Identifying migratory pathways of Nathusius' pipistrelles ( Pipistrellus nathusii ) using stable hydrogen and strontium isotopes

migratory corridors of exact corridors for elusive animals bats. While migrating along the coastline, Nathusius' pipistrelles ( Pipistrellus nathusii are regularly killed at wind turbines. we explored the paths taken on their annual journey. We used isotope ratio mass spectrometry to measure stable hydrogen and strontium isotope ratios in fur keratin of 59 Nathusius' pipistrelles captured on three offshore islands. Samples were pre-treated before analysis to report exclusively stable isotope ratios of non-exchangeable hydrogen. We generated maps to predict summer origins of bats using isoscape models. Results: Bats were classified as long-distance migrants, mostly originating from Eastern Europe. Hydrogen analysis suggested for some bats a possible Fennoscandian origin, yet additional information from strontium analysis excluded this possibility. Instead, our data suggest that most Nathusius' pipistrelles migrating along the German coastline were of continental European summer origin, but also highlight the possibility that Nathusius' pipistrelles of Baltorussian origin may travel offshore from Fennoscandia to Germany. Conclusions: Our findings demonstrate the benefit of using complementary isotopic tracers for analysing the migratory pathways of bats and also potentially other terrestrial vertebrate species. Furthermore, data from our study suggest an offset of fur strontium isotope ratios in relation to local bedrock.


| INTRODUCTION
In the era of the Anthropocene, human-induced changes to the environment, such as land use and global climate changes, threaten a number of species, leading ultimately to a worldwide biodiversity crisis. [1][2][3] Migratory species are particularly vulnerable to anthropogenic changes because they depend not only on intact breeding and wintering locations, but also on a sequence of suitable and intact habitats along their migratory routes. 2,4,5 Thus, conservation efforts need to be established beyond political borders to protect suitable corridors and habitats for these highly mobile species. 6,7 Migratory behaviour is observed in a range of vertebrate taxa, including ungulates, birds, cetaceans, sea turtles and bats. 8 Considering their small size, bats are outstanding among terrestrial mammals in their ability to cover long distances when migrating. 9,10 Yet, efficient conservation measures for the protection of migratory bats are impaired by the lack of information on migratory corridors and stopover sites. 11 Currently, it is widely assumed that coastal habitats and river valleys represent suitable pathways for migratory bats in Europe. 8,[12][13][14][15] In addition, peninsulas have been identified as suitable stopover sites. 16 Other than that, our understanding of where bats migrate remains largely speculative. This lack of knowledge has emerged as a major conservation problem since thousands of migratory bats are killed by wind turbines, 6,17,18 which are increasingly erected along coastal habitats, both onshore and offshore. 13 Thus, understanding where and when bats migrate is essential for deciding where to avoid wind parks and where to establish adequate mitigation schemes.
The European bat, Nathusius' pipistrelle (Pipistrellus nathusii), holds the world record for long-distance migration among bats, covering more than 2200 km one-way between the Baltic countries and southwestern Europe. 8,9,19,20 Large banding campaigns along the presumed migratory routes helped to identify wintering areas based on the recapture of banded bats. 9,19,20 These banding efforts support the existence of a coastal migratory pathway of Nathusius' pipistrelles with a northeastern origin (e.g. Russia, Baltic countries, Belarus) to central, western and southwestern Europe (e.g. Spain, France, Benelux countries, Germany) in late summer 9 (Figure 1). Alternatively, acoustic recordings suggest a possible migratory pathway of Fennoscandian individuals across the Baltic Sea from Sweden via Denmark to Germany 8,21,22 (Figure 1). Recently, it was also suggested that Baltorussian bats move via Finland, Sweden and Denmark to Germany. 13,14 Migratory pathways between Sweden and Germany would conflict with recent offshore wind turbine developments in the Baltic Sea. Despite evidence of these migratory routes, the relative abundance of Nathusius' pipistrelles of Fennoscandian origin at the German coastline is as yet unresolved.
Stable isotope analysis provides a powerful tool to expand our understanding of migratory pathways and the likely origin of migratory animals. Stable isotopes have been repeatedly used as environmental tracers to shed light on the geographic origin of animals using isoscape origin models, i.e. models based on the spatial distribution of stable isotopes across continents. [23][24][25] Isoscape origin models involving bats are usually based on stable hydrogen isotope ratios of fur, since it is a biologically inert matrix that, once formed, carries the same isotopic composition from the moulting area to the wintering area without changing until the next moulting event occurs prior to summer migration. [26][27][28] In the past, isoscape origin models were based mostly on stable hydrogen isotope ratios (depicted in the delta notation δ 2 H in relation to an international standard), since the δ 2 H values of meteoric water follow latitudinal gradients. 29 Yet, the explanatory power of these models is limited because they return F I G U R E 1 Map of potential migratory pathways of Nathusius' pipistrelles along the coast of the Baltic Sea. Blue lines highlight the pathway from Baltorussian populations to Poland and Germany. Red lines highlight the pathways of Fennoscandian populations via Denmark to Germany. Dashed blue lines represent alternative pathways of Baltorussian bats via Fennoscandia to Germany. Western offshore islands in Germany (Heligoland and Fehmarn) are indicated by red circles and the eastern island (Greiswalder Oie) by a blue circle relatively large regions of potential origin, encompassing usually several hundreds of kilometres on the latitudinal scale. 23 Figure 1). This assumes that bats of Fennoscandian origin cross Denmark and the Baltic Sea along a northeast-southwest trajectory between Sweden and Germany.
Alternatively, Nathusius' pipistrelles observed at the western capturing sites could originate from Baltorussian populations but make use of the Fennoscandian route to reach Germany via Denmark.

