although Todd & Carriker ( 1922 ) indicated that these two forms occupy distinct elevations separated by a distributional gap, more recent data suggest they are mostly parapatrically distributed but with partial sympatry, with H. l. bangsi ranging from c. 600 to 2100 m, and H. l. anachoreta from c. 1800 to 3600 m (Hilty & Brown, 1986 ). Prior to this study, however, the distribution of these forms had been described based only on data from a few specimens and nonquantitative field observations. Therefore, the existence and extent of a zone of elevational overlap had been inferred only tentatively and the pattern of elevational replacement was not clear. Hence, we avoid referring to the different subspecies names owing to the uncertainty in their distributions and to the fact that, a priori, we were unable to rule out the possibility that variation along the elevational gradient was clinal. By studying patterns of morphological, vocal, genetic and behavioural variation along the elevational gradient in the SNSM, we can test predictions of the hypothesis of parapatric ecological speciation and also determine whether patterns of variation are consistent with those seen in similar studies in Ecuador (Dingle et al., 2008 , 2010 ), which would be suggestive of parallel adaptive differentiation in separate mountain systems.

Field sampling

To characterize patterns of genetic, phenotypic and vocal variation in San Lorenzo, we collected data along an elevational transect ranging from c. 1100 to 2810 m (the location of the San Lorenzo transect in the SNSM and the Neotropics is shown in Fig. 1). Sampling efforts were concentrated in three main areas: (1) around Finca La Victoria (1100–1360 m), (2) El Dorado Natural Bird Reserve (1780–2200 m) and (3) around the headquarters of the Sierra Nevada de Santa Marta National Park (2250–2810 m). In each of these areas, we collected samples for genetic analyses, took morphological measurements and recorded songs from birds in 20 territories. We also captured birds at other elevations to collect morphological data and samples for genetic analyses, seeking to cover the elevational gradient as thoroughly as possible.

Testing the geographic pattern of differentiation

To determine whether the two forms existing in the SNSM originated as a result of parapatric divergence from a single ancestor along the elevational gradient, or whether they colonized the area independently implying their divergence involved an allopatric phase (i.e. secondary contact), we examined their phylogenetic relationships with respect to other populations of H. leucophrys. We based our phylogenetic analyses on sequences of the mitochondrial ATPase 6 and ATPase 8 genes (842 base pairs) owing to the existence of previous and ongoing extensive phylogeographic studies of the genus Henicorhina using these markers (Dingle et al., 2006 , 2008 J.L. Pérez-Emán et al., unpubl. data).

Samples from 101 individuals captured using mist-nets were collected along the elevational gradient in the SNSM from June to July 2009. A sample of c. 0.05 mL of blood from the brachial vein was obtained with a heparinized capillary tube and stored in lysis buffer (White & Densmore, 1992 ). These samples were supplemented with five tissue samples associated with voucher specimens collected previously, for a total of 106 individuals. We extracted genomic DNA from blood or tissue using a DNeasy Kit (QIAGEN, Valencia, CA, USA), and we amplified and sequenced ATPase 6 and 8 using standard approaches (Cadena et al., 2007 ). We were able to generate ATPase sequences for 100 of the 106 samples available.

Preliminary phylogenetic analyses considering our 100 sequences from the SNSM and sequences of more than 300 individuals from populations thoroughly covering the distribution range of H. leucophrys and of the other three species in the genus Henicorhina indicated that all individuals from the SNSM are assignable to one of two strongly supported monophyletic groups nested within a South American clade, and that these populations are most closely related to neighbouring populations from Northern South America. Thus, to test whether the forms occurring in the SNSM are sister taxa (as predicted by the hypothesis of parapatric divergence) or not (a scenario favouring the hypothesis of double colonization), we conducted phylogenetic analyses using a database including only the five individuals from the SNSM with associated voucher specimens plus sequences from populations selected to represent biogeographic areas relevant to address the question of interest (Fig. 1, Table 1). Such areas corresponded to localities in Venezuela (Serranía de Perijá, Mérida Andes, Cordillera de la Costa, n = 5 individuals), Colombia (Serranía de Perijá and all three Andean cordilleras, n = 10), Panama (Chiriquí, Bocas del Toro, n = 2) and Ecuador and Northern Peru (localities on both slopes of the Andes, n = 5). We also included in analyses one sequence of H. leucophrys from Mexico and considered two sequences of the White-breasted Wood-wren (H. leucosticta, from Belize and Ecuador), together with representatives of the genera Cyphorhinus, Cantorchilus and Pheugopedius, as outgroup taxa (Table 1).

ID Taxon Catalogue No. Accession Reference Locality
1 Henicorhina leucophrys FMNH 393980 KC209528 This study Mexico, Jalisco, Sierra de Manantlan
2 H. leucophrys STRI JTW 133 AY304314 Dingle et al., 2006 Panama, Bocas del Toro, Palo Seco
2 H. leucophrys LSUMZ B-26442 KC209529 This study Panama, Chiriqui, Fortuna
3 H. leucophrys IAvH BT5224 KC209530 This study Colombia, Antioquia, Páramo Frontino
4 H. leucophrys IAvH 2151 KC209531 This study Colombia, Antioquia, El Encanto
5 H. leucophrys ANDES-BT AMC 739 KC209532 This study Colombia, Antioquia, Angelópolis
6 H. leucophrys IAvH- 4516 KC209533 This study Colombia, Risaralda, La Linda
7 H. leucophrys IAvH 12499 KC209534 This study Colombia, Valle del Cauca, Chicoral
8 H. leucophrys IAvH 13944 KC209535 This study Colombia, Cundinamarca, Los Robles
9 H. leucophrys ICN 32612 KC209536 This study Colombia, Meta, Serranía Aguas Claras
10 H. leucophrys ANDES-BT CDC 079 KC209537 This study Colombia, Magdalena, SNSM, San Lorenzo, 2000 m
10 H. leucophrys ANDES-BT AMR 016 KC209538 This study Colombia, Magdalena, SNSM, San Lorenzo, 2000 m
10 H. leucophrys ANDES-BT CDC 080 KC209539 This study Colombia, Magdalena, SNSM, San Lorenzo, 2400 m
10 H. leucophrys ANDES-BT CDC 081 KC209540 This study Colombia, Magdalena, SNSM, San Lorenzo, 2400 m
10 H. leucophrys ANDES-BT AMR 017 KC209541 This study Colombia, Magdalena, SNSM, San Lorenzo, 2400 m
11 H. leucophrys ANDES-BT AMC 1038 KC209542 This study Colombia, Cesar, Perijá, Manaure, San Antonio
11 H. leucophrys ANDES-BT NGP 036 KC209543 This study Colombia, Cesar, Perijá, Manaure, El Cinco
12 H. leucophrys ANDES-BT 0910 KC209544 This study Colombia, Norte de Santander, Tamá
13 H. leucophrys COP IC-807 KC209527 This study Venezuela, Zulia, Serranía de Perijá, Las Lajas
13 H. leucophrys COP IC-827 KC209526 This study Venezuela, Zulia, Serranía de Perijá, Las Lajas
14 H. leucophrys COP 07N0446 KC209525 This study Venezuela, Mérida, La Mucuy
15 H. leucophrys COP 07N0195 KC209524 This study Venezuela, Lara, PN Yacambú, El Blanquito
16 H. leucophrys COP JP408 KC209523 This study Venezuela, Yaracuy, Sierra de Aroa
17 H. leucophrys NA EU022425 Dingle et al., 2008 Ecuador, Pichincha, Bellavista
18 H. leucophrys NA EU022434 Dingle et al., 2008 Ecuador, Napo, Yanayacu
19 H. leucophrys CTR 02N9307 AY304309 Dingle et al., 2006 Ecuador, Morona-Santiago, PN Sangay
20 H. leucophrys NA EU022433 Dingle et al., 2008 Ecuador, Zamora-Chinchipe, Romerillos
21 H. leucophrys LSUMZ B-32605 AY304306 Dingle et al., 2006 Peru, Cajamarca
Henicorhina leucosticta CTR 00N0009 AY304334 Dingle et al., 2006 Belize, Cayo District, Chaa Creek
Henicorhina leucosticta CTR 00N0627 AY304331 Dingle et al., 2006 Ecuador, Loreto, Orellana
Cyphorhinus aradus CTR 00N3310 AY304300 Dingle et al., 2006 Ecuador, Napo, Loreto
Cantorchilus nigricapillus NA AY103284 Dingle et al., 2006 Panama
Pheugopedius rutilus LSUMZ 163699 AY103274 González et al., 2003 Panamá, Old Gamboa Road
Troglodytes aedon NA AY115237 Ricklefs & Bermingham, 2001 Lesser Antilles

