I examined hypotheses of Aphelocoma jay phylogeny derived from allozyme data. Results from various algorithms differ in details, but the overall patterns are consistent: Scrub Jays (A. coerulescens) and Unicolored Jays (A. unicolor) were derived independently from different populations of Gray-breasted Jays (A. ultramarina). Within Scrub Jays, the californica subspecies group was derived from the populations of interior North America (woodhouseii group). One Unicolored Jay population and two Scrub Jay populations, all strongly differentiated, are placed consistently at the base of the phylogeny, but phenotypic, biogeographic, and theoretical evidence suggests that these populations represent rapidly evolving populations derived from within populations of their respective species. Because analyses of rates of molecular evolution demonstrate significant rate heterogeneity, I suggest that the application of a molecular clock to date-splitting events in the Aphelocoma jays is not a valid approach. Received 18 February 1991, accepted 3 July 1991.
Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois 60637, USA
THE THREE species of Aphelocoma jays range
throughout western and southern North Amer-
ica and northern Central America (Fig. 1; Pi-
telka 1951). Scrub Jays (A. coerulescens) range
from Oregon and Wyoming south to the Isth-
mus of Tehuantepec, with disjunct populations
on Santa Cruz Island off the coast of southern
California and in peninsular Florida. The 15
subspecies form five groups, each characterized
by unique combinations of plumage, morpho-
logical, and behavioral characters (Fig. 1; Pi-
telka 1951, Peterson 1991a): (1) woodhouseii group
(Wyoming and southeastern Oregon south
through Great Basin and along both sides of
Rocky Mountains, and then along interior slopes
of Sierra Madre Oriental and Sierra Madre Oc-
cidental of northern Mexico to southern Chi-
huahuan Desert and vicinity of Mexico City);
(2) californica group (western Oregon, Califor-
nia, and Baja California); (3) sumichrasti group
(southern Mexico); (4) coerulescens group (pen-
insular Florida); and (5) insularis group (Santa
Cruz Island). The Gray-breasted Jay (A. ultra-
marina) ranges throughout the mountains of
northern and central Mexico and the south-
western United States. The seven subspecies fall
into three groups characterized by unique com-
binations of morphological and behavioral
characters (Fig. 1; Pitelka 1951 ): (1) pot osina group
(Sierra Madre Oriental); (2) wollweberi group (Si-
Present address: Department of Zoology, Field
Museum of Natural History, Roosevelt Road at Lake
Shore Drive, Chicago, Illinois 60605, USA.
erra Madre Occidental); and (3) ultramarina group
(Transvolcanic Belt). Finally, the Unicolored Jay
(A. unicolor) consists of five allopatric popula-
tions, each a separate subspecies, in southern
Mexico and northern Central America (Pitelka
1951).
The Aphelocoma jays have been the subject of
numerous comparative studies. Evaluations of
social systems in the genus have led to advances
in understanding ecological factors important
in the evolution of sociality (Woolfenden and
Fitzpatrick 1984, Fitzpatrick and Woolfenden
1986, Brown 1987, Peterson and Burt, in press).
Differential habitat use in Scrub Jays (Peterson
and Vargas 1992) is correlated with geographic
variation in beak shape, suggesting that beak
shapes have responded to natural selection (Pe-
terson, in prep.). Integration of phylogenetic
information into such investigations will allow
an important new dimension of understanding
(Brooks and McLennan 1990). Hence, I have
attempted to estimate the phylogeny of the dif-
ferentiated forms in the genus.
Rates of molecular evolution.--Since the pub-
lication of the influential paper of Zuckerkandl
and Pauling (1962), the idea of a "molecular
clock" has been controversial in molecular bi-
ology and systematics. The clock concept is based
on the assumption of a uniform rate of molec-
ular evolution in a group. If the uniform rate
assumption were correct, the accumulation of
genetic differentiation between sister taxa would
be time-invariant, and divergence times could
be estimated from genetic distances.
The clock concept is an important feature of
many sectors of molecular systematics. At-
tempts have been made to calibrate clocks for
a variety of taxa and biochemical data sets (e.g.
Sarich 1977, Wilson et al. 1977, Sibley and
Ahlquist 1981). Several commonly used tree-
building algorithms depend on the assumption
of a clock (Felsenstein 1982, 1989). The molec-
ular clock is now a common method of dating
divergence times between taxa (e.g. Zink 1982,
Nevo et al. 1987, Johnson and Marten 1988).
Hence, a uniform rate of molecular evolution
and the consequent existence of a clock can be
important assumptions in molecular systemat-
ics.
However, the assumption that evolutionary
rates are homogeneous often goes untested. If
molecular evolutionary rates differ among lin-
eages, the clock becomes unreliable. Equality of
rates in sister lineages can be tested with the
relative-rate test: in the three-taxon statement
((A, B), C), A and B should show similar degrees
of differentiation from C (Wilson et al. 1977).
Significant departure from equality indicates
that A and B have evolved at different rates.
Rate uniformity has been tested in a number of
taxa, with some studies documenting homo-
geneity (e.g. Sibley and Ahlquist 1983, Bledsoe
1987), and others finding heterogeneity (e.g.
Britten 1986, Sibley et al. 1987, Sheldon 1987,
Springer and Kirsch 1989). A uniform rate and
molecular clock are commonly and uncritically
assumed in systematic studies, so a test of the
assumption of rate uniformity was desirable in
my study.
METHODS
Sampling design.--During 1986-1989, I studied and
collected Aphelocoma jays at 35 sites in the United
States and Mexico. Tissue from the endangered Flor-
ida population of Scrub Jays was salvaged from re-
cently dead individuals at nests and roadsides. Sam-
ples of heart, liver, and pectoral muscle from each
individual were stored in cryotubes in liquid nitro-
gen. All tissue was deposited in the Frozen Tissue
Collection of the Field Museum of Natural History.
