Migratory Behavior as a Factor Influencing the Evolution of

Avian Brain Organization

Roman Fuchs1, Jeremy D. Ross2, Daniel Witek1, Hans Winkler3, Barbara Helm4 Gustav

Bernroider1 & Verner P. Bingman2

1University of Salzburg, Austria; 2Bowling Green State University, Ohio, USA; 3 KLI,

Academy of Sciences, Austria; 4MPI Ornithology, Seewiesen, Germany

Abstract. Numerous bird species have their annual behavioral cycles punctuated

twice each year by often dramatic migrations of thousands of kilometers. The habit of

migration is a conspicuous component of avian life history and has served as a selective

force shaping the evolution of migratory species, and of particular relevance, the

evolution of a brain organization adapted to the challenges of migration. In the present

study, we have measured a number of brain morphometric and neurochemical features in

migratory and non-migratory populations of the old world stonechat (Saxicola torquata)

and the new world lark sparrow (Chondestes grammacus). For stonechat populations,

characterized by detectable genetic differences and subspecies status, the data reveal the

smallest telencephalic volume in long-distance Asian populations, larger telencephalic

volume in shorter distance, European migrants and the largest telencephalic volume in

resident, African populations. Looking at specific brain regions, one structure, the medial

striatum (previously called the lobus parolfactorius), which may correspond to portions of

the mammalian basal ganglia, shows the same trend in becoming smaller as migratory

distance increases. By contrast, migrant (Nebraska) and non-migrant (Texas) lark

sparrow populations, characterized to date by the absence of detectable genetic variation

and subspecies status, fail to demonstrate any differentiation in telencephalic volume.

The stonechat data promote the hypothesis that the energetic challenges of migration

have led to a reduction in the size of medial striatum. Such an adaptation would reduce

the energetic cost of maintaining a larger brain, resulting in a savings that could be

applied to the energetic cost of migration. However, the absence of similar differences in

lark sparrows, correlating with the lack of genetic differentiation, suggests that migratory

behavior has only recently evolved in lark sparrows, and insufficient time has past for the

different populations to accumulate readily detectable genetic variation and associated

brain morphometric differences.

Introduction

One of the most engaging aspects in the life history of many species of birds is

their often spectacular seasonal migrations. However, with migration come challenges

not experienced by non-migrant relatives. Examples of such challenges include precise

navigation on a global scale, meeting energetic demands and, in nocturnal migrants,

overcoming sleep deprivation, The suite of behavioral as well as energetic adaptations

that evolved in parallel with a migratory habit would necessarily reflect adaptations in

brain organization. The goal of our research collaboration is to identify differences in

brain organization between migrant and non-migrant populations of the same species, and

to a lesser extent closely related migrant and non-migrant species, and to understand

those differences in an evolutionary context. As a first approach, identifying differences

in brain and brain region volume that co-vary with migratory habit would be followed by

higher resolution comparisons in brain organization, e.g., neurotransmitter density, to

reconstruct the suite of brain adaptations that evolved in parallel with migratory behavior.

In many ways, the initial approach we are taking is one that has been employed,

in part, to understand differences in the volume of the hippocampus, which is known to

be important for vertebrate spatial cognition, in food-caching and non-food-caching

species of birds. Specifically, hippocampal volume in food-caching birds is larger than in

related species that do not cache (e.g., Krebs et al., 1989). The evolutionary/adaptive

explanation for this difference has been that the greater spatial memory demands

associated with the need to remember cache sites over extended periods of time acted as a

selective force for a larger hippocampus (and presumably greater spatial memory

capacity/duration). It was only a matter of time, therefore, that a similar research strategy

was directed at comparing migratory and non-migratory species, which also differ in their

spatial behavior. However, the results for migrant/non-migrant hippocampal comparisons

have not been as tidy as the food-caching comparisons. In a first study, Healy and

Gwinner (1996) carried out a complex analysis of hippocampal volume in a migratory

and non-migratory species of European warbler (Sylvia sp.). That analysis revealed a

tenuous larger hippocampus in the migrant species. More recently, Cristol et al. (2003)

working with migratory and non-migratory subspecies of juncos (Junco hyemalis), and

Pravosudov et al. (2006) working with migratory and non-migratory subspecies of white-

crowned sparrows (Zonotrichia leucophrys), presented data indicating that the

hippocampus of the migratory subspecies was different from the non-migratory

subspecies with respect both neuronal density (higher) and relative volume (larger).

Generalizing, the “superior” hippocampus of the migrant subspecies can be used to

explain the considerably longer memory retention found in a migrant species of European

warbler compared to a non-migrant species (Mettke-Hofmann and Gwinner, 2003).

