Migratory strategies, fat loads and torpor use in migrating hummingbirds

North American bee hummingbirds frequently enter torpor during autumn migrations to save energy. The frequency with which they use torpor is mediated by fat loads. To estimate frequencies and temporal patterns of torpor use, we measured lipid loads and feather hydrogen stable isotope ratios (δ2H) in 942 individuals of four species of migrating bee hummingbirds: Selasphorus rufus (Rufous Hummingbird), Selasphorus calliope (Calliope Hummingbird), Selasphorus platycercus  (Broad-tailed Hummingbird), and Archilocus alexandri (Black-chinned Hummingbird), at a stopover site in Southwestern New Mexico. In a subsample of juvenile individuals of each species, we constructed functions relating lipid loads and feather δ2H values to torpor incidence, using respirometry. We used these functions, along with lipid load and δ2H data from the remaining captured juvenile birds to estimate their temporal patterns of torpor use. Lipids were significantly higher in adult S. rufus and S. platycercus than in juveniles but did not differ with age in S. calliope and A. alexandri. In S. rufus, lipid loads decreased with capture date, while in A. alexandri they increased. There was no effect of capture date on lipid loads in S. platycercus and S. calliope. In all species torpor incidence decreased with increasing fat loads in a predictable fashion. A large fraction (70%) of S. calliope and a smaller fraction (30%) of S. rufus individuals entered torpor, but these fractions remained constant over time. In contrast, the fraction of S. playtcercus individuals that entered torpor increased with capture date, whereas that of A. alexandri decreased. Our results revealed similarities, but also unexpected interspecific differences in fat loads and torpor incidence in migratory bee hummingbirds. Some of the correlates of these differences remain unexplained but represent an opportunity to better understand the causes and consequences of torpor-mediated migration.

Methods
We captured 942 adult and juvenile hummingbirds of four species at a stopover site near Mimbres, NM, USA, between July and September 2022. A Quantitative Magnetic Resonance scanner was used to non-invasively measure lipid content in each bird. A Push Flow-through Respirometry System was used to measure overnight metabolic parameters and use of nocturnal torpor in 94 adult and juvenile birds. Respirometry metabolic data were recorded using Sable Systems ExpeData version 1.9.27 software. A retrix (R1) feather was collected from each bird, and feather hydrogen stable isotope ratios were measured on 942 specimens, at the University of New Mexico Center for Stable Isotopes. Results for each species were compiled into analyses of capture date, body mass, fat-free mass, fat mass, molt/natal origin, torpor probability, and torpor fat thresholds. Results of a subset of 63 juvenile birds tested in respirometry were used to predict torpor probabilities in the larger dataset of 573 juvenile birds, in which we measured both lipids and feather hydrogen stable isotopes, but not metabolic parameters. All birds were fed and released at the capture site following measurements. Microsoft Excel and JMP Pro 18 Statistical Software were used to perform data analyses. We conducted statistical analyses to determine how lipid-loads and torpor patterns are related to natal origins and migration patterns.

北美迁徙蜂鸟常在秋季迁徙期间进入蛰伏（torpor）以节约能量。它们使用蛰伏的频率受脂类负荷（lipid loads）调控。为估算蛰伏使用的频率与时间模式，我们在新墨西哥州西南部的一处中途停歇地（stopover site），对4种迁徙蜂鸟的942个个体开展了脂类负荷与羽毛氢稳定同位素比值（δ²H）的测定，这4种蜂鸟分别为：棕煌蜂鸟（Selasphorus rufus）、星蜂鸟（Selasphorus calliope）、宽尾煌蜂鸟（Selasphorus platycercus）以及黑颏蜂鸟（Archilochus alexandri）。

我们针对每个物种的幼年个体亚样本，采用呼吸代谢测定法（respirometry）构建了脂类负荷、羽毛δ²H值与蛰伏发生率之间的关联函数，并结合其余捕获幼年鸟类的脂类负荷与δ²H数据，利用上述函数估算了它们蛰伏使用的时间模式。

研究结果显示：成年棕煌蜂鸟与宽尾煌蜂鸟的脂类水平显著高于幼年个体，但星蜂鸟与黑颏蜂鸟的脂类水平无年龄差异。棕煌蜂鸟的脂类负荷随捕获日期推移而降低，黑颏蜂鸟则相反，脂类负荷随捕获日期升高；宽尾煌蜂鸟与星蜂鸟的脂类负荷不受捕获日期影响。所有物种的蛰伏发生率均随脂肪负荷增加以可预测的方式下降。星蜂鸟有70%的个体、棕煌蜂鸟有30%的个体进入蛰伏，且该比例随时间保持稳定。与之相反，宽尾煌蜂鸟进入蛰伏的个体比例随捕获日期增加而升高，黑颏蜂鸟的该比例则随捕获日期降低。本研究揭示了迁徙蜂鸟在脂类负荷与蛰伏发生率上既有共性，也存在出人意料的种间差异。这些差异的部分关联因素尚未得到解释，但为深入理解蛰伏介导的迁徙的成因与后果提供了研究契机。

方法

我们于2022年7月至9月间，在美国新墨西哥州明布莱尔斯附近的一处中途停歇地，捕获了4种蜂鸟共942只成体与幼体。使用定量磁共振扫描仪（Quantitative Magnetic Resonance scanner）无创测定每只鸟类的脂类含量；采用推流式呼吸代谢系统（Push Flow-through Respirometry System）测定94只成体与幼体的夜间代谢参数与夜间蛰伏使用情况，代谢数据通过Sable Systems ExpeData 1.9.27版本软件记录。我们从每只鸟类采集尾羽（R1），并于新墨西哥大学稳定同位素中心完成942个样本的羽毛氢稳定同位素比值测定。

我们将各物种的结果整合，开展了捕获日期、体质量、去脂体重、脂肪质量、换羽/出生地起源、蛰伏概率以及蛰伏脂肪阈值的分析。利用经呼吸代谢测定的63只幼年鸟类的子集结果，在包含573只幼年鸟类的更大数据集（该数据集仅测定了脂类与羽毛氢稳定同位素比值，未测定代谢参数）中预测其蛰伏概率。所有鸟类在测定完成后均于捕获地喂食并放飞。数据分析采用Microsoft Excel与JMP Pro 18统计软件完成，旨在明确脂类负荷与蛰伏模式如何与出生地起源及迁徙模式相关联。