In total, I analyzed 615 Aphelocoma jays (458 Scrub
Jays, 138 Gray-breasted Jays, and 19 Unicolored Jays).
Specimens of Scrub, Gray-breasted, and Unicolored
jays were collected at 24, 8, and 3 sites, respectively,
in the United States and Mexico (Table 1, Fig. 1). I
attempted to obtain samples of 20 individuals at each
site to maximize tree stability (Archie et al. 1989):
Scrub Jay samples averaged 19.2 (range 13-27) indi-
viduals; Gray-breasted Jay samples averaged 17.3
(range 7-22) individuals; and Unicolored Jay samples
Fig. 1. Summary of collecting localities and sub-
species groups of three species of Aphelocoma jays;
Florida range indicated in upper right-hand corner.
(A) Scrub Jays: open circle, Santa Cruz Island (ACINS);
closed circles; californica group (CO CAL); closed tri-
angles, northern woodhouseii group (CO WOO); open
triangles, southern woodhouseii group (CO MEX);
squares, sumichrasti group; dotted circle, Florida
(ACCO2). (B) Gray-breasted Jays: triangles, wollweberi
group (UL WOL); circles, potosina group (UL POT);
squares, ultramarina group (UL ULT). (C) Unicolored
Jays: triangles, east slope populations (UN E); circle,
west slope population (ANGUE).
TABLE 1. Sample localities and sample sizes (complete locality data in Peterson 1990).
Sample
Group Sample Locality size
Scrub Jays
CO CAL
CO MEX
CO WOO
CO SUM
Gray-breasted Jays
UL WOL
UL POT
UL ULT
Unicolored Jays
UN E
ACCAL CA, Monterey Co., Bradley 24
ACCAU CA, Trinity Co., Hyampom 16
ACHY3 BCS, Sierra de la Laguna 16
ACIMM OR, Adair Village 15
ACOBc CA, Orange Co., Silverado Canyon 16
ACOBk BCN, Rosa de Castilla (Ojos Negros) 20
ACOOC CA, San Ramon 16
ACSP1 CA, Alturas 22
ACSP3 CA, Bodfish 19
ACCY1 COA, E1 Diamante Pass 21
ACCY2 SLP, Sierra de Bledos 21
ACGR! DUR, Villa Ocampo 21
ACGR3 JAL, Lagos de Moreno 20
ACNE! NV, Toiyabe Mountains 14
ACNE4 NV, Mt. Charleston 27
ACNE6 AZ, Drake 25
ACTEX TX, Edwards Plateau, Carta Valley 22
ACWO! CO, Front Range, Gardner ! 1
ACWO3 NM, Manzano Mountains 26
ACWO4 TX, Davis Mountains 21
ACREM GRO, Xocomanatl/n 13
ACSM2 OAX, Sierra de los Mijes, Zempoaltpetl 20
ACINS CA, Santa Cruz Island 14
ACCO2 FL, Archbold Biol. Station 18
AUAZ1 AZ, Pefia Blanca Lake 16
AUGRA JAL, Sierra de Bolafios 7
AUWO1 ZAC, Valparaiso 20
AUCO2 COA, Sierra E1 Carmen 22
AUPO2 SLP, Sierra de Bledos 22
AUPO3 HID, Jacala 20
AUULT MOR, Huitzilac 14
AUCOL JAL, Sayula 17
ANGUE GRO, Sierra de Atoyac 7
ANOAX OAX, Sierra de los Mijes, Zempoaltpetl 10
ANCON HID, Sierra de la Huasteca, Tlanchinol 2
averaged 6.3 (range 2-10) individuals. To avoid prob-
lems with nonindependence of individual genotypes
when related individuals (e.g. parents and offspring,
multiple siblings) were collected, I included only one
member of each potentially related pair of individ-
uals.
For convenience in referring to population sam-
ples, I assigned each a five-letter code, in which the
first letter indicates the genus, the second indicates
the species, and the remaining three indicate sub-
species and (if necessary) the sample from that sub-
species (e.g. ACNE2 is the second sample of A__phelo-
coma coerulescens nevadae; Table I). For reference to
larger infraspecific divisions, I divided the species in
a manner equivalent to the subspecies groups of Pi-
telka (1951), except that the woodhouseii group was
divided into northern (United States populations) and
southern (Mexican populations) parts (Fig. I, Table
1).
To provide outgroups for phylogenetic analyses, I
included 70 individuals of 17 other corvid species. To
maximize detection of plesiomorphic alleles for po-
tentially closely related genera (Hardy 1969, Hope
1989), I included all material available (up to 20 in-
dividuals) of each of the following species: Cyanocitta
cristata, C. stelleri, Cyanolyca mirabilis, C. cucullata, C.
viridicyana, and Gymnorhinus cyanocephalus. For less
closely related corvid genera, I included one or two
individuals of each species available: Calocitta formosa,
C. colliei, Cyanocorax violaceus, C. cyanomelas, C. yncas,
Corvus brachyrhynchos, C. cryptoleucus, Pica pica, P. nut-
tallii, Nucifraga columbiana, and Perisoreus canadensis.
