Coordinated beak–tongue mechanics enable dexterous seed manipulation in songbirds

Reviewer #1 (Public review):

Summary:

The authors quantified and compared the 3D kinematics of bill and tongue movements between two seed-eating bird species: one that specializes on soft seeds, and one that is more adapted to feeding on hard seeds. Their goal was to determine specifically what the role of the tongue was for processing (e.g., dehusking) seeds, and to understand how differences in biting strength between species affect other aspects of seed processing. The authors provided intricate (visual) details of seed processing movements, and showed how coordination between the tongue and cranial kinesis (i.e., mobility of the upper bill relative to the cranium) is both critically important for properly positioning seeds to enhance feeding efficiency. Many studies have detailed how seed-eating birds process seeds, but this study has elevated those to a new level of quantification and visualization for readers to fully experience firsthand. Furthermore, the authors established that the force-velocity trade-off that has been observed between bill functions (e.g., feeding and singing) is largely driven by the contractile properties of the muscles. The conclusions are well supported by the results, and the authors placed the results more broadly into the context of manual grasping, making the argument that these birds achieve high levels of dexterity with far fewer degrees of freedom, which could have potential biomimetic applications.

Strengths:

This study builds upon - and advances - our understanding of the feeding mechanics of seed-eating birds using cutting-edge 3-dimensional modeling and kinematics. Their quantitative analyses of upper and lower bill, tongue, and seed displacements are complemented by elegant visualizations of seed processing in each species. Their comprehensive Bayesian modeling statistical framework tackles the issue of small sample sizes (i.e., few subjects) with volumes of data for each (i.e., lots of sequential kinematic variables) that plague comparative biomechanics studies, principally because (a) it is difficult to gather these high resolution XROMM and muscle contractile data on more than just a few subjects, and (b) these data streams are inherently very large, as they are gathered at high frame and sampling rates. Furthermore, I believe their approach to statistically testing for differences between species sets a new standard for our field that could (perhaps should?) be implemented in other similar types of studies. Another strength is in how the results were packaged: each subsection indicated how the objectives were addressed, and there were concluding statements trailing each subsection that helped deliver the key takeaways.

Weaknesses:

A potential weakness is one that the authors themselves mentioned, regarding the body (and skull) size differences between species. Because gape size limits bite force, and given the force-velocity tradeoff in muscle function, there could be limitations on the rapid manipulation of relatively large seeds for similar reasons in the smaller finches. I see that the small finches appear to overcompensate in their beak rotations, but it's not clear how those compensatory movements might affect their seed processing kinematics with their preferred seed sizes. This does not nullify the authors' conclusions, but the results for the smaller finches might not be entirely representative of seed processing mechanics in smaller species.

Reviewer #2 (Public review):

Summary:

This study investigates coordinated beak-tongue movements in seed manipulation, biting, and dehusking in songbirds. A comparative analysis of the seed-eating process in two songbird species with different biting forces, the domestic canary and Java sparrow, was conducted using high-speed XROMM with anatomical marker tracking and quantitative behavioral analysis. The authors have done a great job analyzing upper and lower beak rotation and translation, seed orientation and movement speed, and tongue kinematics.

Strengths:

The methodological approach of using high-speed (500 fps) X-ray reconstruction for 3D kinematic tracking in small animals is novel and powerful. It enables high temporal resolution tracking of orofacial movements and could potentially inspire future orofacial research in mammals, including mice and marmosets. Moreover, this study encompasses a wide range of anatomical components involved in seed manipulation behavior, including the upper and lower beak, the tongue, and jaw muscles. The behavioral quantification of these components is solid. The findings that both the upper and lower beaks contribute to seed processing, that the lower beak exhibits greater up-and-down and left-to-right flexibility than the upper beak during seed processing, and that the tongue plays an important role in transporting seeds into the mouth are all solid conclusions consistent with observations of bird feeding behavior. Nevertheless, it is valuable to confirm and quantitatively characterize these observations experimentally. The videos are excellent and very informative.

Weaknesses:

(1) The paper often resorts to qualitative descriptions (e.g., "a high positive correlation of tongue velocity and seed velocity", "Compared to positioning, the measured velocities of both seed and tongue were much lower") instead of providing exact quantitative measurements or statistical results. The authors stated that temporal autocorrelation biases standard statistical analyses (lines 205-210), but this rationale does not justify the absence of statistical validation. Suggestion: use appropriate methods for time-series data, such as a permutation test, to test the significance of correlations between variables and avoid false positives.

(2) (Minor) The marker-tracking image shown in Figure 1B could benefit from the inclusion of a higher-contrast, zoomed-in frame of the head showing the metal markers without the red tracking points, alongside the same frame with the red tracking points overlaid, to provide readers with a clearer view of the X-ray image and the methodology and its precision.

(3) (Minor: possibly soften the mechanistic claim). The proposed mechanism of lingual papillae on the tongue surface may aid food manipulation and food movement towards the posterior region of the mouth is interesting, yet the evidence describing their morphology is not strong enough to support the claim about their functional roles. Furthermore, the claim that papillae orientation affects food transport in lines 294-296 lacks supporting experimental evidence. In addition, the roles of extrinsic and intrinsic tongue muscles in controlling dexterous tongue shape changes and movements are not discussed.

Author response:

We would like to express our gratitude for the thorough evaluation of our manuscript by the editors and reviewers. We are grateful for the overall positive assessment. The suggestions for improvement are reasonable, and we are certain that addressing these points will improve the clarity, accessibility, and scientific integrity of the study. Thus, we plan to conduct a revision of the manuscript, addressing all the points raised. The most important planned adjustments are outlined below.

(1) Improving the accessibility of the probabilistic modeling framework

Reviewer 1 kindly stated that our Bayesian modeling framework for testing for species differences 'sets a new standard for our field.' As a new standard, however, the method should be explained in a more accessible way. Hence, we plan to provide additional explanations for the statistical workflow, e.g., by providing comprehensible visuals, to make the workflow easier to understand and easier to apply.

(2) Statistical validation of qualitative claims

We acknowledge that a statistical validation of qualitative claims regarding the relationship between seed and tongue movements and between upper and lower beak movements would considerably strengthen the validity of our findings. We thank Reviewer 2 for bringing permutation tests to our attention for quantifying the correlation between time series. Since permutation tests involving index-shuffling of one of the data sets are generally not valid for time-series data [1, 2], we'll consider a variant of a trial-swapping permutation test, such as a permute-match test [3]. Alternatively, the truncated time shift (TTS) test [2] might be an option, as also this method is valid for auto-correlated time series data. At this point, we can't tell yet which method we'll use for the revised manuscript. We need more time to assess the requirements of each method and evaluate which test is most appropriate to answer our specific research questions and best fits our kind of data.

(3) Adjustments in the discussion

Following the suggestion by Reviewer 1, we'll refine our discussion on the effects of skull size differences, putting more emphasis on the implications of potential effects for feeding kinematics in small species.

Furthermore, as suggested by Reviewer 2, we'll soften our discussion on potential functions of lingual papillae in seed processing, as the current literature lacks experimental evidence for the claimed mechanistic roles.

References

(1) Yuan, A. E., & Shou, W. (2022). Data-driven causal analysis of observational biological time series. Elife, 11, e72518.

(2) Yuan, A. E., & Shou, W. (2024). A rigorous and versatile statistical test for correlations between stationary time series. PLoS biology, 22(8), e3002758.

(3) Yuan, A. E., & Shou, W. (2025). Permute-match tests: Detecting significant correlations between time series despite nonstationarity and limited replicates. eLife, 14.