So far so good, but a larger hippocampus was only found relative to overall

telencephalic volume in migrating juncos and white-crowned sparrows; there was no

difference in absolute hippocampal volume between migrant and non-migrant subspecies

(which have the same body size). An important parallel finding was that overall

telencephalic volume was found to be smaller in a sample of migrant compared to non-

migrant species (Winkler et al., 2004) as well as in the migrant-sub-species of white-

crowned sparrows (Pravosudov et al., 2007). The latter results have nurtured the

hypothesis that in challenging environments (e.g., arctic/temperate winters) smaller-

brained migrant species are compelled to seasonally leave the area because of their

inferior cognitive abilities, while larger-brained non-migrant species can “figure out”

how to survive (Sol et al., 2005a,b). However, recent evidence suggesting that sedentary

populations of white-crowned sparrows evolved from migrant populations, at the very

least, weakens the generality of this hypothesis.

What the available data suggest is that the hippocampus does not hold a

privileged position in the suite of brain organizational adaptations associated with

migration. In the present study we present preliminary findings examining differences in

the organization of the telencephalon and selected brainstem regions among long, short

and non-migrating Eurasian stone chats (Saxicola torquata) and migrating and non-

migrating North American lark sparrows (Chondestes grammacus).

Methods

The subjects were hand-reared stone chats that were held at the Max Planck

Insititute of Ornithology in Seewiesen, Germany. The stone chats originated from

populations in Russia (long-distance migrants), Germany (shorter distance migrants) and

Africa (non-migrants). The lark sparrows were wild caught and taken from populations in

Texas, USA (non-migrants) and Nebraska, USA (migrants).

For tissue preparation, deep narcosis was induced with an ip injection of

Nembutal (Sodium Pentobarbital, 0.3mg/g body weight), 0.15-0.25 ml for ~25g birds like

the stone chats and lark sparrows. This led to prolonged narcosis and subsequent

respiratory paralysis within a few minutes. Birds were then transcardially perfused with

phosphate buffer followed by 4% paraformaldehyde. The brains were removed from the

skull and stored in 4% PFA for at least 24h. Sagittal sections of the brain hemispheres

were sliced with a vibratome at a section thickness of 60µm. Subsequently, they were

mounted on coated slides in distilled water and dried at 4°C in the refrigerator for at least

24h. Finally, the whole section series of each hemisphere was stained utilizing the Nissl-

technique (toluidine-blue) and coverslipped with Neomount. Images of serial sections

were made at 10-fold magnification with a digital camera (Olympus CV III) mounted on

a stereo microscope (Leica Wild).

For volume reconstruction of overall telencephalon and regional subdivisions,

every fourth image was selected resulting in a total thickness of 240µm per layer. The

image series were manually aligned using “Reconstruct” (Fiala, 2005). Because

automatic image segmentation was not feasible, forebrain structures were also manually

traced.

In addition to the volumetric analyses, we also examined the number of tyrosine

hydroxylase (TH) positive neurons (catecholaminergic neurons) in the area of the

substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and substantia

grisea (SN) of the mesencephalic tegmentum. Cell counts were performed on tyrosine

hydroxylase immuno-stained saggital, total brain, vibratom sections (section thickness 70

um). Rabbit anti-tyrosine hydroxylase (affinity purified polyclonal antibody, AB 152,

Chemicon) was visualized through immuno-gold silver staining after 48 hours of free

floating incubation at 4 o C in the primary antibody solution. Sections were dehydrated

and embedded without counterstain. Cell counts were performed on digitized images

after thresholding the visualization signal. Only cells with a complete nucleus contained

within the section thickness were counted within the manually traced outline of the SNpc

and SG aggregates. Contours of these outlines were used to calculate areas and volumes

and subsequently used for 3D image reconstruction (using the package ‘Reconstruct’,

Fiala 2005).

All work was conducted in accordance under the guidelines of the National

Institutes of Health (USA), and the most recent edition of the Guidelines for the

Treatment of Animals in Behavioural Research and Teaching published by the

Association of Animal Behaviour.

Results

Summarized in Figure 1 are the observed differences in absolute brain volume as

a function migratory distance in migratory and non-migratory stone chats and lark

sparrows. Striking is that while the stone chats show a clear and significant reduction in

brain volume as migratory distance increases, no such relationship is seen in the lark

sparrows; there might even be a trend for brain volume to be larger in the migrant

population.

migration distance in 1000 km

0 1 2 3 4 5 6 7

bra

in v

olu

me

in c

m3

0,3

0,4

0,5

0,6

0,7

0,8

0,9

Saxicola torquata

Chondestes grammacus

Figure 1. Absolute brain size as a function of migratory distance in stone chats

(red squares) and lark sparrows (yellow triangles).

With respect to the telencephalon, two observations are noteworthy. First, in lark

sparrow females, hippocampal volume did not vary between migrants and non-migrants

(Figure 2).

Figure 2. Hippocampal volume relative to overall telencephalic volume in

migratory (Nebraska) and non-migratory (Texas) female lark sparrows.

