Coevolution between crossbills and black pine: the importance of competitors,forest area and resource stability

Studies of predator‐prey interactions have found that geographically structured coevolution has played an important role in the adaptive diversification of crossbills (Loxia spp.). We extend those studies by considering common crossbills (L. curvirostra) in the Mediterranean where they rely on seeds in the cones of black pine (Pinus nigra). On the continent, where tree squirrels (Sciurus vulgaris) are present, enhanced defenses against crossbills were most evident in larger areas of pine forest. On islands in the absence of tree squirrels, crossbills and black pine have coevolved in a predator‐prey arms race on Cyprus but not Corsica. In contrast to other conifers that island endemic crossbills rely upon, black pine does not hold seeds in its cones year round. Consequently, key to the strong crossbill–pine interaction on Cyprus is likely the presence of an alternative conifer that provides seeds during early summer when black pine seeds are scarce.     

Temporal variation in breeding and cone size selection by three species of crossbills Loxia spp. in a native Scots pinewood

The breeding biology and cone size selection of crossbills was studied mainly during 1995 to 2002 at Abernethy Forest, Scotland, an ancient native Scots pine Pinus sylvestris wood, where only a single crossbill species, the Scottish crossbill Loxia scotica, was assumed to occur and to be adapted to feed on seeds in Scots pine cones. However, three crossbill species (common Loxia curvirostra, Scottish and parrot crossbills Loxia pytyopsittacus) nested in some years, with the parrot crossbill being the most abundant. Most nests were in old large pines, with the three crossbill species not differing in their use of tree size or stand density for nesting. The mean clutch and brood sizes were 3.8 and 2.9, and their mean survivals were 86 and 74%, respectively, with no significant differences among species. The timing of breeding differed between species, with parrot crossbills breeding earliest (median date 21 March, including second attempts) and common crossbills breeding last (median date 21 April), probably in response to the differing accessibility of Scots pine seeds to these species. The difference in the time of breeding may reduce mixed mating. Crossbills foraged preferentially on trees with small cones when the cones were closed. Small cones had thinner scales than large cones, suggesting that the preference for small cones was related to higher feeding rates on these cones when cones are closed. Such a preference was also found for captive crossbills with the Scottish crossbill showing a more pronounced preference for smaller cones than the larger‐billed parrot crossbill. However, crossbills selected larger cones within trees and trees with larger cones once the cones opened in April. Such a shift occurred presumably because variation in scale thickness has little impact on seed accessibility once cones open, and larger cones have larger and more seeds. The greater ability of parrot crossbills to exploit seeds in closed Scots pine cones allowed parrot crossbills to start breeding earlier and to have young when seeds were most accessible. Only after the cones opened were the smaller‐billed common crossbills able to easily access seeds and to start breeding. The time of breeding of Scottish crossbills was intermediate between common and parrot crossbills, and they probably had an intermediate ability to exploit Scots pine cones. The reason why there were few Scottish crossbills nesting in Abernethy Forest remains a puzzle, considering that native pine wood is assumed to be the ancestral habitat to which the Scottish crossbill is adapted. The breeding season for all crossbills ended in June, when most of the seed from a given cone cohort was shed. This is when starved broods were found, not associated with bad weather.     

AUTOMATIC FEATURE EXTRACTION AND CLASSIFICATION OF CROSSBILL (LOXIA SPP.) FLIGHT CALLS

ABSTRACT

In this paper a new method for the automatic classification of bird sounds is presented. Our method is based on acoustic parameters (features) taken from the first harmonic component computed from the sound spectrogram. The features are based on a line segment approximation of the first harmonic component. The final feature vectors, consisting of 16 real numbers, are then classified using a self-organizing map (SOM) neural network. Flight calls of four crossbill species (Loxia spp.) are used as a test example. In the first phase, an unsupervised network was trained and tested using common crossbill L. curvirostra flight calls recorded mainly in the Netherlands. The network was tested using two-barred L. leucoptera, Scottish L. scotica and parrot L. pytyopsittacus crossbill flight calls in the second phase. Finally, the results were validated applying the same network to flight calls of common crossbills and parrot crossbills recorded in Finland. The method automatically separated common crossbill flight calls from those of parrot crossbills. The classification accuracy of the Dutch recordings was 58% in the first phase and 54% in the second phase. The Finnish recordings were classified with 54% accuracy.     