Phylogenetic analyses were conducted using maximum-parsimony and maximum-likelihood methods. Maximum-parsimony analyses consisted of a heuristic search with 100 stepwise-addition replicates and 1000 bootstrap replicates in PAUP*4.0b10 (Swofford, 2002 ). For maximum-likelihood analysis, we implemented the TrN+I+G model of nucleotide substitution, selected as the best-fit to the data according to the Akaike and Bayesian information criteria in jmodeltest 0.1.1 (Posada, 2008 ). We ran 100 bootstrap replicates to assess branch support. We also used the program Network 4.5.0.0 (Bandelt et al., 1999 ) to generate median-joining haplotype networks to visualize relationships within major clades using data for all 100 individuals from the SNSM.

To examine statistical support for predictions of our alternative phylogenetic hypotheses (parapatric divergence predicts populations from the SNSM to be sister taxa, double colonization does not), we implemented an approximately unbiased test (Shimodaira, 2002 ) using PAUP*. Because phylogenetic analyses appeared to support the double-colonization hypothesis (see below), we tested whether a tree in which the forms from the SNSM were forced to be sister to each other was significantly less likely than the maximum-likelihood tree.

Testing for phenotypic divergence along the elevational gradient

To examine phenotypic variation along the elevational gradient in fitness-related traits (Smith et al., 2005a Milá et al., 2009 ), we measured standard morphological features with dial callipers (to the nearest 0.1 mm) from all individuals captured. Measurements included tarsus length, wing chord, tail length, exposed culmen length, total culmen length, bill depth and bill width (measured at the proximal edge of nostrils and at the commissures). We also recorded body mass to the nearest 0.25 g using a spring scale. All individuals were banded using numbered aluminium bands. Because we only considered data from adult birds, not all genotyped individuals were included in morphological analyses accordingly, the sample size was 81 individuals.

We used a principal components analysis (PCA) to reduce morphometric data to an uncorrelated set of variables, and then used factor scores obtained following varimax rotation to characterize variation along the elevational gradient. Visual inspection of patterns of morphometric variation as summarized by the first principal component (PC1) revealed the existence of two groups along the elevational gradient. To formally test the existence of two morphological groups of wood-wrens defined by PC1, we used a maximum-likelihood classification approach based on mixture models implemented in the mclust package for R (Fraley & Raftery, 2002 Fraley et al., 2012 ). Because this analysis confirmed the existence of two separate morphological groups (see Results), we obtained the probability that each individual bird belonged to either group using mclust and plotted this probability with respect to elevation.

From March to June 2009, we recorded five to ten ‘fast solo’ songs (Dingle et al., 2008 ) for 20 individuals in each of three different elevational zones: the two extremes of the gradient (1100–1360 m and 2270–2810 m) and intermediate elevations (1780–2200 m), where we suspected the two forms of wood-wren would overlap (Hilty & Brown, 1986 ). Recordings were made using a Marantz PMD661 Portable Solid State Recorder and a ME 67 Senheiser shotgun microphone with a K6 Sennheiser Power Supply. Song recordings were digitized at a sampling rate of 44 kHz, and sonograms were generated with a 5 ms frame-length using 1 ms time-steps in the software Luscinia (Lachlan, 2007 ). These settings resulted in a spectral resolution of 43 Hz for single recordings and 10.5 Hz for the individual means.

We measured spectral and temporal characteristics of songs on sonograms to examine vocal differentiation. We measured the following parameters for each song (Dingle et al., 2008 ): maximum and minimum frequency, maximum and minimum peak frequency (frequency with highest amplitude in a note) of songs, overall peak frequency (average of maximum and minimum peak frequency), song duration, duration of within-song silent interval and note duration in order to calculate the delivery rate (number of notes per second). We then reduced these variables using PCA and plotted scores with respect to elevation to characterize vocal variation along the gradient.

Examining patterns of genetic divergence

Because different kinds of genetic markers might introgress to different extents between populations owing to various evolutionary processes and to their different modes of inheritance (Sætre & Sæther, 2010 ), we examined the variation at multiple molecular markers along the elevational gradient. Specifically, we attempted to obtain allele or sequence data for all individuals sampled for: six unlinked autosomal microsatellite loci (Bowie et al., 2012 ), mitochondrial DNA (the ATPase 6 and 8 genes Eberhard & Bermingham, 2004 ), an autosomal nuclear intron (β-fibrinogen-5 Fuchs et al., 2004 ) and a sex-linked nuclear intron (CHDZ Griffiths & Korn, 1997 ). Because phylogenetic analyses of mtDNA data indicated that some individuals from the SNSM were closely related to individuals from populations in the Serranía de Perijá (see below), we also obtained microsatellite and β-fibrinogen-5 sequences for seven individuals from this area.

Microsatellite analyses were based on six polymorphic loci (10–25 alleles per locus Bowie et al., 2012 ). Allele sizes were estimated using GeneMapper 3.7 (Applied Biosystems, Foster City, CA, USA). Estimates of the frequency of null alleles and extent of large allelic dropout events were obtained using Micro-checker (Van Oosterhout et al., 2004 ). Assumptions of Hardy–Weinberg equilibrium and linkage disequilibrium were tested in Genepop 4.0.10 (Raymond & Rousset, 1995 ). No evidence of significant null-allele frequency or large allelic dropout events was found, and deviations from Hardy–Weinberg equilibrium and linkage disequilibrium were not significant for any locus (Bowie et al., 2012 ).

As described above, sequencing the mitochondrial ATPase 6 and ATPase 8 genes was based on standard protocols, which were also used with minor modifications (available upon request) to sequence the β-fibrinogen-5 and CHDZ nuclear introns. It was not necessary to phase sequences because all individuals were homozygous.