Electrophoresis.--I homogenized approximately 0.4 g
each of heart, muscle, and liver from each sample
in a 1 ram disodium EDTA/100 ram Trizma base/0.2
ram NAD, NADP, and ATP buffer. Homogenates were
centrifuged at 12,000 rpm for 30-45 min. Superna-
tants were drawn into capillary tubes, and stored at
-76øC for later use. Gels for electrophoresis were
made of 10-12% starch in the appropriate buffer. After
electrophoresis, gels were sliced, and stained differ-
entially following Shaw and Prasad (1970) and Harris
and Hopkinson (1978). Loci studied included ACON
(enzyme number 4.2.1.3; Harris and Hopkinson 1978),
ACP (3.1.3.2), ADA (3.5.4.4), ADH (1.1.1.1), AK (2 loci,
2.7.4.3), CK (2 loci, 2.7.3.2), ES (2 loci, 3.1.1.1), FUM
(2 loci, 4.2.1.2), GDA (3.5.4.3), GOT (2.6.1.1), GPD
(1.1.1.8), GPI (5.3.1.9), IDH (1.1.1.42), LDH (2 loci,
1.1.1.27), MDH (2 loci, 1.1.1.37), MPI (5.3.1.8), PEP
(leucyl-alanine, 2 loci; leucyl-aminopeptide, 1 locus;
3.4.11), PGD (1.1.1.44), PGM (2.7.5.1), SOD (1.15.1.1),
and SORDH (1.1.1.14). Details of electrophoretic con-
ditions are given in Peterson (1990). Loci showing
difficult-to-interpret patterns of variation were ex-
cluded. Stained gels were digitized with an optical
imaging system, and visual images were stored in
computer files; gels were later discarded.
To assure correct assignment of homologies, I in-
cluded reference individuals on each gel. Alleles from
different populations were not considered equivalent
until found indistinguishable in side-by-side com-
parisons on the same gel. I rechecked hornology as-
signments for each locus under different buffer con-
ditions and/or at maximum migration distances (an
average of two recheck runs per variable locus; Pe-
terson 1990).
Tree-building analyses.--I employed a variety of
methods to estimate phylogenetic relationships of the
Aphelocoma jays. I began with simple methods with
restrictive assumptions; then, with successively more
complex approaches, I attempted to relax these as-
sumptions. I initially analyzed relationships of all 35
population samples, but later combined individual
samples and investigated relationships of the groups
of samples described above.
Although an explicit test of the monophyly of the
Aphelocoma jays has yet to be conducted, several lines
of evidence indicate that the group is monophyletic.
Several characters of plumage, vocalizations, and
morphology are characteristic of Aphelocoma jays (Pi-
telka 1951). Hope (1989) found that Aphelocoma, Cy-
anocitta, and Gymnorhinus form a well-supported group
based on phylogenetic analysis of qualitative and
mensural osteological characters. Aphelocoma and Cy-
anocitta share a synapomorphy (a new bony element
in the sclerotic ring; Curtis and Miller 1938) not pre-
sent in Gymnorhinus (Peterson, unpubl. data). While
Cyanocitta has a number of unique features, Aphelo-
coma is not so well defined. Because it seems unlikely
that Cyanocitta was derived from within Aphelocoma,
I assume for the purposes of this paper that Aphelo-
coma is monophyletic.
I performed phenetic analyses on matrices of mod-
ified Rogers' genetic distances (as modified by Wright
1978), which were calculated using programs devel-
oped by Scott M. Lanyon. Allele-frequency data and
genetic-distance matrices are available in Peterson
(1990) or on request from the author. Exploratory
analyses based on other genetic distance measures
either produced similar results (e.g. Cavalli-Sforza
chord distance) or were difficult to interpret (e.g. Nei's
genetic distance), so I report only results from Rogers'
distances. Two different clustering methods, the un-
weighted pair-group method (UPGMA) and single-
linkage cluster analysis, were applied to the genetic
distance matrix using NTSYS-pc (version 1.40; Rohlf
1988).
One potential problem with strictly phenetic meth-
ods is the assumption of equal evolutionary rates in
each lineage (Felsenstein 1982). A first step in relaxing
this assumption is the Fitch-Margoliash algorithm,
which allows for some variation in evolutionary rates
(Fitch and Margoliash 1967, Felsenstein 1989). I used
Steller's Jays (Cyanocitta stelleri, CYSTE) as the out-
group, because it is sufficiently close phylogenetically
to be informative, and the sample size (20) was suf-
ficiently large to permit accurate estimation of gene
frequencies. Because of limited computer memory,
the entire data set could not be analyzed at one time,
so I analyzed subsets of the data in two ways. I either
assumed species to be monophyletic and analyzed
each separately, or I used representative populations
from each of the subspecies groups. All trees were
checked for stability using a iackknife manipulation
(Lanyon 1985), in which the data were reanalyzed
sequentially omitting each ingroup taxon and the re-
sulting trees combined by strict consensus using pro-
grams developed by Scott M. Lanyon to complement
those of PHYLIP (Felsenstein 1989).
Cladistic analysis of electrophoretic data is difficult
both logically and methodologically, chiefly because
of problems with the treatment of polymorphic char-
acters (Buth 1984, Swofford and Berlocher 1987, Swof-
ford and Olsen 1990). Computer packages such as
PAUP deal with the problem by selecting the most-
parsimonious character state from among the possi-
bilities and ignoring potentially conflicting infor-
mation in other alleles present in the population (D.
Swofford, pets. comm.). Because loci in my study were
highly polymorphic (Peterson 1990), coding loci as
characters would not be very informative; instead, I
chose to code alleles as characters.
Using the heuristics/subtree-pruning and regraft-
ing branch-swapping option of PAUP (version 3.0;
TAIIE 2. Matrix of character states representing presence and absence of 38 phylogenetically informative
alleles at 29 loci in 35 Aphelocoma jay populations and a composite outgroup.