Genome divergence and diversification within a geographic mosaic of coevolution

Despite substantial interest in coevolution's role in diversification, examples of coevolution contributing to speciation have been elusive. Here, we build upon past studies that have shown both coevolution between South Hills crossbills and lodgepole pine (Pinus contorta), and high levels of reproductive isolation between South Hills crossbills and other ecotypes in the North American red crossbill (Loxia curvirostra) complex. We used genotyping by sequencing to generate population genomic data and applied phylogenetic and population genetic analyses to characterize the genetic structure within and among nine of the ecotypes. Although genome‐wide divergence was slight between ecotypes (F_ST = 0.011–0.035), we found evidence of relative genetic differentiation (as measured by F_ST) between and genetic cohesiveness within many of them. As expected for nomadic and opportunistic breeders, we detected no evidence of isolation by distance. The one sedentary ecotype, the South Hills crossbill, was genetically most distinct because of elevated divergence at a small number of loci rather than pronounced overall genome‐wide divergence. These findings suggest that mechanisms related to recent local coevolution between South Hills crossbills and lodgepole pine (e.g. strong resource‐based density dependence limiting gene flow) have been associated with genome divergence in the face of gene flow. Our results further characterize a striking example of coevolution driving speciation within perhaps as little as 6000 years.     

Assortative mating and patterns of inheritance indicate that the three crossbill taxa in Scotland are species

There are three breeding taxa of crossbills in highland Scotland, the common crossbill Loxia curvirostra , parrot crossbill L. pytyopsittacus and endemic Scottish crossbill L. scotica . These taxa show no genetic differentiation in neutral DNA (microsatellites and mitochondrial DNA), but do show some differentiation in morphology and have distinct calls. Mating patterns and bill size inheritance were examined to test four different hypotheses for their genetic similarity: (1) the three taxa belong to the same species and differences in bill sizes are caused by phenotypic plasticity, (2) the taxa are a result of genetic polymorphism at a single locus, (3) the taxa largely act as species but occasionally cross-breed, such that gene flow results in genetic homogenisation of part of the genome, and (4) the taxa are fully reproductively isolated species but diverged only recently, such that insufficient time has passed for neutral DNA to differentiate by genetic drift. Hypotheses 1 and 2 could be excluded because wild crossbills mated assortatively with respect to bill depth (a measure of bill size) and bill depth was highly heritable; narrow-sense heritabilities of 0.58 for female and 0.71 for male Scottish crossbills. Only two of 46 pairs (4.3%) were not assortative by bill size. Pairing was strongly assortative by flight calls (50 out of 52 pairs, 96.2%) and excitement calls (88 out of 93 pairs, 94.6%). Therefore, the three crossbill taxa do not cross-mate freely, nor do they seem totally reproductively isolated (excluding hypothesis 4). Thus, the results mostly favour hypothesis 3. The small amount of gene flow among crossbill taxa may contribute to the lack of genetic differentiation in neutral DNA. However, this appears to be insufficient not to classify them as species, as long as the calls can be regarded as diagnostic.     

A coevolutionary arms race causes ecological speciation in crossbills

We examined three ecological factors potentially causing premating reproductive isolation to determine whether divergent selection as a result of coevolution between South Hills crossbills (Loxia curvirostra complex) and Rocky Mountain lodgepole pine (Pinus contorta latifolia) promotes ecological speciation. One factor was habitat isolation arising because of enhanced seed defenses of lodgepole pine in the South Hills. This caused the crossbill call types (morphologically and vocally differentiated forms) adapted to alternative resources to be rare. Another occurred when crossbills of other call types moved into the South Hills late in the breeding season and feeding conditions were deteriorating so that relatively few non-South Hills crossbills bred ("immigrant infecundity"). Finally, among those crossbills that bred, pairing was strongly assortative by call type (behavioral isolation). Total reproductive isolation between South Hills crossbills and the two other crossbills most common in the South Hills (call types 2 and 5) summed to .9975 and .9998, respectively, on a scale of 0 (no reproductive isolation) to 1 (complete reproductive isolation). These extremely high levels of reproductive isolation indicate that the divergent selection resulting from the coevolutionary arms race between crossbills and lodgepole pine is causing the South Hills crossbill to speciate.     