To examine whether two genetically distinct groups of wood-wrens exist in the SNSM with no hybridization or whether there was evidence for gene flow, we compared the patterns of variation across markers and related such patterns to elevation. We used structure 2.3.3 (Pritchard et al., 2000 ) to determine the number of genetically defined populations existing in the SNSM and to compute the posterior probability of assignment of individuals to each population using microsatellite data. structure analyses were conducted with an admixture model (α = 1) with a run length of 1 000 000 generations following a burn-in of 100 000 steps. To determine the number of populations (i.e. the most likely number of clusters, K), we used a heuristic approach (Evanno et al., 2005 ) by considering variation in likelihood between K = 1 through 10. The results of ten replicate analyses for each value of K were collectively summarized using the Greedy algorithm of CLUMPP 1.1.2 (Jakobsson & Rosenberg, 2007 ).

Based on patterns observed using mtDNA and β-fibrinogen-5 data, which revealed the existence of distinct lineages, we also assigned each sample to a clade for each of these markers. Because nuclear variation was reduced, we used a median-joining haplotype network for this purpose. Owing to the clear population structure observed (see below), we were able to assign each individual to mtDNA and nuclear DNA lineages unambiguously. With these data, we examined membership to different genetic groups along the elevational gradient.

Testing divergence in song perception

To determine whether vocal variation along the elevational gradient was related to patterns of conspecific recognition or aggressive response, we conducted song playback experiments following the design of Dingle et al. ( 2010 ). We conducted a total of 60 playback trials in three experiments. The first two experiments compared the behavioural response of territorial males of higher elevations (2270–2810 m) and lower elevations (1100–1360 m) to playback of their own songs (i.e. songs recorded in their elevational zone) and to foreign songs (i.e. songs from the other elevational zone). The third experiment compared the behavioural response to songs from high-elevation and low-elevation populations of males occurring at mid-elevations (1780–2200 m), where we expected the two forms to overlap.

We selected the best 10 ‘fast solo’ song recordings, each belonging to a different individual, to prepare 10 playback stimuli of the high-elevation population, and we did the same to prepare 10 playback stimuli of the low-elevation population. We applied a balanced reciprocal design in each of three playback experiments: at low-, mid- and high-elevation populations as defined above. Each experiment consisted of 20 trials, each conducted in one of 20 different territories. Our set-up limited the potential impact of pseudoreplication (Kroodsma, 1989 Slabbekoorn & Bouton, 2008 ) while optimally exploiting the available recordings. Playback trials lasted 30 min. The first 5 min was devoted to observations of ‘baseline behaviour’, followed by 5 min during which we recorded ‘response behaviour’ during and after a 2-min period of stimulus playback. Then, there was silence for 10 min, followed by the same procedure for the other stimulus. We used a Marantz PMD661 Portable Solid State Recorder and a portable field speaker SME-APS for song playback. Playback levels were standardized at 85 dB (A) at 1 m from the speaker, as measured with a Sphynx digital Sound Pressure Level meter. Speakers were located c. 1 m above the ground at locations near to sites where we had previously heard or seen territorial displays during monitoring visits.

We measured the strength of response to playback by evaluating three response measures: the approach-to-speaker distance, the total duration of vocal response and the response delay time. The approach distance was measured as the minimum distance between subject and speaker during the subsequent response periods in a trial. Distance categories were as follows: 0 = >16 m 1 = 8–16 m 2 = 4–8 m 3 = 2–4 m and 4 = <2 m (Nelson & Soha, 2004 Dingle et al., 2010 ). The duration of vocal response was measured as the total amount of time singing. The response delay time was defined as the time elapsed between start of playback and the initial response.

To investigate whether there was a difference in response to playback stimuli in the three populations, we first reduced all response variables using PCA to a single variable, indicating the aggressiveness of the response. Wilcoxon signed-ranks tests were used to test for significant differences in response to different stimuli at the different elevations using SPSS ( 2007 ).


MATERIALS AND METHODS

Crickets

Crickets, Gryllus assimilis (Fabricius 1775), were reared in plastic containers (60×40×44 cm) at 27°C, on a 14 h:10 h light:dark cycle, with ad libitum access to Purina cat chow and water. Males and females were separated before the final moult. One to two days before experiments, the crickets were individually isolated in inverted mesh-covered plastic cups, with food and water ad libitum. We used 3- to 10-day-old virgin females and 1- to 2-week-old males in all experiments.

Song recording and analysis

A male and a female were placed into a cylindrical (14×13 cm) open-top arena, the floor of which was a 15 cm Petri dish covered by a paper towel, and the walls of which were formed from aluminum screening. A microphone (Brüel and Kjær, type 4134, 0.5 inch Nærum, Denmark) was placed at a height of 5–6 cm from the top of the arena. The output of a measuring amplifier (Brüel and Kjær 2610) was digitized (100 kHz sampling rate) using a National Instruments multi-function interface (USB-6212, Austin, TX, USA), controlled by MATLAB (MathWorks, Natick, MA, USA) programs. Temporal parameters and power spectra of the songs were analyzed with CoolEdit (Syntrillium, Seattle, WA, USA) and TurboLab 4.0 (Bressner Technology, Gröbenzell, Germany). We analyzed songs of 21 males. We measured 11 temporal and three frequency parameters (Table 1), each measured for 10 instances of the relevant parameter in the same song.

Behavioral experiments

Trials were performed within the first 4 h of scotophase in an anechoic chamber at 24–26°C, illuminated by a red light. Courtship consists of a series of stereotypical behaviors, including production of a courtship song, that, if successful, culminates in mounting of the male by the female (Loher and Dambach, 1989 Adamo and Hoy, 1994). We introduced a male into the arena and after he appeared calm (in 3–5 min), we introduced a female. If no contact occurred within 5 min, or if the male failed to produce courtship song within 5 min after contact, the trial was discarded. If the female failed to mount the male within 5 min after the beginning of courtship the trial was scored as ‘no mounting’. We used each female only once males were used in up to four trials. After each trial, the arena was rinsed with 70% ethanol and the paper towel was replaced to remove any olfactory cues that might have been left by the crickets.


4 DISCUSSION

Our models suggest that under all projected SLR scenarios, and without adaptation by BSSP or accommodation by humans, near complete loss of BSSP habitat is likely throughout the SCB under high SLR scenarios. Carpinteria, currently the smallest study site, could support the last remaining BSSP population within fully tidal basins due to its relatively high-elevation marsh.

Our results are consistent with projected declines in other mid to high salt marsh species. Seaside sparrow habitat in Georgia is expected to decline between 2025 and 2050 (Hunter et al., 2016 ). Under high SLR scenarios, two high-elevation salt marsh birds, the Common Yellowthroat (Geothlypis trichas) and Marsh Wren (Cistohorus palustris), will likely become extirpated from the SFBE salt marsh within a century (Veloz et al., 2013 ). Small mammals, such as the salt marsh harvest mouse (Reithrodontomys raviventris), could be extirpated from areas currently dominated by pickleweed as sea-levels rise and that habitat disappears (Shellhammer, 1989 Swanson et al., 2014 ). High to moderate SLR, coupled with low sediment supply and insufficient area for shoreward retreat, could reduce habitat for many species besides BSSP.

BSSP might not readily disperse to better sites as suitable habitat is lost. Heavy industrialization and urbanization of the landscapes of southern California might further reduce BSSP dispersal by limiting connectivity between habitats. In a 1995–1997 study, BSSP were shown to have high site fidelity all monitored BSSP stayed within their current salt marsh (Powell & Collier, 1998 ). Furthermore, in the following year, 45.5% of banded male BSSPs in that same site occupied the same territory that they occupied when they were originally banded, highlighting their site fidelity. Reduced dispersal will make restoration more difficult if local populations are extirpated.