| Data collection
We captured 458 Nathusius' pipistrelles during the non-migratory period for δ 2 H reference material, and 10 for 87 Sr/ 86 Sr reference material; and 59 during the migratory period with unknown origin.
Bats were captured with mist nets and from bat boxes. δ 2 H reference material bats were captured in several European sites (Table SM1, supporting information) and 87 Sr/ 86 Sr reference material bats were captured in Engure Lake, Latvia (57 09 0 44.3 00 N 23 13 0 11.0 00 E; Table SM2, supporting information). For the reference materials, we defined July 15 th as the end of moulting period, 27 and thus included only fur samples of animals that were captured a few weeks before that date. However, we acknowledge that individual and geographic variation may cause deviations from this date.

| Stable isotope analysis
We analysed samples for stable hydrogen isotope ratios (reference material and unknown origin samples) at the Stable Isotope  The Sr separation procedure was based on the micro-Sr column chemistry method designed for samples with small amounts of Sr available. 53 The microcolumns (200-μL) were made from shrinkable Teflon.
The separation of Sr was achieved by Sr spec resin (Eichrom technologies LLC, Lisle, IL, USA) that was thoroughly precleaned with 6 M HCl, 0.5 M HNO 3 and H 2 O. 53 All reagents had a blank contribution less than 1 pg Sr blank. With each batch of samples, one total procedural blank was determined. The average Sr total procedural blank was low (6.6 ± 3.3 pg; 2 standard deviations, n = 4).
Samples were loaded together with TaF 5 activator 54 57 The transfer function defines how data from bats with unknown origin are related to the environment where fur was collected, providing the basis for a δ 2 H isoscape with predicted sample values. It also accounts for potential geospatial assignment errors. We compared the slope and the uncertainty around values of the previously established transfer function of common noctule bats 58 with our newly developed transfer function for Nathusius' pipistrelles using a t-test.
Using the δ 2 H isoscape with predicted sample values, we assigned every individual a p-value for each of the 0.03 km grid cells to test the putative summer origin. 56 Any location with p ≤ 0.05 was not considered to be a likely place of origin (i.e. the δ 2 H value of the individual fell outside the 95% confidence interval of the possible origin location). Individuals were classified as long-distance migrants when the sampling location was not considered a possible origin location (i.e. p ≤ 0.05). All other animals were considered regional bats. To simplify the description of the migratory origin of Nathusius' pipistrelles, we categorized individuals into groups based on their Previous studies tested for a possible fractionation of strontium isotopes in consumer tissue samples and overall observed no significant deviation from local bedrock chemistry. 37,59-61 It has thus been generalized that strontium isotope ratios do not fractionate in relation to endogenous sources such as diet. 37 Yet, most previous studies investigated bone collagen and dentine in mammals, or chitin in insects. 37 We also ran two-way analysis of variance models to test the influence of location and sex on δ 2 H and 87 Sr/ 86 Sr values, followed by a post hoc Tukey's test. All analyses were conducted in R. 65 3 | RESULTS