Matching habitat choice in nomadic crossbills appears most pronounced when food is most limiting

Of the various forms of nonrandom dispersal, matching habitat choice, whereby individuals preferentially reside in habitats where they are best adapted, has relatively little empirical support. Here, I use mark‐recapture data to test for matching habitat choice in two nomadic ecotypes of North American Red Crossbills (Loxia curvirostra complex) that exist in the lodgepole pine (Pinus contorta) forests in the South Hills, Idaho, every summer. Crossbills are adapted for foraging on seeds in conifer cones, and in the South Hills the cones are distinctive, favoring a relatively large bill. During a period when seed was most limiting, only the largest individuals approximating the average size of the locally adapted ecotype remained for a year or more. During a period when seed was less limiting, proportionately more individuals remained and the trend for larger individuals to remain was weaker. Although matching habitat choice is difficult to demonstrate, it likely contributed to the observed patterns. Otherwise, nearly unprecedented intensities of natural selection would be needed. Given the nomadic behavior of most crossbill ecotypes and the heterogeneous nature of conifer seed crops, matching habitat choice should be favored and likely contributes to their adaptation to alternative conifers and rapid diversification.     

Beak morphology predicts apparent survival of crossbills: due to selective survival or selective dispersal?

Dozens of morphologically differentiated populations, subspecies and species of crossbills (genus Loxia) exist. It has been suggested that this divergence is due to variation in the conifer cones that each population specialises upon, requiring a specific beak size to efficiently separate the cone scales. If so, apparent survival should depend on beak size. To test this hypothesis, we undertook multievent capture–recapture modelling for 6844 individuals monitored during 27 years in a Pyrenean common crossbill L. curvirostra population in a forest of mountain pine Pinus uncinata. Apparent survival was indeed related to beak width, resulting in stabilizing selection around an optimum that was close to the observed mean beak width, indicating that local crossbill beak morphology is adapted to the conifer they feed upon. Both natural selection (selective mortality) and selective emigration of maladapted individuals may explain our findings. As is often the case in capture–recapture analyses but rarely recognised, we could not formally decompose apparent survival into selective mortality versus selective permanent emigration. Nonetheless, there are several indications that selective permanent emigration should not be fully excluded. First, natural selection by itself would have to be unusually strong compared to other empirical estimates to create the observed pattern of apparent survival. Second, the observed mean beak width was a bit lower than the estimated optimum beak width. This can be explained by immigration of crossbills with smaller beaks originating from southern populations, which may subsequently have left the study area permanently in response to low food intake. This is in line with a detected transient effect in the data, yet apparently little influx from crossbills from northern Europe. When permanent emigration is phenotypically selective this will have ecological and evolutionary consequences, so this possibility deserves more attention in general.     

Morphological divergence among Spanish Common Crossbill populations and adaptations to different pine species

Crossbills (Loxia spp.) provide a classical avian model of ecological specialization on food resources. Previous studies have suggested that morphometric, genetic and vocal diversification among Common Crossbill Loxia curvirostra populations is better explained by ecological distance (use of different conifers) than by geographical distance, indicating that populations have diverged adaptatively. We tested for adaptive divergence in Iberian crossbills using bill and body size measurements of 6082 crossbills from 27 sites, each consisting of a dominant or single pine (Pinus) of four possible species. Crossbills using different pines differed significantly in body size and bill size and shape. There was no correlation between geographical and morphological distance among sampling sites, consistent with the hypothesis that the morphological divergence of Iberian crossbills is shaped by their ecological differences (foraging on alternative conifers) rather than geographical distance. However, for unknown reasons, Common Crossbills foraging on Pinus sylvestris in Iberia have on average much smaller bills than Parrot Crossbills Loxia pytyopsittacus feeding on the same pine species in northern Europe. The extent to which crossbills specialize on Iberian P. sylvestris remains to be established. Specialization on conifers with overlapping geographical distributions may be facilitated by matching habitat choice of crossbills as a function of their local intake rates.     