BSSP extirpation could occur before all habitats are submerged. For example, salt marshes smaller than 10 ha have been shown not to support BSSP breeding populations (Powell & Collier, 1998 Zembal et al., 1988 ). As habitat shrinks in area due to increasing inundation, it may also decline in quality, which might lead to breeding failure before all habitats are lost. Based on this threshold, extirpations could occur at Carpinteria and Seal Beach under a moderate SLR scenario by 2100 and 2040. Thus, a patchy distribution of marginal breeding habitat might preclude nesting well before our model predicts full breeding habitat loss.

Habitat change also depends on the extent to which that upland habitat will convert to salt marsh. Historically, this would have been a normal consequence of SLR. However, as the SCB has become more urbanized, BSSP are closer to the urban edge where they tend to do poorly. Perched BSSP react to pedestrians at distances between 47 and 63 m in southern California sites thus, increased SLR may increase disturbance rates (Fernandez-Juricic, Zahn, Parker, & Stankowich, 2009 ). Furthermore, increasing proximity to upland habitats could increase the frequency of interactions with upland predators such as red fox (Vulpes fulva) and raccoons (Procyon lotor), species that have been detected on the edge of Carpinteria Salt Marsh (Zembal et al., 2015 ). Common raven (Corvus corax) and American crow (Corvus brachyrynchus) are known nest predators of several threatened and endangered species in California (Liebezeit & George, 2002 ), and these impacts could also increase if BSSP habitat concentrates near uplands.

Future marsh elevation and associated habitat change depend on the extent that sediment supply will make up for SLR. Large storm events in the SCB have been known to rapidly increase elevations in mudflats and low marsh zones. For example, in Tijuana, high sedimentation rates during storms have led to an increase in elevation, and low to high marsh zone habitat conversion (Ward, Callaway, & Zedler, 2003 ). The same is true of Mugu, where low elevation areas have been repeatedly filled with sediment during storm episodes (Onuf, 1987 ). The potential for extreme sedimentation and transgression is different for each of these sites. For example, although catastrophic sedimentation from the rugged Santa Ynez Mountain watersheds have buried sections of Carpinteria under 20 cm of inorganic sediments, urban development has eliminated most of the upper marsh (Callaway, Jones, Ferren, & Parikh, 1990 ) and has altered connectivity to freshwater sources through concrete channelization (Sadro et al., 2007 ). Because sediment availability is dependent on infrequent storm events that are difficult to predict (Warrick & Farnsworth, 2009 ), future management of sediment supply and adjacent land use will play an important role in current habitat stability. Seal Beach provides a testing ground for managing BSSP through habitat restoration and increasing tidal marsh elevation by adding dredge spoils. At a 10 ha test site, dredge materials were applied to increase elevation suitable for cordgrass (Spartina foliosa), however, elevations and substrate may be more suitable for pickleweed habitat in the near future.

Our analyses suggest that the recent increase in BSSP counts in the SCB (Zembal et al., 2015 ) will likely reverse in the near future. Even before Pacific Coast salt marshes are completely submerged in 2110 (Thorne et al., 2018 ), our modeling predicts that there will be no suitable habitat for BSSPs under a high SLR scenario. Although habitat suitability could temporarily increase in two of the six salt marshes we studied under low SLR scenarios, local extirpations may occur. These losses could possibly be ameliorated with management intervention, restoration, and increasing transgression upland refugia habitat.


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Introduction

Ewing sarcoma is an aggressive soft tissue malignancy of children and adolescents, which is characterized by the chromosomal translocation leading EWS to fuse to FLI1 (1–4). Although about 70% of children with Ewing sarcoma can be cured by surgery and chemotherapy either with or without radiotherapy, only 30% of those with metastasis can be cured (5). Thus, new effective therapies are needed. Many pediatric solid tumors have activation of tyrosine kinases (TK), which play important roles in Ewing sarcoma biology (6, 7). For example, EWS–FLI1 fusion protein promotes the activities of TKs, including FAK, PDGFR, and IGF1R (8–11). Notably, targeting IGF1R by either small-molecule inhibitors or antibodies has enhanced patients' survival in several clinical trials (12).

Spleen tyrosine kinase (SYK) is a nonreceptor TK that is highly expressed in hematopoietic cells and regulates cellular adaptive immune responses (13). SYK also promotes cancer cell survival in leukemia and pediatric retinoblastoma (14). Small-molecule inhibitors of SYK (PRT062607 and GS-9973) have shown antineoplastic properties in these tumor types (15–17).

In this study, two unbiased high-throughput screens, a TK-focused siRNA library (18) and a small-molecule inhibitor library (19), were performed to identify signaling pathways which were valuable for therapeutic interventions. Through a series of functional investigations, we established a novel signaling pathway involving SYK/c-MYC/MALAT1 in the setting of Ewing sarcoma biology, and further showed its potential for therapeutic intervention for this pediatric malignancy.


Introduction

High-altitude environments pose numerous challenges to animal life. The physical environment changes dramatically on ascent, with declines in oxygen availability, temperature, air density and humidity. Despite these challenges, many animals live successfully in the high mountains. Birds are particularly diverse in montane regions – many live at over 4000 m above sea level and some surmount the world's highest mountain peaks during their migration (Fig. 1). Although some species are unique to high elevation, others are found across broad elevational gradients (McCracken et al., 2009b).

The decreases in total barometric pressure (hypobaria) and O2 partial pressure (hypoxia) at high altitude are inescapable, unlike elevational declines in temperature and humidity, which can be buffered by local climatic variation. Hypobaria has unique consequences for flying animals, because the mechanical power output needed to sustain lift increases in thin air (Altshuler and Dudley, 2006). This amplifies the already high metabolic rates needed for flapping flight (Chai and Dudley, 1995) in an environment where the O2 available to fuel metabolism is limited. According to Tucker, “some birds perform the strenuous activity of flapping flight at altitudes in excess of 6100 m, an altitude at which resting, unacclimated man is in a state of incipient hypoxic collapse” (Tucker, 1968). How then can O2 supply processes meet the high O2 demands of flight at high altitudes? What unique physiological characteristics allow the highest-flying species (Fig. 1) to sustain the most metabolically costly form of vertebrate locomotion at elevations that can barely support life in many other animals?

In order to properly address these questions, one must consider the properties of the pathway that transports O2 from the environment to the sites of O2 demand throughout the body. This pathway is composed of a series of cascading physiological ‘steps’ (Fig. 2): (1) ventilation of the lungs with air (2) diffusion of O2 across the pulmonary gas-exchange surface, from the air to the blood (3) circulation of O2 throughout the body in the blood (4) diffusion of O2 from the blood to mitochondria in tissues (the pectoralis muscle is the primary site of O2 consumption during flight) and (5) metabolic utilization of O2 to generate ATP by oxidative phosphorylation. Although not a strict component of the O2 transport cascade, properties of intracellular ATP turnover will also have important consequences for matching O2 supply and O2 demand. Not surprisingly, the answer to how birds fly at high altitudes lies, at least partly, in the characteristics of this pathway.

The objective of this Commentary is to review the importance of both ancestral and derived characteristics in the O2 transport pathway of birds that fly at high altitudes. Many features of birds in general probably endowed high fliers with numerous exaptations (also known as pre-adaptations), but many uniquely derived and presumably adaptive traits also appear to be important for high-altitude flight.