Sample 1 11 21 31
OUTGR 7110010110 0100011000 0000001000 00100000
ACCAL 0000010000 0001110000 0100111000 00000110
ACCAU 0000000000 1000110100 0000010000 00000100
ACCO2 0100000000 0000010000 0010000000 01000000
ACCY1 0100000000 0000010001 0000011001 00011111
ACCY2 0100000000 0010010001 0100011000 10000100
ACGR1 0000110100 1010010001 0100011000 10000110
ACGR3 0000000000 0000010000 0101011000 10000110
ACHY3 0000010000 0000110000 0110011000 00110010
ACIMM 0000000000 0000110000 0000011000 00000010
ACINS 0000000000 0000010000 0000000000 00000000
ACNE1 1000010110 0000010000 0010011110 00000110
ACNE4 0000010100 0000010101 0110011111 00000110
ACNE6 0000000010 0000010000 0010011000 00000110
ACOBc 1000000001 0000110000 1010011000 00000111
ACOBk 0010000000 0000110000 0000010000 00001010
ACOOC 0000100000 1000010001 0000111000 00000110
ACREM 0000000010 0000010000 0000011000 01000000
ACSM2 0000100010 0000010010 0000011000 01101000
ACSP1 1000000000 1000110100 0000011010 00000111
ACSP3 0000000000 0000110000 0000011000 00001110
ACTEX 0000000000 0000010000 0000011000 00000010
ACWO1 1000000000 0000010001 0001001010 00000110
ACWO3 0000000001 0010010001 0010011010 10000110
ACWO4 1000000000 0010010101 0000001110 00000110
AUPO3 0000001000 0000010010 0000011100 00000000
AUGRA 0000001000 0000011000 0000001000 00000000
AUCOL 0000000000 0100001000 0000001000 00000000
AUULT 0000000000 0000001000 0000011000 00100000
AUPO2 0011000000 0000010010 1000011100 00000000
AUWO1 0000001000 0000011010 0000001000 00000000
AUAZ1 0000000000 0000001000 0000000000 00000000
AUCO2 1001000000 0000011010 0000011100 00000000
ANCON 0000000000 0001001000 0000000000 00000000
ANOAX 0000000000 0100001000 0000011000 00000000
ANGUE 0000000000 0000001000 0000000000 00000000
Swofford 1989), I analyzed a binary coding of 102
alleles from 29 loci (Table 2). Of these alleles, 38 were
potentially informative about relationships among
ingroup taxa. This approach can have the undesirable
consequence of hypothetical ancestors lacking alleles
at particular loci (Buth 1984, Swofford and Berlocher
1987). However, this problem can be ignored if pop-
ulation samples are large enough that the presence
or absence of one allele in the sample can be assumed
to be independent of the presence of other alleles. In
other words, the alleles under study were indeed
lacking in those problematic hypothetical ancestors,
and some other allele is assumed to have been present
at that particular locus.
If the assumption of independence of alleles at a
locus were unreasonable, weighting alleles so that
each locus contributes equally to the analysis would
be desirable. In this particular data set, however, pre-
liminary runs indicated that weighting serves only
to overemphasize loci with few alleles and deem-
phasize apparently informative alleles at loci with
more alleles. Hence, succeeding analyses were based
on unweighted presence-and-absence data for alleles.
I used a composite outgroup representing allele pres-
ences and absences in all outgroup taxa to root re-
sulting trees. All data sets were tested for significant
phylogenetic information with the randomization tests
of Archie (1989). To assess the stability of nodes on
the resulting trees, a jackknife manipulation (Lanyon
1985) was conducted, in which the analyses were re-
peated sequentially omitting each of the ingroup taxa.
In each repetition, the heuristic search was allowed
to run to completion, with up to 2,500 trees stored
for branch-swapping, and the resulting 35 sets of 13
to 2,500 equally-parsimonious trees were combined
using a majority-rule consensus.
One problem plaguing cladistic analyses of elec-
trophoretic data is failure to detect alleles actually
present in populations because of inadequate sam-
pling (Swofford and Berlocher 1987). This nondetec-
T^SLE 3. Matrix of character states representing presence and absence of 33 phylogenetically informative
alleles at 29 loci in eight subspecies groups of Aphelocoma jays and a composite outgroup.
Sample 1 11 21 31
OUTGR 7110010110 1001100000 0000000100 000
CO CAL 1011100011 0011010111 1010100111 111
CO WOO 1000101110 0101010101 1111111000 110
CO MEX 0101101001 0101000101 0110011011
CO SUM 0001000100 0001001000 0010000101 000
UL POT 1010010000 0001101010 0011000000 000
ULWOL 0000010000 0001101000 0000000000 000
UL ULT 0000000000 1000100000 0010000100 000
UNICE 0000000000 1010100000 0010000000 000
tion causes extra homoplasy in the resulting trees in
the form of "reversals," which are actually undetected
synapomorphic alleles. In my study, several popu-
lation samples were available from most of the sub-
species groups, which reduced considerably the prob-
ability of nondetection of alleles. I conducted a cladistic
analysis of relationships among regions within the
three species (i.e. subspecies groups; Fig. 1, Table 1)
by combining all alleles found in populations of each
region into a subspecies group (Table 3). I found 33
alleles to be phylogenetically informative about re-
lationships of ingroup taxa (Table 3). Results based
on other phylogeny-estimation algorithms, such as
the polymorphism parsimony of Felsenstein (1979),
yielded similar results, and are not presented below.
Rate tests.--I tested for evolutionary rate uniformity
by focusing on three populations previously identi-
fied as distinctive in studies of genetic differentiation
(Peterson 1990): Scrub Jays from Florida; Scrub Jays
from Santa Cruz Island; and Unicolored Jays from
Guerrero. The hypothesis tested was that historical
independence of lineages could account for zones of
extreme genetic differentiation among populations of
Aphelocoma jays. Using three-taxon statements from
the phylogenetic results, I conducted relative-rate tests
that compared genetic distances in all representatives
of the two taxa connected by the interior node of these
trees to all representatives of the third taxon. Because
at least two of the groups are represented by numer-
ous population samples, replicate comparisons were
possible.