Replicated population divergence caused by localized coevolution? A test of three hypotheses in the red crossbill‐lodgepole pine system

Several lines of evidence support the hypothesis that local populations of red crossbills (Loxia curvirostra complex) enter into a predator-prey arms race with lodgepole pine (Pinus contorta latifolia) in the absence of competing pine squirrels (Tamiasciurus hudsonicus). Nevertheless, the alternative hypotheses that neutral evolution or factors other than squirrels have caused crossbill population differentiation have not been thoroughly tested. We compared crossbill and pine cone morphology between island populations where squirrels are absent or present, and mainland sites where squirrels are present, in order to distinguish among these hypotheses. All comparisons supported an effect of squirrel absence, not island status, on crossbill and cone morphology. Hence our results provide further evidence that strong localized coevolutionary interactions in a geographic mosaic have driven adaptive population differentiation. In addition, vocal differentiation of crossbills was related to the absence of squirrels, but not to island status. As morphological and vocal differentiation is correlated with reproductive isolation in crossbills, the geographic mosaic of coevolution also seems to promote ecological speciation.     

Is the nominate subspecies of the common crossbill Loxia c. curvirostra polytypic? II. Differentiation among vocal types in functional traits

Vocally differentiated common crossbill Loxia curvirostra populations ('vocal types') in North America show a high degree of morphological and ecological specialization coupled with significant genetic differentiation and appreciable levels of assortative mating in sympatry. Similar vocally differentiated common crossbill vocal types have recently also been uncovered in Europe. These vocal types frequently overlap in space and time, and preliminary data indicate strong assortatively mating in sympatry. These observations suggest that the European nominate subspecies also consists of several independent evolutionary lineages, but so far morphological and ecological support for this view has been lacking. Bill morphology of crossbills is tightly linked to resource use, and we earlier showed that average morphology (of birds of unknown vocal type) indeed differed among years, consistent with the possibility that vocal types are morphologically differentiated if the proportion of each type caught varies among years. Here we test specifically for morphological differences between birds assigned to vocal type, at two sites and during two independent influxes. Differentiation between the two vocal types studied is highly significant, with the same pattern uncovered at each site and for each influx. Of eight traits investigated, in both univariate and multivariate analyses, the trait that differs most between the two types is the ecologically important bill depth. The difference in average bill depth between these two European vocal types (0.26 mm) is equivalent to the difference between some ecologically specialised North American vocal types. These results provide further evidence that the nominate subspecies of the common crossbill consists of several ecologically distinct populations, if not cryptic species.     

Ecological differentiation in relation to bill size amongst sympatric, genetically undifferentiated crossbills Loxia spp.

Recent evidence of genetic homogeneity across morphologically diverse crossbill taxa Loxia spp. suggests that strong directional natural selection sustains morphological differences. If so, we would expect that, in sympatry, persistent crossbill morphs will be associated with the ecological circumstances that select for particular features. Here we report on a field study of niche differentiation in sympatric crossbills, showing correlation between bill size and habitat use, foraging and movements. In Deeside, north-east Scotland, crossbills occupied three ecological niches. Small-billed birds L. curvirostra were itinerant and migratory. They switched between conifer species in relation to the phenology of cone ripeness, feeding on spruce or larch from summer through winter and Scots Pine Pinus sylvestris in spring and early summer. Large-billed birds L. pytyopsittacus were more sedentary, feeding on pine seed year round in semi-natural pine forest. Birds with intermediate bills L. scotica were also sedentary but switched seasonally between conifer species. Deeside crossbills thus occupied three niches in line with the current designation of three species, but in the study years (1990–1997) there was no shortage of conifer seed and no evidence of strong selection for optimal bill size. Bill sizes did not fall precisely into three distinct modes so other factors were involved. These could have included the immigration of two sizes of L. curvirostra , and introgression (and possibly phenotypic plasticity) amongst the more sedentary larger-billed birds. The origin of L. scotica is discussed within the context of novel habitat, introgression, niche shift and competition for pine seed.     

