Chancellor. R. I). & B.-l'. Moyburg eds. 2004
Raptors Worldwiclo
W WCi BP/MM H

Phylogenetic Differentiation of theOsprey Pandion haliaetus inferredfrom nucleotide sequences of themitochondrial cytochrome b gene
Michael Wink, Hedi Sauer-Giirth and Hans-Hinrich Witt
ABSTRACTTho mitochondrial cytochrome b gene of three of the four subspecies e)f theOsprey (P. Ii. Iinlinctus, P. h. cnrolinensis, and P. h. cristatus) was amplified by
PCR and sequenced. Ospreys cluster as a monophyletic group within theFalconifonnes and show a clear and unequivocal phylogeographic patternaccording to the recogmseel subspecies. Genetic distances between the subspecies
are between 1» and 3.89r, i.e. in the range of "goeni" species. Together withmorphological characters and distribution pattern the genetic data indicate thatthe three osprey subspecies appear to represent distinct species.

INTRODUCTION
The Osprey Pnndion Iuiliiwtns is a unique raptor that feeds entirely on fishand has a world-wide distribution. It has boon placed in a monotypic family,
the Pandiomdae, within the Falconiformes (Sibley & Monroe 1990) and has
been subelivieleel at present into four geographically defined subspecies:•nominate /'. /;. Iinliaeliis ( Palaearctic. incl. Cape Verde Islands; migratory).
• P. Ii. cnrolinensis (North America: migratory).
•
P. h. rid^wnyi (Caribbean subregion; non-migrator) ).
• P. h. cristatus (Australia, from Sulawesi and Java to New Caledonia; nonmigratory)
When Ospreys wore first described between 1822 (Vieillot 1822) and 1837
(Gould 1837). three species were recognised that were placed in the genus
Pnndion. Lumping started in 1874 (Sharpe 1874) and ended with Knowiton &Ridgway (1909) (summary Provost 1983). In the following decades disputes
mainly concerned the recognition of additional subspecies.
511

Ospreys vary in size, both geographically and by sex. Usually females arelarger than males, about 20% larger in body mass, and S-IOI larger in winglength. Some overlap is possible, but this is limited; in North America, forexample, nearly all males weigh less than 1600 grams, while nearly all females
weigh more. Yet at a distance such differences are obscured and it is often
difficult to distinguish between the sexes in the field, even though females tend
to have darker breast plumage.
The overall habits and behaviour of the four subspecies are very similar,
although a few characters have been described that differ (del Hoyo et al. 1994).
The subspecies show only slight differences, mostly in plumage and size, withIialiaetus and caroliiietisis the largest and darkest, and ridgwayi essentially a pale
form of caroliiietisis. The Australian cristatus is a small osprey with dark breastband and a relatively pale crown (del Hoyo et al. 1994).
Table 1 summarises a few plumage characters and Table 2 differences inwing length as an indicator of size (Weick 1980; Prévost 1983; del Hoyo et al.
1994). The osprey s of the northern hemisphere appear to share a number ofcharacters and differ from the Australasian subspecies P. h. cristatus, which isthe smallest of the four subspecies.

Table 1. Morphological characters of the Pandion taxa
Sample sizes are in brackets (after Prévost 1983).

PalaearcticCape VerdeRed Sea
North AmericaBahaman
Australasian

Breast 'Average

Male
Crown 2average

Breastaverage

FemaleCrown
average

2.7 (72)
2.2 ( 6)
1.6(5)
1.4 (49)
1.0(4)
2.9 (42)2.5 (32)
2.5 ( 6)
2.0(5)
2.6 (49)
1.0(3)
1.5 (42)

3.4 (65)
2.3(4)
1.7(3)
2.4 (47)1.0(3)
3.7 (50)

2.7 (41)
2.5 ( 4)
1.7(3)
2.8 (46)
1.0(3)
1.3 (50)