To visualize patterns of nonuniformity of rates, I
also estimated branch lengths and their variability
for two subspecies-group phylogenies with a resam-
pling technique. The resampling manipulation was
an attempt to estimate how robust branch-length es-
timates are to choice of populations for sampling. I
used the user-tree option of the FITCH program in
PHYLIP to estimate branch lengths in 30 replicate
analyses. Each replicate included ACINS, ANGUE,
ACCO2, and randomly selected population samples
representing each of the remaining eight subspecies
groups (Table 1). The wollweberi group of Gray-breast-
ed Jays was excluded from some analyses because it
is part of an unresolved trichotomy in some trees, and
the user-tree option of PHYLIP requires dichotomous
branching at internal nodes. Branch lengths from the
replicate analyses were compared using Mann-Whit-
ney U-tests.
RESULTS
Phenetic methods.--The two phenetic analyses
differed in the placement of a few groups and
in the degree of resolution (Fig. 2). However,
both established one cluster that consisted of
populations of the californica group of Scrub Jays,
and another of populations of the woodhouseii
and sumichrasti groups of Scrub Jays (Pitelka
1951). The only exceptions are the inclusion of
the populations of the southern tip of Baja Cal-
ifornia and northwestern California (ACHY3
and ACCAU) as lineages basal to both of the
clusters in the single-linkage method, and the
inclusion of four populations of Gray-breasted
Jays at various points within these clusters. The
remaining populations of Gray-breasted Jays and
the two east slope Unicolored Jay populations
(ANCON and ANOAX) form a sister cluster to
the two just described. Scrub Jay populations
from Florida and Santa Cruz Island, and the
Guerrero Unicolored Jay population fall outside
all of these clusters.
Fitch-Margoliash analyses.--Analyses of the re-
lationships of the 24 population samples of Scrub
Jays (Fig. 3A) showed basal branches leading to
the Santa Cruz Island and Florida populations
(ACINS and ACCO2), and then branches to
populations from the interior western United
States, northern Mexico, and southern Mexico
(woodhouseii and sumichrasti groups). Derived
from within the latter group are the populations
of the californica group. A jackknife manipula-
tion of this data set preserves the same basic
ACCAL
ACOOC
A COBC
A CIMM
ACOBk
A CSP3
ACSP 1
ACCY1
ACGR 1
ß ACCY2
A CGR3
ACNE6
ACW03
ACW01
' ACNE4
ACNE 1
ß ACTEX
A CREM
-F--- ACSM2
AUP03
AUP02
ACW04
A CHY3
A CCAU
AUGR
AUC02
z AUCOL
AUAZl
ANCON
AUUL T
AUWO 1
ANOAX
A CC02
ANGUE
A ClNS
Single-linkage
UPGMA
0.3 0.2 0.1 0.0 0.3 0.2 0.1
A CCAL
ACOOC
ACOBc
A CIMM
A COBk
A CSP3
A CSP 1
A CCAU
ACHY3
ACCY1
A CGR I
ACCY2
A CGR3
A CNE6
ACW03
ACW01
-- ACNE1
ACNE4
ACTEX
ACREM
A CSM2
AUP03
AUP02
ACW04
AUGRA
AUC02
AUCOL
AUAZ 1
ANCON
-- AUUL T
AUWO 1
ANOAX
ACCO2
AClNS
ANGUE
0.0
Modified Rogers' Genetic Distance
Fig. 2. Phenetic relationships among 35 populations of Aphelocorna jays based on (A) single-linkage cluster
analysis and (B) UPGMA. See Table 1 for abbreviations.
structure, with 8 of the 23 nodes lost due to
instability of the trees (Fig. 3A). Removal of
populations with very long branches (Santa Cruz
Island, ACINS, and Florida, ACCO2), as sug-
gested by Swofford and Olsen (1990), preserves
the same branching pattern in the remaining
populations.
The eight populations of Gray-breasted Jays
divided into two main lineages, one consisting
of the two southern populations and AUAZ1,
and the other of the eastern populations plus
AUGRA and AUWO1 (Fig. 3B). None of the
nodes was unstable under a jackknife manip-
ulation. Analyses of the three Unicolored Jay
populations indicated that the east-slope pop-
ulations (ANOAX and ANCON) are sister taxa,
well differentiated from the west-slope popu-
lation (ANGUE, Fig. 3C).
The analysis of representatives from each
subspecies group in the genus yielded unex-
pected results. Inclusion or exclusion of the bas-
al-split populations ANGUE, ACINS, and
ACCO2 did not change the placement of other
taxa, so these populations were excluded from
analysis. In nine replications (all jackknifed) of
randomly chosen representatives of each sub-
species group (for three examples, see Fig. 4),
the southern Gray-breasted Jay population
(AUCOL or AUULT) clustered with the Uni-
colored Jay population (ANCON or ANOAX),
and the eastern Gray-breasted Jay population
(AUPO2, AUPO3, or AUCO2) clustered with
the various Scrub Jay populations. The western
Gray-breasted Jay population (AUAZ1, AU-
GRA, or AUWO1) joined with the southern
Gray-breasted Jay-Unicolored Jay group, joined
with the Scrub Jay-eastern Gray-breasted Jay
group, or separated in a basal trichotomy. A1-
Fig. 3. Fitch-Margoliash analysis of relationships among populations within each of three species of
Aphelocoma jays: (A) Scrub Jays; (B) Gray-breasted Jays; and (C) Unicolored Jays. Asterisks indicate nodes
unstable to a jackknife manipulation. Numbers indicate fitted branch lengths; unmarked branches sized
proportionally. CYSTE is Cyanocitta stelleri, the outgroup; see Table ! for other abbreviations.
though the outgroup (CYSTE) is linked to the
network in a fairly consistent position, it could
be argued that inaccurate rooting could produce
the paraphyly in Gray-breasted Jays. In this case,
however, the two-branched form of the phy-
logeny makes any possible rooting lead to para-
phyly in that species. From these results, I sug-
gest that Gray-breased Jays represent a
paraphyletic taxon that gave rise to both Scrub
Jays and Unicolored Jays.