Diversifying Coevolution between Crossbills and Conifers

Coevolution between granivorous crossbills (Loxia spp.) and conifers has been a prominent process in the diversification of crossbills. A striking example occurs in western North America where coevolution between crossbills and Rocky Mountain lodgepole pine (Pinus contorta latifolia) is ongoing in isolated ranges without the crossbill’s dominant competitor for seeds, the red squirrel (Tamiasciurus hudsonicus). Preferential foraging by crossbills on lodgepole pine cones in the South Hills and Albion Mountains, two small mountain ranges in southern Idaho where red squirrels are absent, has led to the evolution of larger, thicker-scaled cones than in nearby ranges where red squirrels are present. This in turn has favored the evolution of larger-billed crossbills that have diverged from other crossbills in the region. However, such diversifying coevolution, resulting from geographic variation in the distribution of strongly interacting species, is vulnerable to species introductions. For example, the introduction of red squirrels caused the precipitous decline and perhaps extinction of the Newfoundland crossbill and perhaps a crossbill endemic to the Cypress Hills, Canada. In general, species introductions act to reduce the geographic variation in species interactions, which may be critical for the diversification of many taxa.     

When directional selection reduces geographic variation in traits mediating species interactions

Although we often focus on the causes of geographic variation, understanding processes that act to reduce geographic variation is also important. Here, we consider a process whereby adaptive foraging across the landscape and directional selection exerted by a conifer seed predator, the common crossbill (Loxia curvirostra), potentially act to homogenize geographic variation in the defensive traits of its prey. We measured seed predation and phenotypic selection exerted by crossbills on black pine (Pinus nigra) at two sites in the Pindos Mountains, Greece. Seed predation by crossbills was over an order of magnitude higher at the site where cone scale thickness was significantly thinner, which was also the cone trait that was the target of selection at the high predation site. Additional comparisons of selection differentials demonstrate that crossbills exert selection on black pine that is consistent in form across space and time, and increases in strength with increasing seed predation. If predators distribute themselves in relation to the defensive traits of their prey and the strength of selection predators exert is proportional to the amount of predation, then predators may act to homogenize trait variation among populations of their prey in a process analogous to coevolutionary alternation with escalation.     

CONSISTENCY AND VARIATION IN PHENOTYPIC SELECTION EXERTED BY A COMMUNITY OF SEED PREDATORS

Phenotypic selection that is sustained over time underlies both anagenesis and cladogenesis, but the conditions that lead to such selection and what causes variation in selection are not well known. We measured the selection exerted by three species of predispersal seed predators of lodgepole pine (Pinus contorta latifolia) in the South Hills, Idaho, and found that net selection on different cone and seed traits exerted by red crossbills (Loxia curvirostra) and cone borer moths (Eucosma recissoriana) over 10 years of seed crops was similar to that measured in another mountain range. We also found that the strength of selection increased as seed predation increased, which provides a mechanism for the correlation between the escalation of seed defenses and the density of seed predators. Red crossbills consume the most seeds and selection they exert accounts for much of the selection experienced by lodgepole pine, providing additional support for a coevolutionary arms race between crossbills and lodgepole pine in the South Hills. The third seed predator, hairy woodpeckers (Picoides villosus), consumed less than one‐sixth as many seeds as crossbills. Across the northern Rocky Mountains, woodpecker abundance and therefore selective impact appears limited by the elevated seed defenses of lodgepole pine.     

Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (lineage GRW2): the effects of the co-infection on experimentally infected passerine birds

The effects of avian malaria parasites of the genus Plasmodium on their hosts are insufficiently understood. This is particularly true for malarial co-infections, which predominant in many bird populations. We investigated effects of primary co-infection of Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (GRW2) on experimentally infected naive juveniles of siskin Spinus spinus, crossbill Loxia curvirostra and starling Sturnus vulgaris. All siskins and crossbills were susceptible but starlings resistant to both these infections. A general pattern of the co-infections was that heavy parasitemia (over 35% during peaks) of both parasites developed in both susceptible host species. There were no significant effects of the co-infections on mean body mass of the majority of infected birds. Mean haematocrit value decreased approximately 1.5 and 3 times in siskins and crossbills at the peak of parasitemia, respectively. Mortality was recorded among infected crossbills. We conclude that co-infections of P. relictum and P. ashfordi are highly virulent and act synergetically during primary infections in some but not all passerine birds.     