The benefits of being avian

The hypoxia tolerance of birds has frequently been suggested to be greater than that of mammals. Although some ectothermic vertebrates are even more tolerant of hypoxia, birds possess a relatively high tolerance when considering the increase in metabolic demands associated with endothermy. Early work showed that lowland house sparrows (Passer domesticus) behaved normally, and could even fly for short periods in a wind tunnel, at a simulated altitude of 6100 m (Tucker, 1968). In contrast, mice were comatose and unable to maintain body temperature at the same simulated altitude. Comparisons of the few species for which tolerance (survival) data are available also support the suggestion that birds are more tolerant of hypoxia than mammals (Thomas et al., 1995). However, this issue has yet to be addressed with rigorous phylogenetic comparisons that incorporate species in both groups that are adapted to hypoxia.

Although most birds live and fly at relatively low altitudes, species from several avian orders live, migrate or occasionally ascend much higher. These include multiple species of raptor, waterfowl, crane, passerine, hummingbird and others. The highest flight altitudes reported from various sources in the literature are shown here (Eastwood and Rider, 1965 Swan, 1970 Faraci, 1986 Faraci, 1991 del Hoyo et al., 1999 Kanai et al., 2000 McCracken et al., 2009b).

Although most birds live and fly at relatively low altitudes, species from several avian orders live, migrate or occasionally ascend much higher. These include multiple species of raptor, waterfowl, crane, passerine, hummingbird and others. The highest flight altitudes reported from various sources in the literature are shown here (Eastwood and Rider, 1965 Swan, 1970 Faraci, 1986 Faraci, 1991 del Hoyo et al., 1999 Kanai et al., 2000 McCracken et al., 2009b).

The O2 transport pathway of birds has several distinctive characteristics that should support a greater capacity for vigorous activity and aerobic metabolism during hypoxia (Fig. 2). Increases in breathing (i.e. ventilation) are an important response of the respiratory system to hypoxia, and the magnitude of this response is dictated primarily by the partial pressures of O2 and CO2 and the pH of arterial blood (Scott and Milsom, 2009). The decline in arterial O2 tension (hypoxaemia) drives the increase in ventilation, whose secondary consequence is an amplification of CO2 loss to the environment. This causes hypocapnia (low partial pressure of CO2 in the blood), which reflexively inhibits breathing and causes an acid–base disturbance. It has been suggested that birds have a higher tolerance of hypocapnia than mammals (Scheid, 1990), which could arise from an ability to rapidly restore blood pH in the face of CO2 challenges (Dodd et al., 2007) and from the hypocapnic insensitivity of the brain vasculature (see below). The significance of this tolerance is that it would allow birds to breathe more before depletion of CO2 in the blood impairs normal function, thus enhancing O2 transport to the gas-exchange surface.

The structure and function of the lungs is perhaps the best-known advantage of avian respiratory systems. The many distinctive features of bird lungs are the subject of an extensive literature that unfortunately can be dealt with only briefly here (Piiper and Scheid, 1972 Scheid, 1990 Maina, 2006). Air flows in one direction through the gas-exchange units of avian lungs (parabronchioles) and the arrangement of airway and vascular vessels creates a functionally cross-current gas exchanger (Fig. 3A). This differs substantially from the lungs of most other terrestrial vertebrates, in which gases flow in and out of terminal gas-exchange units (alveoli in mammals) such that capillary blood equilibrates with air having a uniform gas composition (uniform pool gas exchanger Fig. 3B). The important consequence of this difference is that avian lungs can attain a superior efficiency for gas exchange in normoxia and moderate hypoxia (as explained in Fig. 3), although their advantage diminishes as hypoxia becomes severe (Scheid, 1990). The capacity for pulmonary O2 diffusion is also greater in birds because of the exceptional thinness and large surface area of the exchange tissue. Nevertheless, the diffusion barrier appears to be mechanically stronger in birds than in mammals, so pulmonary blood flow and pressure can increase without causing stress failure (West, 2009). Each of these distinctive features of avian lungs should improve O2 loading into the blood during hypoxia.

The capacity for delivering O2 throughout the body in the systemic circulation may be higher in birds than in other vertebrates. Birds have larger hearts and cardiac stroke volumes than mammals of similar body size (Grubb, 1983), suggesting that birds are capable of higher cardiac outputs. If this were indeed the case, birds would have an enhanced capacity for convective delivery of O2 in the blood during hypoxia. Cardiac output increases sevenfold to eightfold during flight (Peters et al., 2005) and threefold or more during hypoxia at rest (Black and Tenney, 1980), but maximum cardiac output has yet to be determined in birds, particularly during flight in hypoxia.

The distribution of blood flow throughout the body has consequences for hypoxia tolerance, and the mechanisms regulating this distribution are altered in birds compared with mammals. Hypoxaemia per se causes a preferential redistribution of O2 delivery towards sensitive tissues like the heart and brain and away from more tolerant tissues (e.g. intestines). However, increases in O2 delivery to the brain are offset in mammals at high altitudes because of the respiratory hypocapnia induced by increases in breathing. This causes a constriction of cerebral blood vessels that can completely abolish the hypoxaemic stimulation of cerebral blood flow. In contrast, the cerebral vessels of birds are insensitive to hypocapnia, such that blood flow is allowed to increase and O2 delivery is maintained (Faraci, 1991). This and possibly other distinctive features of the avian cerebral circulation (Bernstein et al., 1984) should improve brain oxygenation during hypoxia. Coupled with the inherently higher tolerance of avian neurons to low cellular O2 levels (Ludvigsen and Folkow, 2009), the central nervous system of birds appears to be well protected from cellular damage induced by a lack of O2. Nevertheless, an intriguing question that has yet to be addressed is whether heightened blood flow increases intracranial pressure in birds, as frequently occurs in humans (Wilson et al., 2009). If so, birds may face the secondary challenge of avoiding or tolerating cerebral oedema and other neurological syndromes that can result from excessive intracranial pressure in mammals.

The transport of O2 occurs along several steps of a cascading physiological pathway from atmospheric air to the mitochondria in tissue cells (e.g. muscle fibres). The effectiveness of this pathway at transporting O2 during hypoxia is imperative for flight at high altitudes, which depends upon several distinctive characteristics of birds in general and many unique features that have evolved in high fliers. The properties of O2 utilization and ATP turnover in the flight muscle are also important to consider in high fliers, such as how ATP equivalents are moved between sites of ATP supply and demand [which can occur via phosphocreatine (PCr) by virtue of the creatine kinase shuttle see text]. Cr, creatine.

The transport of O2 occurs along several steps of a cascading physiological pathway from atmospheric air to the mitochondria in tissue cells (e.g. muscle fibres). The effectiveness of this pathway at transporting O2 during hypoxia is imperative for flight at high altitudes, which depends upon several distinctive characteristics of birds in general and many unique features that have evolved in high fliers. The properties of O2 utilization and ATP turnover in the flight muscle are also important to consider in high fliers, such as how ATP equivalents are moved between sites of ATP supply and demand [which can occur via phosphocreatine (PCr) by virtue of the creatine kinase shuttle see text]. Cr, creatine.