Cladistic analyses.--In cladistic analyses of re-
lationships of the 35 individual populations,
PAUP found at least 2,500 trees of 108 steps. A
majority-rule concensus of these trees is shown
in Figure 5. Of the 12 nodes in the concensus-
tree, eight were retained in all jackknife pseu-
doreplicate analyses. The randomization test of
Archie (1989) indicated the existence of signif-
icant phylogenetic information in the data (P
-< 0.03).
These trees have several interesting features.
First, they identify several pairs and trios of
populations as sister taxa: central California and
southern Baja California (ACCAL and ACHY3);
northern and central Great Basin populations
(ACNE1 and ACNE4); northern and southern
Rocky Mountain populations (ACWO1 and
ACWO4); southern Mexican populations of
Scrub Jays (ACREM and ACSM2); and central-
western Mexican (AUGRA and AUWO1) and
eastern Mexican (AUPO2 and AUCO2 and then
AUPO3) populations of Gray-breasted Jays.
Many of these groupings correspond to those
described based on morphology and geography
(Pitelka 1951). Within the woodhouseii group,
northern (ACNE1) and southern (ACNE4) neva-
dae populations are sister taxa, and northern
(ACWO1) and southern (ACWO4) woodhouseii
are also sister taxa. This finding confirms geo-
graphic patterns described by Pitelka (1951), and
ANCON i
AUCOL
AUGRA
AUPO2
ACCY2
ACREM
ACWO3
ACOBk
ANCON i
AUAZ1
AUULT
AUCO2
ACCY1
ACSM2
ACNE1
ACSP3
ANOAX i
AUULT
AUWO1
AUPO3
ACGR3
ACSM2
ACNE6
ACCAL
Fig. 4. Three replicate Fitch-Margoliash analyses
with randomly chosen representative populations of
eight subspecies groups of Aphelocoma jays. Bars in-
dicate species membership: white, Scrub Jay; gray,
Gray-breasted Jay; and black, Unicolored Jay. See Ta-
ble 1 for abbreviations.
contradicts patterns described by Phillips (1986),
who suggested that southern Great Basin
(southern nevadae) and southern Rocky Moun-
tain (southern woodhouseii) populations are more
closely related to each other than to populations
to the north.
In the original consensus tree and in 33 of 35
of the jackknife pseudoreplicates, the eastern
ACCAL
ACHY3
ACCAU
ACSP1
ACCY1
ACNE1
ACNE4
75-99% ACWO1
' 50-74% ACWO4
ACOOC
length 108 steps
ACCY2
c.I. = 0.352 ACGR1
ACGR3
AClMM
.............
^co.
ACSP3
ACTEX
ACREM
ACSM2
AUPO3
AUPO2
AUCO2
ACCO2
ACINS
ANCON
ANGUE
AUAZ1
AUGRA
AUWO1
AUCOL
AUULT
ANOAX
Fig. 5. Cladistic analysis of 35 Aphelocoma jay pop-
ulations, based on binary coding of 38 alleles. A ma-
jority-rule consensus of 2,500 108-step trees shown
by line type. Nodes marked with asterisk found to be
unstable to a jackknife manipulation. See Table 1 for
abbreviations. Consistency index (c.i.) indicated.
populations of Gray-breasted Jays are united
with the Scrub Jay clade, making Gray-breasted
Jays as a taxon paraphyletic (Fig. 5). In addition,
in all trees examined, a northwestern Gray-
breasted Jay population sample (AUAZ1) groups
with two Unicolored Jay populations (but see
Discussion). Again, the exact position of the root
does not change the paraphyly in Gray-breasted
Jays. Therefore, the currently recognized spe-
cies Gray-breasted Jay appears to be a paraphy-
letic taxon, with the other two recognized spe-
cies derived from different populations within
it.
Inspection of patterns of character evolution
on this tree indicates that the Scrub Jay clade
(minus ACTEX, ACREM, and ACSM2) is sup-
ported by two synapomorphies, and that sev-
eral other internal nodes are supported by syn-
apomorphic alleles. Because the GDA locus
shows an odd pattern of variation in Gray-
breasted Jay populations (Peterson 1990), I be-
came concerned that it might be responsible for
the patterns of paraphyly in Gray-breasted Jays.
This locus might be suspect because the two
alleles observed differed in mobility by only
about 2 mm. (Several rechecks of allele homol-
ogies at maximum mobility failed to distinguish
more than two alleles at this locus.) ! reran the
analyses omitting the information at the GDA
locus. Although the strict consensus of the re-
suiting 2,500 most-parsimonious trees is less well
resolved than the overall analysis, AUAZ1 was
still more closely related to two Unicolored Jay
populations than to other Gray-breasted Jay
populations (but see Discussion). A node unit-
ing eastern Gray-breasted Jay populations with
Scrub Jays was found in 69% of the trees. There-
fore, support exists for the hypothesis of para-
phyly of Gray-breasted Jays at loci other than
GDA.
In analyses of relationships of the subspecies
groups, PAUP found one 58-step tree (Fig. 6),
nine 59-step trees, and many longer trees. The
shortest tree is almost identical to those pro-
duced by the Fitch-Margoliash analyses (Fig. 4),
but differs in that eastern and western Gray-
breasted Jays group together; it provides better
resolution among populations of Scrub Jays. The
59-step trees differ in placement of a few
branches, but the basic structure is similar. Sev-
eral alleles provide support for most nodes, in-
cluding 10 alleles that represent synapomor-
phies for all Scrub Jay populations except the
sumichrasti group, and one to five synapomor-
phies that support other nodes (Fig. 6). A jack-
knife manipulation of the data set preserved
the general structure of the tree, with two un-
stable nodes. The randomization test of Archie
(1989) indicated the presence of significant phy-
logenetic information in the data set (P < 0.01).
Omitting the two GDA alleles from analysis did
not change the tree structure, except in that two
nodes are unresolved.