| Isoscapes
We established a hydrogen isoscape for the summer locations of

| Capture and stable isotope data
In total, we captured 59 Nathusius' pipistrelles at three places in northern Germany: one island in the North Sea (Heligoland: 9 males/2 F I G U R E 2 European isoscapes of stable hydrogen and strontium isotope ratios from precipitation and bioavailable strontium data, respectively. The highlighted polygon is the current breeding range of Nathusius' pipistrelles in Europe (IUCN report, adjusted in Eastern Europe based on pers. comm. Dekker J, Keribirou C, Kravchenko K). We used a non-linear colour pallet for the 87 Sr/ 86 Sr isoscape, following 42 , because the distribution of 87 Sr/ 86 Sr values is heavily skewed. Dots represent Heligoland (blue), Fehmarn (green) and Greifswalder Oie (orange). We also report density plots for both isotope ratios females) and two islands in the Baltic Sea (Greifswalder Oie: spring:  Table SM3 (supporting information).

| Geographic assignment based on keratin stable isotope ratios
Nathusius' pipistrelles were assigned to three isotopic bins based on pipistrelles captured on Heligoland were assigned to ranges 1 and 2 (45% and 55%, respectively; Figure 5).    1 and 2). This is consistent with migration pathways established in banding studies (i.e. a northeastern and southwestern pathway). We did not find a consensus for derived hydrogen and strontium enriched probability maps, since presumed places of origin in western Europe (France and British Isles) were inconsistent between the strontium data. These contradictory results require additional studies to ensure that accurate conclusions are reached with these data.
Our isotopic approach was impeded by the large variation in δ 2 H values observed in non-migratory Nathusius' pipistrelles across Europe, which resulted in broad isotopic bins (i.e. most of central Europe was covered in range 2). Indeed, the transfer function established for Nathusius' pipistrelles deviated largely from the previously established transfer function of a sympatric aerial-hawking species, the common noctule bat. 66 This is probably caused by a larger contribution of food items of aquatic origin, such as chironomid flies, in the diet of Nathusius' pipistrelles than, for example, in the diet of common noctule bats and other bats. 12,76,77 A previous isotopic study with a focus on non-migratory European bat species showed that δ 2 H values varied largely with the relative contribution of aquatic to terrestrial food items to the diet of bats. 78 By these Nathusius' pipistrelles consuming a mixture of insects of aquatic and terrestrial origin, the δ 2 H values in their fur might become highly variable within local populations, which may explain why the transfer function of this species shows an offset and a larger variation of δ 2 H values for a given location than the transfer function previously established for non-migratory bats.
It is noteworthy that we documented a likely origin of Nathusius' pipistrelles in various areas of Poland, Russia and Ukraine. This would imply that populations from these countries head northward before changing to the southwestern direction of their wintering sites.
Although our isotopic evidence for this scenario is weak, acoustic recordings also suggest a northward movement of migratory Nathusius' pipistrelles from Baltic countries to Finland. 13 Migratory movements of Nathusius' pipistrelles may be more complex than previously assumed, especially when animals perform indirect migration. Isotopic assignments of animals may be insufficient to offer unambiguous insights into the exact migratory corridors used by bats when moving along or across the Baltic Sea. We suggest engaging in large-scale radio-tracking campaigns using miniaturized radio transmitters and automated receiver stations to unravel the exact locations of migratory corridors. At this point, our data do not argue against an influx of Nathusius' pipistrelles migrating from Sweden to Germany; however, these individuals may originate from Baltorussian populations.