The capacity for O2 to diffuse from the blood into the tissues is higher in birds compared with mammals and other vertebrates. The best evidence for this difference is the systematically higher ratio of capillary surface area to muscle-fibre surface area in the flight muscle of birds compared with the locomotory muscles of mammals (Mathieu-Costello, 1990). At least two factors account for this difference: (1) the tight mesh of capillaries surrounding avian muscle fibres, due to a high degree of branching between longitudinal vessels, and (2) the smaller aerobic fibres of birds compared with similar-sized mammals (Mathieu-Costello, 1990). The heart and brain also have higher densities of capillaries in birds compared to mammals (Faraci, 1991). Diffusion of O2 from the blood to the mitochondria in various tissues should therefore be higher in birds than in other vertebrates during hypoxaemia.

Although these distinctive characteristics of birds should enhance hypoxia tolerance by improving the overall capacity for O2 transport, being avian is not in itself sufficient for flight at high altitudes. The flight muscle of birds has a very high aerobic capacity, by virtue of fast-contracting oxidative fibres (type IIa) that have abundant mitochondria (Mathieu-Costello, 1990 Scott et al., 2009b), and the high rates of metabolism during flight are supported primarily by lipid fuels (Weber, 2009). Lipid oxidation is essential for supporting long-duration flight, but it amplifies the amount of O2 required to produce a given amount of ATP when compared with carbohydrate oxidation. The metabolic demands of flight are further intensified at high altitudes by hypobaria, which requires that birds flap harder to produce lift (Chai and Dudley, 1995). The implication of these factors is that high-altitude flight requires very high rates of O2 transport when very little O2 is available. This is clearly not possible for most lowland birds – many species cannot tolerate severe hypoxia (Black and Tenney, 1980) and some fly long distances to avoid high-elevation barriers during their migration (Irwin and Irwin, 2005). What then are the uniquely derived attributes that differentiate the high fliers?

The unique attributes of high fliers

The physiology of birds that fly at high altitudes differs in many ways from that of lowland birds. The basis for this conclusion comes largely from studying the bar-headed goose [Anser indicus (Latham 1790)], a species that can tolerate severe hypoxia [∼21 Torr or ∼2.8 kPa (1 Torr=133 Pa), equivalent to 12000 m elevation] (Black and Tenney, 1980) and has been seen flying over the Himalayas at nearly 9000 m elevation during its migration between South and Central Asia (Swan, 1970) (Fig. 1). Studies of bar-headed geese have revealed many important insights into the physiological basis for high-altitude flight and, when coupled with comparative phylogenetic approaches, its evolutionary origins. My discussion of the unique attributes of high fliers will focus largely – out of necessity – on this species, but will also highlight work in other species when possible. Most of the previous work looking for inherent differences between high- and low-altitude birds compared animals in a common environment at sea level. This will be the case in the following discussion unless otherwise stated.

It is useful to begin this discussion by outlining the most influential steps in the O2 transport pathway during exercise in hypoxia. We have assessed this issue in waterfowl using theoretical modeling to calculate the physiological control coefficient for each step in the pathway (Fig. 4) (Scott and Milsom, 2006 Scott and Milsom, 2009). This approach allows physiological traits to be altered individually so that their influence on the whole O2 pathway can be assessed without compensatory changes in other traits. A physiological trait with a larger control coefficient will have a greater influence on flux through the pathway, so an increase in the capacity of this trait will have a greater overall benefit. Interestingly, the proportion of control vested in each step was dependent on the inspired O2 (Fig. 4). At sea level (inspired O2 tensions ∼150 Torr) and in moderate hypoxia (∼90 Torr, equivalent to ∼4500 m elevation), circulatory O2 delivery capacity (which incorporates both maximum cardiac output and blood haemoglobin concentration) and the capacity for O2 diffusion in the muscle retained most of the control over pathway flux (Fig. 4). In contrast, ventilation and the capacity for O2 diffusion in the lungs became much more influential in severe hypoxia (∼40 Torr, roughly 9000 m), whereas muscle diffusion remained important and circulatory O2 delivery capacity became less so (Fig. 4). These results suggest that every step in the O2 transport pathway can be influential and that the relative benefit of each step changes with altitude.

Schematics of (A) the cross-current model of gas exchange in the avian lung and (B) the uniform pool model of gas exchange in the lungs of mammals and most other terrestrial vertebrates. In the cross-current model, inspired air flows through rigid parabronchioles that are oriented perpendicular to blood capillaries. The partial pressure of O2 (PO2) in the parabronchioles (PPO2) declines along their length as O2 diffuses into the blood, such that capillaries leaving the exchanger near the entrance of airflow (right side of figure) take up more O2 than capillaries leaving near the exit (left side). The contents of all capillaries mix to dictate the PO2 of arterial blood (PaO2), which can have a higher PO2 than expired air (PEO2). In the uniform pool model, gas flows in and out of terminal alveoli. Capillary blood flowing past these alveoli extract O2, such that capillary PO2 rises and alveolar PO2 (PAO2) declines uniformly to less than the PO2 of gas that entered the alveoli. Arterial blood leaving the lungs has a PO2 that is at best equal to PAO2 (but is generally slightly less), which is less than the average PEO2. The cross-current model is therefore considered to be more efficient at gas exchange than the uniform pool model (Piiper and Scheid, 1972). PIO2, inspired PO2 PVO2, PO2 of venous blood.

Schematics of (A) the cross-current model of gas exchange in the avian lung and (B) the uniform pool model of gas exchange in the lungs of mammals and most other terrestrial vertebrates. In the cross-current model, inspired air flows through rigid parabronchioles that are oriented perpendicular to blood capillaries. The partial pressure of O2 (PO2) in the parabronchioles (PPO2) declines along their length as O2 diffuses into the blood, such that capillaries leaving the exchanger near the entrance of airflow (right side of figure) take up more O2 than capillaries leaving near the exit (left side). The contents of all capillaries mix to dictate the PO2 of arterial blood (PaO2), which can have a higher PO2 than expired air (PEO2). In the uniform pool model, gas flows in and out of terminal alveoli. Capillary blood flowing past these alveoli extract O2, such that capillary PO2 rises and alveolar PO2 (PAO2) declines uniformly to less than the PO2 of gas that entered the alveoli. Arterial blood leaving the lungs has a PO2 that is at best equal to PAO2 (but is generally slightly less), which is less than the average PEO2. The cross-current model is therefore considered to be more efficient at gas exchange than the uniform pool model (Piiper and Scheid, 1972). PIO2, inspired PO2 PVO2, PO2 of venous blood.

High capacities at several steps in the O2 transport pathway have been shown to distinguish high-flying birds from their lowland cousins (Fig. 2), confirming the theoretical predictions. The first step of this pathway, ventilation, appears to be enhanced in high-altitude birds to improve O2 uptake into the respiratory system. Bar-headed geese breathe more than low-altitude waterfowl when exposed to severe hypoxia (inspired O2 tensions ∼23–35 Torr or ∼3.1–4.7 kPa) (Black and Tenney, 1980 Scott and Milsom, 2007) and the magnitude of their ventilatory response is greater than in any other bird species yet studied (Scott and Milsom, 2009). Bar-headed geese also breathe with a more effective breathing pattern, taking much deeper breaths (i.e. higher tidal volumes) than low-altitude birds during hypoxia. There are at least two mechanistic causes for these differences: (1) ventilatory insensitivity to respiratory hypocapnia and (2) a blunting of the metabolic-depression response to hypoxia (Scott and Milsom, 2007 Scott et al., 2008). These differences increase the amount and partial pressure of O2 that ventilates the pulmonary gas-exchange surface during hypoxia. Bar-headed geese also have enlarged lungs (Scott et al., 2011), as do numerous other highland species sampled at high altitudes (Carey and Morton, 1976), which should enhance the second step of the O2 transport pathway by increasing the area of the gas-exchange surface. The respiratory system of high-altitude birds therefore seems capable of loading more O2 into the blood during hypoxia than that of lowland birds.