Although the tree of the eight subspecies
groups in the three species is well resolved,
placement of the remaining three distinct lin-
eages is equivocal. The Scrub Jays of Santa Cruz
Island (ACINS) and Florida (ACCOE), and the
Unicolored Jays of Guerrero (ANGUE) are in-
OUTGR
!- Apomorphy
[]- Parallelism
- Revereal
length 58 steps
c.I. = 0.569 3
!
!
2
!
2 1
10 I 21 _ CO CAL
co woo
* L CO MEX
1
I CO SUM
21
I I la UL POT
UL WOE
UL ULT
I *2
UN E
Fig. 6. Cladistic analysis of eight subspecies groups
of Aphelocoma jays and outgroup showing character-
state changes and support for nodes. Nodes marked
with asterisk found to be unstable to a jackknife ma-
nipulation. See Table 1 for abbreviations; consistency
index (c.i.) indicated.
variably placed as basal offshoots of the trees
(Fig. 7B). For reasons outlined below, this ar-
rangement seems unlikely, so a placement of
these three populations that agrees more closely
with geography (Fig. 7A) is also considered in
the analyses of rate uniformity.
Rates of molecular evolution.--Because the phy-
logenetic studies described above are equivocal
as to the placement of three populations (ACINS,
ANGUE, and ACCO2), ! used both trees of Fig-
ure 7 in the rate tests. ! conducted a relative-
rate test based on the following three-taxon
statements (from Figs. 7A and 7B): ((ACINS, cal-
ifornica group), woodhouseii group); ((ANGUE,
east-slope unicolor), southern Gray-breasted
Jays); ((ACCO2, rest of Scrub Jays), eastern ul-
tramarina); ((ACINS, rest of ingroup), CYSTE);
((ANGUE, rest of ingroup), CYSTE); and
((ACCO2, rest of ingroup), CYSTE). In the six
tests, apparent departures from rate equality ex-
ist; ACINS, ANGUE, and ACCO2 in every com-
parison had greater genetic distances and pa-
0 x 0 x _
oo
z z O O O O
G.I. = 1.0
PLUMAGE TYPE
ß Unicolored Jay
[] Gray-br. Jay
[] Scrub Jay
Fig. 7. Two possible phylogenies combining the results of Figures 2-6, showing most-parsimonious re-
constructions of patterns of plumage evolution. Abbreviations follow Table 1; consistency index (c.i.) indicated.
tristic distances to the third taxon than did their
sister lineages (sign test; all P < 0.001). Presum-
ably, higher rates of molecular evolution have
characterized these three lineages relative to
the rest of the group.
Analyses involving resampling of popula-
tions revealed a high degree of heterogeneity
of rates among lineages (Fig. 8). Statistical com-
parisons indicate that evolutionary rates are sig-
nificantly different in every pair of sister lin-
eages (Mann-Whitney U-test; all P < 0.05). The
validity of these statistical tests depends on the
effectiveness of the resampling manipulation in
estimating true levels of branch-length vari-
ability. The manipulation employed here eval-
uates error introduced by choice of sample lo-
cality, but not sampling error in estimation of
genetic distances. Nevertheless, it is clear that
molecular evolutionary rates have been uneven
in the history of the Aphelocoma jays.
DISCUSSION
The consistency with which geographically
related populations clustered together indicates
that these data contain phylogenetic informa-
tion. For example, in most analyses, the 10 wood-
houseii-group populations cluster together, as do
the nine californica-group populations. Below I
discuss the unexpected features of the trees in
an attempt to understand what factors, meth-
odological or biological, led to those patterns.
Paraphyly of the Gray-breasted Jay.--In all anal-
yses, the Gray-breasted Jay is a paraphyletic tax-
on. Unicolored Jays invariably group with the
southern Gray-breasted Jay lineage, and Scrub
Jays with the eastern Gray-breasted Jay lineage.
The position of the western Gray-breasted Jay
lineage is less clear. On the whole, it appears
that the Gray-breasted Jay is a relatively old
lineage that has been in the mountains of north-
ern Mexico for a long period of time, and that
Scrub Jays and Unicolored Jays were derived
independently from different parts of the Gray-
breasted Jay complex.
Although unexpected (Pitelka 1951), this idea
receives some support from other evidence. First,
Gray-breasted Jays are intermediate in habitat
use between the other two species. Where the
three species' ranges overlap in central Vera-
cruz, Scrub Jays live on dry interior slopes and
Unicolored Jays on humid coastal slopes. Gray-
breasted Jays inhabit the pine-oak forests at
higher elevations intermediate between the two,
and so direct invasion of either habitat would
have been possible. Second, Gray-breasted Jays
in the southern and western portions of the
range show delayed maturation of beak color,
a character present in rudimentary form in Uni-
colored Jays and otherwise rare in birds (Pe-
terson 199 lb). Eastern Gray-breasted Jays do not
delay beak color maturation, which is similar
to the ontogenetic patterns of Scrub Jays (Pe-
terson, in press). Similarly, western and south-
ern Gray-breasted Jays do not give the female-
specific "rattle" vocalization, which is found in
eastern Gray-breasted Jay and all Scrub Jay pop-
ulations (Brown and Horvath 1989). Therefore,
the hypothesis of independent derivations of
Scrub and Unicolored jays from Gray-breasted
Jays is supported by other characters. Taxonom-
ic implications of these findings will be treated
elsewhere (Peterson, in prep.).
Origin of the californica group of Scrub Jays.-
In a number of Fitch-Margoliash trees (e.g. Fig.
3), the californica group of Scrub Jays is derived
from within the woodhouseii group of Scrub Jays.
Some support for this hypothesis is also found
in cladistic results although these nodes are not
preserved in the consensus analyses (Fig. 5).
This pattern is to be expected based on a prob-
able Central American/Mexican origin of the
species with subsequent invasion into the Great
Basin and then into California (Peterson 1990).