Coevolution,local adaptation and ecological speciation

Coevolution is one of the major processes organizing the earth's biodiversity, but it remains unclear when and how it may generate species diversity. The study by Parchman et al. ( 2016 ) in this issue of Molecular Ecology provides the clearest evidence to date that divergent local adaptation in a coevolving interaction may lead to speciation on one side of an interaction but not necessarily on the other side. Red crossbills in North America have diversified into ecotypes that specialize on different conifer species, use different calls and vary in the extent to which they are nomadic or sedentary. This new study evaluated genomic divergence among nine crossbill ecotypes. The authors found low overall genomic divergence among many of the ecotypes, but the sedentary South Hills crossbills, which are specialized to eat the seeds of a unique population of lodgepole pines, showed substantial divergence from other crossbills at a small number of genomic regions. These results corroborate past studies showing local coadaptation of the morphological traits of South Hills crossbills and lodgepole pines, and premating isolation of the South Hills crossbills from other populations. Together, the past and new results suggest that local coevolution with lodgepole pines has led to reduced gene flow between South Hills crossbills and other crossbills.     

Diversifying coevolution between crossbills and black spruce on Newfoundland

Coevolution is increasingly recognized as an important process structuring geographic variation in the form of selection for many populations. Here we consider the importance of a geographic mosaic of coevolution to patterns of crossbill (Loxia) diversity in the northern boreal forests of North America. We examine the relationships between geographic variation in cone morphology, bill morphology, and feeding performance to test the hypothesis that, in the absence of red squirrels (Tamiasciurus hudsonicus), black spruce (Picea mariana) has lost seed defenses directed at Tamiasciurus and that red crossbills (L curvirostra) and black spruce have coevolved in an evolutionary arms race. Comparisons of cone morphology and several indirect lines of evidence suggest that black spruce has evolved defenses in response to Tamiasciurus on mainland North America but has lost these defenses on Newfoundland. Cone traits that deter crossbills, including thicker scales that require larger forces to separate, are elevated in black spruce on Newfoundland, and larger billed crossbills have higher feeding performances than smaller billed crossbills on black spruce cones from Newfoundland. These results imply that the large bill of the Newfoundland crossbill (L. c. percna) evolved as an adaptation to the elevated cone defenses on Newfoundland and that crossbills and black spruce coevolved in an evolutionary arms race on Newfoundland during the last 9000 years since glaciers retreated. On the mainland where black spruce is not as well defended against crossbills, the small-billed white-winged crossbill (L leucoptera leucoptera) is more efficient and specializes on seeds in the partially closed cones. Finally, reciprocal adaptations between crossbills and conifers are replicated in black spruce and Rocky Mountain lodgepole pine (Pinus contorta ssp. latifolia), with coevolution most pronounced in isolated populations where Tamiasciurus are absent as a competitor. This study further supports the role of Tamiasciurus in determining the selection mosaic for crossbills and suggests that a geographic mosaic of coevolution has been a prominent factor underlying the diversification of North American crossbills.     

The geographic selection mosaic for ponderosa pine and crossbills: a tale of two squirrels

Recent research demonstrates how the occurrence of a preemptive competitor (Tamiasciurus) gives rise to a geographic mosaic of coevolution for crossbills (Loxia) and conifers. We extend these studies by examining ponderosa pine (Pinus ponderosa), which produces more variable annual seed crops than the conifers in previous studies and often cooccurs with tree squirrels in the genus Sciurus that are less specialized than Tamiasciurus on conifer seed. We found no evidence of seed defenses evolving in response to selection exerted by S. aberti, which was apparently overwhelmed by selection resulting from inner bark feeding that caused many developing cones to be destroyed. In the absence of S. aberti, defenses directed at crossbills increased, favoring larger-billed crossbills and causing stronger reciprocal selection between crossbills and ponderosa pine. However, crossbill nomadism in response to cone crop fluctuations prevents localized reciprocal adaptation by crossbills. In contrast, evolution in response to S. griseus has incidentally defended cones against crossbills, limiting the geographic range of the interaction between crossbills and ponderosa pine. Our results suggest that annual resource variation does not prevent competitors from shaping selection mosaics, although such fluctuations likely prevent fine-scale geographic differentiation in predators that are nomadic in response to resource variability.