Physiological control analysis of flux through the O2 transport pathway in waterfowl. The influence of the respiratory system (ventilation, V, and the capacity for pulmonary O2 diffusion, Dp) on O2 transport increases and that of circulatory O2 delivery capacity (Q the product of maximum cardiac output and 4× blood haemoglobin concentration) declines as hypoxia becomes more severe. The capacity for O2 diffusion in the muscle (Dm) has a large influence on O2 transport at all partial pressures of inspired O2. Control coefficients were calculated using theoretical modelling of the respiratory system with a haemoglobin P50 that is typical of highland birds (25 Torr or 3.3 kPa), and are defined as the fractional change in O2 transport rate divided by the fractional change of any given step in the O2 transport pathway. Expressed as a percentage, the control coefficients for all steps in the pathway will sum to 100. Modified from Scott and Milsom (Scott and Milsom, 2006 Scott and Milsom, 2009).

Physiological control analysis of flux through the O2 transport pathway in waterfowl. The influence of the respiratory system (ventilation, V, and the capacity for pulmonary O2 diffusion, Dp) on O2 transport increases and that of circulatory O2 delivery capacity (Q the product of maximum cardiac output and 4× blood haemoglobin concentration) declines as hypoxia becomes more severe. The capacity for O2 diffusion in the muscle (Dm) has a large influence on O2 transport at all partial pressures of inspired O2. Control coefficients were calculated using theoretical modelling of the respiratory system with a haemoglobin P50 that is typical of highland birds (25 Torr or 3.3 kPa), and are defined as the fractional change in O2 transport rate divided by the fractional change of any given step in the O2 transport pathway. Expressed as a percentage, the control coefficients for all steps in the pathway will sum to 100. Modified from Scott and Milsom (Scott and Milsom, 2006 Scott and Milsom, 2009).

The circulatory delivery of O2 throughout the body is also enhanced in high-altitude birds. The most pervasive mechanism for sustaining the circulation of O2 in hypoxia is an alteration in the O2-binding properties of haemoglobin in the blood. Numerous high-altitude birds, such as the bar-headed goose (Fig. 5), Andean goose (Chloephaga melanoptera) (Black and Tenney, 1980), Tibetan chicken (Gallus gallus) (Gou et al., 2007) and Ruppell's griffon (Gyps rueppellii) (Weber et al., 1988), are known to possess haemoglobins with an increased O2 affinity. This can dramatically increase O2 delivery and pulmonary O2 loading in hypoxia by increasing the saturation of haemoglobin (and thus the O2 content of the blood) at a given O2 partial pressure (Fig. 5A), and can, in doing so, greatly improve flux through the O2 transport pathway (Scott and Milsom, 2006). The genetic and structural bases for haemoglobin adaptation to high altitude have been resolved in many species. For example, the bar-headed goose possesses a major (HbA) and minor (HbD) form of haemoglobin, whose α subunits contain four (α A ) (Fig. 5B) and two (α D ) uniquely derived amino-acid substitutions, respectively (McCracken et al., 2010). One of the substitutions in α A (Pro-119 to Ala) (green in Fig. 5B) is thought to cause a large increase in O2 affinity (Jessen et al., 1991) by altering the interaction between α and β subunits and destabilizing the deoxygenated state of the protein (Zhang et al., 1996). Parallel genetic changes can sometimes arise in the haemoglobin of different highland species (e.g. Andean waterfowl) (McCracken et al., 2009b). Highland haemoglobin genotypes can even be maintained when gene flow from low altitudes is high, presumably because they are strongly favoured by natural selection (McCracken et al., 2009a).

High-altitude adaptations in the haemoglobin (Hb) of bar-headed geese. (A) The O2 affinity of bar-headed goose Hb is higher than that of lowland waterfowl, as reflected by a leftward shift in the O2 equilibrium curve of blood (measured at a pH of 7.3). Redrawn from Black and Tenney (Black and Tenney, 1980). 1 Torr=133 Pa. (B) The α A subunit of bar-headed goose Hb contains four uniquely derived amino-acid substitutions (blue and green). Ala-119 (green) has a large influence on O2 binding because it alters the interaction between α and β subunits. For simplicity, only one out of two α and β subunits that compose the complete Hb tetramer are shown. This cartoon was drawn in Pymol from the previously published structure of oxygenated Hb (Zhang et al., 1996) (Protein Data Bank ID, 1A4F).

High-altitude adaptations in the haemoglobin (Hb) of bar-headed geese. (A) The O2 affinity of bar-headed goose Hb is higher than that of lowland waterfowl, as reflected by a leftward shift in the O2 equilibrium curve of blood (measured at a pH of 7.3). Redrawn from Black and Tenney (Black and Tenney, 1980). 1 Torr=133 Pa. (B) The α A subunit of bar-headed goose Hb contains four uniquely derived amino-acid substitutions (blue and green). Ala-119 (green) has a large influence on O2 binding because it alters the interaction between α and β subunits. For simplicity, only one out of two α and β subunits that compose the complete Hb tetramer are shown. This cartoon was drawn in Pymol from the previously published structure of oxygenated Hb (Zhang et al., 1996) (Protein Data Bank ID, 1A4F).

The circulation of O2 may also be sustained in hypoxia by specializations in the heart that safeguard cardiac output. Bar-headed geese have a higher density of capillaries in the left ventricle of the heart (Fig. 6A), which should help maintain the O2 tension in cardiac myocytes and thus preserve function when hypoxaemia occurs at high altitudes (Scott et al., 2011). Cellular function could also be challenged if the production of reactive O2 species increases at high altitudes, as occurs in some lowland animals when declining O2 levels at cytochrome c oxidase (COX, the enzyme that consumes O2 in oxidative phosphorylation) shift the electron transport chain of mitochondria towards a more reduced state (akin to a buildup of electrons) (Aon et al., 2010). However, COX from bar-headed goose hearts has a higher affinity for its substrate (cytochrome c in its reduced state) (Fig. 6B), which could allow the electron transport chain to operate in a less reduced state and thus minimize oxidative damage by reactive O2 species (Scott et al., 2011). A possible cause of this difference is a single mutation in subunit 3 of the COX protein, which occurs at a site that is otherwise conserved across vertebrates (Trp-116 to Arg) (green in Fig. 6C) and appears to alter inter-subunit interactions (Scott et al., 2011). These (and likely other) unique specializations may explain how bar-headed geese maintain arterial blood pressure and increase cardiac power output to deeper levels of hypoxia than Pekin ducks (G.R.S. and W. K. Milsom, unpublished). Cardiac specializations in high-altitude birds may have a transcriptional basis, based on a comparison of cardiac gene expression in late-stage embryos of Tibetan chickens and lowland breeds (Li and Zhao, 2009): embryonic hypoxia altered the expression of over 70 transcripts in all chickens, but an additional 12 genes (involved in energy metabolism, signal transduction, transcriptional regulation, cell proliferation, contraction and protein folding) were differentially expressed in only the highland Tibetan breed. Overall, these findings lend some credence to a previous suggestion that the hypoxaemia tolerance of the heart has a strong influence on the ability to fly at high altitudes (Scheid, 1990).