The breast markings were scored from 1 to 4; l=the breast almost white;
2=a band of partially brown feathers < 3 cm wide; 3=a band of partiallybrown feathers > 3 cm wide; 4=a wide band of entirely brown feathers~ Crown markings were scored from 1 to 3 : I=Iess than 10 % black feathers;
2= from 10 to 50% black feathers; 3=over 50% black feathers
Another character is larger underwing coverts. All Palaearctic adult ospreys, andonly these, have the large underwing coverts (one half rufous-brown, one half white).
Although North American ospreys have underwings similar to those of Palaearctic
ospreys, their larger underwing coverts are barred dark brown. In Bahamian ospreysthese coverts are nearly white, whereas in Australasian birds they are nearly all ashy
brown (Prévost 1983).
In this communication we report on a molecular phylogeny and phylogeographyof ospreys based on nucleotide sequences of the mitochondrial b gene covering threeof the subspecies. Experimental evidence is presented that the three taxa represent
genetic lineages isolated for probably more than 1 million years.
512

Table 2. Wing lengths (mm) of the Pandion taxa (after Weick 1980)
P. h. haliaetus
P. IL carolinensis
P. h. ridgwayi
P. h. cristatus

femalesmales

470-510488-518455-495
425-490450-510462-506
433-483426-431

MATERIAL AND METHODS
We have isolated total DNA from feather, blood or tissue samples (seeWink 2000) which had been kindly supplied by several colleagues (B.
Bed'Hom, J. Haapala, D. Ristow, J. Penhallurick, J.-C. Thibault, W. E. Boles,
R. Schodde. 0 . Hadzofe). The cytochrome b gene was amplified by PCR(primer sequences in Wink & Sauer-Gürth 2000) and sequenced by using
AlfExpress ( Amersham Pharmacia Biotech) or ABI 3100 (AppliedBiosystems) instruments. Sequences were aligned manually and analysed withthe software packages PAUP* (Swofford, 2002) and MEGA2 (Kumar et a!.
2001) (see Wink 2000; Wink & Sauer-GUrth 2000; Wink et ai 2002; Broders
et al. 2003 for further details).

RESULTS AND DISCUSSION
Ospreys form a well-supported monophyletic clade (Fig. 1) (Wink &Sauer-Giirth 2000) and cluster within the basal groups of the Accipitridae; inother reconstructions Pandion takes a basal place outside the Accipitridae(which would support its status as a monotypic family). Several taxa of kites
that had not been included into previous phylogenetic studies (Wink 2000;
Wink & Sauer-Gürth 2000) take an even more basal position (Fig. 1) thanhitherto seen for Pandion. However, longer mitochondrial sequences andnuclear sequences are needed to identify the position of Pandion
unequivocally.

Table 3. Informative characters in the Pandion data set

Taxon/NodeP. h. h a lì aelu. s- FL
P.h. Iuiliiietiis Is
P.h. haliaetus Co
P.h. haliaetus Po
P.h. haliaetus F20425
P. h. Iuiliaeliis F20429
P. h. haliaetus F20430
P. h. haliaetus F9263
P.h. carolinensis USA 4252P.h. carolinensis USA 4253
P.h. carolinensis USA 4255
P.h. carolinensis USA 4254
P.h. cristatus AUS 701 1
P.h. cristatus AUS 21009
P.h. cristatus AUS 21008

iuiiii
11222223344445556666677788889999900000CÛ
99242449929246823 9128 9 95 694 6 99144890 0124 5 7
3 6 9756917799759274845049223448829306810831
GGGAATCCCGCGTCGATCGTGTGCACCACTTCGCTTAGTACC
A. . .AA

A. . .GCATTAAACGAG.TA . .C.T . . .GT
CC.A.GTA
AA. .GCATTAAACGAG.TA.ACAT . . . . T . . . A.CC.A.CTA
AA. .GCATTAAACGAG.TA. . C . T . . . GT . . .AACC.A.GTA
A...GCATTAAACGAG.TA.ACAT....T,..AACC.A.GTA.AAG. . . .TA. .C.AGCTAC.C. . G . T . . CCT . . CC
.AAG. . . .TA. .C.AGCTAC.C. .GTT. .CCT . .CCG
.AAG. . . .TA. .C.AGCTAC.C. . G . T . .CCT. . CCG

513

Within the ospreys a cleareut phylogeograpliic pattern was found,irrespective of the methods or outgroups used for phylogeny reconstructions(MP. ML. NJ) (Fig. 2). The Australasian P. h. distants usually takes a basal
position, whereas P. h. carolinensis and P. h. haliaetus cluster as sister taxa in
Ml' reconstructions (this would agree with morphological characters). No
phylogeographic pattern was found between different European populations of
the osprey. Table 3 illustrates the comparably Iaige number of phylogenelicalh
informative characters in the Pandion data set.