Second, in stone chats one telencephalic area found to be smaller in the migrant

populations was the medial striatum (regression Stm % = 23,607 - (0,320 * distance), p =

0,03, r2 = 0,43, Figure 3a). The medial striatum is known to receive a catecholaminergic

projection from the midbrain, and a preliminary analysis has shown (Figure 3b) that there

are fewer TH positive, presumptively dopaminergic, brainstem-projection neurons in the

substantia nigra in migratory stone chats from Asia compared to African non-migrants;

no such difference is found in migrant and non-migrant lark sparrows.

migration distance in 1000 km

0 1 2 3 4 5 6 7

nu

mb

er o

f Th

po

sitiv

e c

ells

in S

Np

c

1000

1200

1400

1600

1800

2000

2200

2400

2600 Sylviinae

Saxicola

Chondestes g.

migration distance in 1000 km

0 1 2 3 4 5 6 7

Stm

(% o

f tele

nce

ph

alo

n) +

- SE

M

20

21

22

23

24

25

Figure 3 (a) Relative size of medial striatum (Stm) as percentage of telencephalon

volume in stonechats as a function of migration distance and (b) number of tyrosine

hydroxylase (dopaminergic) positive neurons in the midbrain substantia nigra of stone

chats (green squares) and lark sparrows (yellow triangles) as a function of migratory

distance. For comparison, grey circles in (b) are from different species of European

warblers, which are not discussed in this paper.

Finally, we wish to highlight that although we have yet to find any brain

differences in populations of migratory and non-migratory lark sparrows, there was

considerable variation across individuals in the pooled sample. Examining Figure 4 it is

apparent that differences in nidopallium volume explains most of the variation in overall

telencephalic volume. The nidopallium in a heterogeneous structure related to audition,

higher order sensory processing and memory. It is therefore premature to speculate on the

importance of the observed correlation.

a b

brain volume in cm3

0,62 0,64 0,66 0,68 0,70 0,72 0,74 0,76 0,78 0,80 0,82 0,84

volu

me o

f tele

ncephalic

stru

ctu

re in

mm

3

0

20

40

60

80

100

120

Nidopallium

Arcopallium

Hippocampal

Hyperpallium

Mesopallium

Hyperpallium denso

Septum

Striatum

Figure 4. Correlation between brain subdivision volume and overall telencephalic

volume in lark sparrows. Note that the nidopallium in particular increases in volume as

overall telencephalic volume increases independent of migratory behavior.

Discussion

The data presented offer only a snapshot of the ongoing analyses we are carrying

out. However, despite their preliminary nature, some interesting relationships have

already been observed. In our opinion, the clear correlation of decreasing brain volume

with migratory distance in stone chats, and the absence of such a correlation in lark

sparrows, is particularly interesting. The origin of this difference likely has a number of

sources, and two possibilities are worth commenting on here. First, even the European

migratory stone chat populations carry out migrations that are considerably longer than

the lark sparrows from Nebraska. If longer migrations are associated with greater

selective pressure for adaptations in support of migration, this could in part explain the

lack of a difference between the two lark sparrow populations (although the lark sparrows

from Nebraska display longer wing lengths, which are typically interpreted as an adaptive

response to migratory behavior). Perhaps more interesting as an explanation, migratory

behavior has evolved independently numerous times among songbirds; stone chats and

lark sparrows certainly evolved their migratory behavior independent of each other. The

different populations of stone chats examined in this study are characterized by genetic

and plumage differences, as well as subspecies status, suggesting a relatively long history

of independent evolution. By contrast, genetic differences in the two lark sparrow

populations have yet to be identified (Ross and Bouzat, personal communication). The

lark sparrow populations also share plumage characteristics and are not considered

subspecies. As such, in stone chats we may be looking at an older evolving system, which

is approaching the terminal stages in the evolution of migratory adaptations. By contrast,

lark sparrows may have only recently (relatively) evolved a migratory habit in some

populations; populations that have yet to become genetically distinct from non-migratory

populations, and therefore, have yet to display brain organizational adaptations to the

same degree found in stone chats.

With respect to the stone chats, the robust correlation of a smaller telencephalon

with increasing migratory distance, coupled with a smaller medial striatum and smaller

dopaminergic projection, suggests one adaptive response to the challenges of migration.

A smaller brain means less energetic cost allowing migrants to expend more energy to

fuel the migratory journey. But what might be the cost of a specific reduction in the size

of the medial striatum? Functional considerations of the medial striatum suggest that it

may be involved in regulating motor coordination as part of the basal ganglia. As such,

one consequence may be a reduced capacity to carry out complex motor sequences in

migrant stone chats compared to non-migrants. Alternatively, other portions of the medial

striatum support reward processes related to associative learning (e.g., Izawa et al., 2003).

It is, therefore, also possible that the smaller medial striatum in stone chats might impact

certain learning processes.

In any event, the preliminary data reported here highlight that the evolution of a

“migrant brain” is complex. Given that songbird migration has evolved numerous times

in different groups, a comparison of brain differences among migrants and non-migrants

in these different groups potentially offers an unmatched basis to assess how conservative

or improvisational natural selection can be in adapting an avian brain for the challenges

of migration.

Acknowledgements

This work was supported by grants from NSF and the J. P. Scott Center for

Neuroscience, Mind and Behavior at Bowling Green State University.

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