Cardiac adaptations to high altitude in bar-headed geese. (A) Capillary density is enhanced in the hearts (left ventricle) of bar-headed geese compared with low-altitude geese. Insets are representative images of capillary staining in bar-headed geese (left), pink-footed geese (centre) and barnacle geese (right). Scale bar, 100 μm. (B) Cytochrome c oxidase (COX) from the hearts of bar-headed geese has a different maximal activity (lower Vmax) and substrate kinetics (lower Km for cytochrome c [Fe 2+ ], cytochrome c in its reduced state) than COX from the two species of low-altitude geese. Asterisk represents a significant difference from both low-altitude species. (C) COX subunit 3 (COX3) of bar-headed geese contains a single amino acid mutation at a site that is otherwise conserved across all vertebrates (Trp-116 to Arg) and is predicted by structural modeling to alter the interaction between COX3 and COX1. Modified from Scott et al. (Scott et al., 2011).

Cardiac adaptations to high altitude in bar-headed geese. (A) Capillary density is enhanced in the hearts (left ventricle) of bar-headed geese compared with low-altitude geese. Insets are representative images of capillary staining in bar-headed geese (left), pink-footed geese (centre) and barnacle geese (right). Scale bar, 100 μm. (B) Cytochrome c oxidase (COX) from the hearts of bar-headed geese has a different maximal activity (lower Vmax) and substrate kinetics (lower Km for cytochrome c [Fe 2+ ], cytochrome c in its reduced state) than COX from the two species of low-altitude geese. Asterisk represents a significant difference from both low-altitude species. (C) COX subunit 3 (COX3) of bar-headed geese contains a single amino acid mutation at a site that is otherwise conserved across all vertebrates (Trp-116 to Arg) and is predicted by structural modeling to alter the interaction between COX3 and COX1. Modified from Scott et al. (Scott et al., 2011).

The capacity for O2 to diffuse from the blood to mitochondria in the flight muscle is also enhanced in high-altitude birds. Andean coot (Fulica americana peruviana) populations that reside and were sampled at high altitudes had a higher capillarity and a smaller fibre size in the flight muscle than populations residing at low altitudes (León-Velarde et al., 1993). Because there were no differences in muscle aerobic capacity between coot populations, the increase in O2 diffusing capacity should serve to improve O2 transport in hypoxia rather than to match differences in cellular O2 demands. Similar differences exist between bar-headed geese and lowland waterfowl from a common environment at sea level (Scott et al., 2009b). Mitochondria are also redistributed closer to capillaries in the aerobic fibres of bar-headed geese (Scott et al., 2009b), which reduces intracellular O2 diffusion distances. These various mechanisms for improving the diffusion capacity for O2 in the flight muscle should help sustain mitochondrial O2 supply when hypoxaemia occurs at high altitudes.

In addition to improvements in the capacity to transport O2 during hypoxia, various features of metabolic O2 utilization and ATP turnover are altered in the flight muscle of high-altitude birds. This does not generally include changes in the inherent metabolic capacity of individual muscle fibres, based on observations in bar-headed geese of the abundance and respiratory capacities of mitochondria as well as the activities of metabolic enzymes (Scott et al., 2009b Scott et al., 2009a). However, inherently higher aerobic capacities can exist for the whole muscle by virtue of increases in the proportional abundance of aerobic fibres (Scott et al., 2009b). Furthermore, the metabolic capacity of individual fibres can sometimes (Mathieu-Costello et al., 1998), but not always (León-Velarde et al., 1993), increase after high-altitude acclimatization. Increases in aerobic capacity, and the associated increases in overall mitochondrial abundance, could be important for counterbalancing the inhibitory effects of low O2 levels on the respiration of individual mitochondria [this strategy is discussed in Hochachka (Hochachka, 1985)]. Mitochondrial ATP production is also more strongly regulated by creatine kinase in bar-headed geese than in low-altitude waterfowl (Scott et al., 2009a) and the expression of mitochondrial creatine kinase is upregulated by hypoxia in Tibetan chickens (Li and Zhao, 2009). A potential consequence of these alterations is that energy supply and demand in the muscle is better coupled via the creatine kinase shuttle, a system important for moving ATP equivalents around the cell [this system is described in Andrienko et al. (Andrienko et al., 2003)]. An interesting possibility is that bar-headed geese developed a more active shuttle to compensate for the redistribution of mitochondria, which moved these organelles closer to capillaries but further from the contractile elements that constitute the major sites of ATP demand in the flight muscle.

Can flapping flight be sustained above the high peaks?

It has been suggested that the iconic migration of bar-headed geese, which takes some individuals of this species over the highest peaks in the Himalayas, is impossible without vertical wind assistance (Butler, 2010). This suggestion was based on the observation that captive bar-headed geese forced to run on a treadmill do not perform as well in hypoxia (inspired O2 tension ∼50 Torr or ∼6.7 kPa) as in normoxia (Fedde et al., 1989). However, it was clearly not an impairment of the cardiorespiratory system at supplying O2 that impaired running performance in this study, as ventilation and cardiac output were both well below what can be sustained by this species during severe hypoxia at rest (Black and Tenney, 1980 Scott and Milsom, 2007). The more parsimonious explanation is that the leg muscles cannot sustain high activity during hypoxaemia, which is not terribly surprising given that this tissue is inactive when bar-headed geese fly at high altitudes. Nevertheless, the possibility that some of the highest-flying birds depend on wind assistance is intriguing and warrants examination with empirical data.

Most birds migrate below 4000 m elevation and, when possible, may alter flight altitude to take advantage of favourable wind, temperature, humidity or pressure (Liechti et al., 2000 Dokter et al., 2011). It is unclear to what extent this strategy is employed by high-altitude birds, but some evidence suggests that favourable conditions are not requisite for flying high. For example, demoiselle cranes (Anthropoides virgo) that were tracked on their southward migration between central and southern Asia flew over the Himalayas at 5000–6000 m elevation into a headwind (Kanai et al., 2000). Bar-headed geese have been tracked at 5000–7750 m elevation while crossing the Himalayan peaks in a single non-stop flight (Köppen et al., 2010 Hawkes et al., 2011) (although personal accounts have verified that at least some individuals of this species can fly over 1000 m higher Fig. 1). We have found that bar-headed geese climbing the southern Himalayan face actually avoid flying in the afternoons when upslope tailwinds could reduce the metabolic requirements of flight, and prefer instead to fly in the stable and colder conditions overnight and early morning when there is a slight downdraft (Hawkes et al., 2011). These data suggest that active flight is indeed possible without wind assistance up to at least 6000 m elevation. A definitive answer to whether flapping flight can be sustained above the highest peaks awaits physiological and biomechanical data for birds flying at even higher altitudes.

Conclusions and perspectives

The ability of birds to fly at high altitudes is critically dependent on the effective transport of O2 from hypoxic air to all of the tissues of the body. Part of this effectiveness comes from many characteristics that distinguish the O2 transport pathway of all birds in general from that of other vertebrates. Although not truly adaptive for high-altitude flight, these characteristics were undoubtedly an important basis upon which high-altitude adaptation could proceed. As it did so, unique specializations appear to have arisen at every step of the O2 transport pathway of high fliers to facilitate their impressive exercise performance. However, it is not yet certain whether the numerous examples above are sufficient to entirely explain high-altitude flight.


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