Figure 1. A molecular phvlogeny of ospreys based on almost complete
nucleotide sequences of the cytochrome b gene

11 lawks, kites, sea eagles.
1harriers• vultures !

eaglesvultures Ilospreys..1,'v!

kites"
A black-shouldered kiteIHaleons
Table 4 summarises the genetic distances between the three osprey taxa. Pdistances are between 2 and 4C4 : assuming a molecular clock of 1'/< = 1 million
years of divergence (Tarr et al.. 1993: Wilson et al. 1987), the taxa havediverged I to 2 million years ago.

514

Table 4. Genetic distances between Pandion taxa
1*

2

3**-

2.1-3.5
2.9-3.8

1.9-2.6

1. P. h. haliaetus
2. P. h. carolinensis

3. P. h. cristatus^distances within European ospreys: 0.1-0.5%; **within Australian birds: 0.3-0.8%

Figure 2. Phylogenetic differentiation within Pandion haliaetus
MP= Maximum parsimony; NJ= Neighbour Joining (Kimura2-distance algorithm)
Numbers at branches are bootstrap values in % (from 1000 replications)GallusGallusP. h. haliaetus Finland 7523

P. h. haliaetus Finland 7523

P.h. haliaetus Corsica

P. h. haliaetus Corsica

P.h. haliaetus France 20425"P. h. haliaetus Israel

P h. haliaetus France 20429

P. h. haliaetus France 20425

P. h. haliaetus France .9263

P. h. haliaetus France 20430

P. h. haliaetus France 20430

P. h. haliaetus France 20429

P. h. haliaetus Portugal

P. h. haliaetus France 9263

P.h. haliaetus Israel

P. h. haliaetus Portugal

P. h. carolinensis USA.4252—

P. h. cristatus Australia 7011

P, h. carolinensis USA.4255/

f P . h. carolinensis USA.4253
P. h. carolinensis USA.4254

Ff:

P. h. cristatus Australia 21008

P. h. cristatus Australia 21009
P. h. carolinensis USA 4252

"P. h. cristatus Australia 070110—10 changes

r~P. h. carolinensis USA 4253

P. h cristatus Australia 21009

P. h, carolinensis USA 4254

P. h. cristatus Australia 21008"P. h. carolinensis USA 4255

—0.005 substitutions/site

Genetic distances between closely related sister species of some eagles (A.
clanga/ A. pomarina; A. heliaca/A. adalberti; H. fasciatus/H.

spilogaster) are in

the range of 1.7 to 2.1% (Table 5). Since the distances within ospreys clearlyexceed this threshold, we suggest that the Pandion taxa represent "good"
species although the morphological differences are small. They are also verysmall in case of the sister species mentioned in Table 5. Thus the old conceptof the early 19th century (Vieillot 1822; Gould 1837), of defining at least three
distinct osprey species, might not have been so wrong at all.
Table 5. Distance between closely related sister pairs of eagles (in %sequence divergence)
1
1 Aquila clanga
2 Aquila pomarina
3 Aquila helìaca
4 Aquila adalberti
5 Hieraaetus fasciatus
6 Hieraaetus spilogaster2
1.7-37.1
7.3-4
7.5
7.7
1.8-

5
7.2
7.1
7.4
7.8
-

6
6.7
6.6
6.6
7.2
2.1
515

CONCLUSIONSGenetic distances between osprey taxa are in a range that is typical for
distinct raptor species. Morphological and genetic differences exist and support
the suggestion, that the geographically defined subspecies may be recognised
as distinct species:•P. haliaetus• P. carolinensis• P. ridgwayi• P. cristatus
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Prof. Dr. Michael Wink
Institut für Pharmazie und Molekulare
Biotechnologie
Universität Heidelberg
INF 364, 69120 Heidelberg, Germany
E-mail: wink@uni-hd.deHedi Sauer-Gürth
Institut für Pharmazie und Molekulare
Biotechnologie
Universität Heidelberg
INF 364, 69120 Heidelberg, Germany

Dr. Hans-Hinrich WittForsthaus Kornberg 134621 Frielendorf
Germany516