Rapid evolution versus old splits.--Three dis-
junct populations--the Scrub Jays of Santa Cruz
Island (ACINS), the Scrub Jays of Florida
(ACCO2), and the Unicolored Jays of Guerrero
(ANGUE)--consistently are placed as basal lin-
eages in the phylogenetic analyses (Fig. 7; in a
few alternative trees, the Florida population
ACCO2 grouped with southern Mexican Scrub
Jay populations based on one allele unique to
the two groups). This result is surprising, given
that each shares obvious similarities of plumage
coloration, shape and size, vocalizations, and
behavior with other members of its species. For
example, ACINS shares several unique plumage
characters with populations of California and,
on that basis as well as on grounds of geograph-
ic proximity, seems to have been derived from
adjacent mainland populations (Pitelka 1951).
Two alternative hypotheses are apparent: (A)
that these lineages are exceptionally divergent
populations derived from within Scrub Jays and
Unicolored Jays, but for nonhistorical reasons
appear as basal splits on the trees (Fig. 7A); or
(B) that these lineages are exceptionally old, and
the characters that each shares with its conspe-
cific populations are actually retained similar-
ities (Fig. 7B). Alternative B is better supported
by the allozyme data than alternative A (tree-
length of 62 steps in B; 73 steps in A).
Still, two biological points argue against al-
co wco
Fig. 8. Trees showing results of resampling anal-
yses of rates of molecular evolution in different lin-
eages, based on two trees of Figure 7. See Table 1 for
abbreviations.
ternative B. First, although placement of these
three populations as basal splits (Fig. 7B) re-
duces hypothesized levels of heterogeneity of
rates of molecular evolution, it leads to prob-
lems in understanding the evolution of plum-
age coloration. For the tree in Figure 7B, plum-
age coloration must have undergone a number
of reversals and episodes of rapid evolution
(plumage coloration mapped onto tree; consis-
tency index = 0.5). Under the view in Figure
7A, plumage coloration shows only two changes:
one at the derivation of Scrub Jays; and another
at the derivation of Unicolored Jays (consisten-
cy index = 1.0). Hence, hypothesis B is less
consistent with patterns of variation in plum-
age-color characters than hypothesis A. Fur-
thermore, these populations share not only as-
pects of plumage coloration, but a wide range
of characters (e.g. body size and shape, ecolog-
ical requirements, vocalizations, behaviors) with
conspecifics (Pitelka 1951), so whole suites of
characters not included in my analyses would
have had to have changed unpredictably.
Second, even if these populations represent
basal splits (alternative B), they still have ex-
ceptional branch lengths. Unequal branch
lengths can result from incorrect choice of out-
group (i.e. an outgroup taxon actually belongs
to the ingroup). Outgroups in this study include
representatives of the entire family Corvidae,
so derivation of outgroup taxa from the ingroup
is unlikely. Nevertheless, if to avoid unequal
branch lengths, the trees are rerooted at the
midpoint of the longest branch (in this case,
that leading to ACINS or ANGUE), not only do
at least two other ingroup taxa remain with ex-
ceptional branch lengths, but the outgroup ap-
pears as an exceptionally divergent branch de-
rived from within ApheIocoma.
Removal of the three long branches from the
tree (after Swofford and Olsen 1990) produces
results that agree with geography and mor-
phology. The populations on the three long
branches all represent small, isolated popula-
tions isolated from the remainder of the species'
ranges. Hence, ! considered features of small
populations that could possibly affect accurate
reconstruction of phylogenies.
A possible resolution of these difficulties may
lie in the extremely reduced levels of within-
population genetic variation in these same three
populations (Peterson 1990). Heterozygosities
for the three populations averaged 1.9, as com-
pared with 3.9 for the remainder of the popu-
lations sampled (Mann-Whitney U-test, P < 0.05;
Peterson 1990). Other measures of within-pop-
ulation variation (e.g. proportion of loci poly-
morphic, number of alleles) are similarly re-
duced in the three populations. Many of the
characters on which the phylogenies are based
are alleles at low or moderate frequencies (e.g.
PGD-e, which is found in all but one of caIifor-
nica-group populations, never at a frequency
>0.136). If a population with low-frequency al-
leles that identify its sister-taxon relationships
loses variation, perhaps due to population bot-
tlenecks, those same low-frequency alleles
would most likely be lost. Consequently, many
of the characters shared with the ingroup (syn-
apomorphies) are secondarily absent. These
changes will move such lineages to the base of
cladograms. Phenetic techniques that assume
rate equality obviously will be sensitive to in-
creases in evolutionary rates in particular lin-
eages and, potentially, also to secondary losses
of alleles. Thus, the tempo and mode of evo-
lution in a group can affect the outcome of phy-
logenetic analyses; in this case, it leads to the
basal placement of ACINS, ANGUE, and
ACCO2.
This problem may affect other parts of the
phylogenetic reconstruction of the ApheIocoma
jays as well. For example, it could be responsible
for the basal placement of ACTEX within Scrub
Jays and the grouping of ANCON, ANGUE, and
AUAZ1, all of which show reduced genetic
variation (Fig. 5). Grouping of Florida and Santa
Cruz Island populations (ACCO2 and ACINS)
in some analyses (a most unlikely outcome geo-
graphically) is almost certainly a result of this
phenomenon. Unexpected features of the phy-
logenies discussed above, such as the derivation
of the caIifornica group from the woodhouseii
group and paraphyly of Gray-breasted Jays, most
likely do not result from this problem because
they do not involve groups that differ markedly
in levels of genetic variability.
The best hypothesis for the position of ACINS,
ACCO2, and ANGUE in the Aphelocoma jay phy-
logeny appears to be that they are exceptionally
derived lineages from within Scrub Jays and
Unicolored Jays. This conclusion is supported
by several logical problems with the alternative
hypothesis. The methodological reason for their
basal placement in the analyses is clearly iden-
tifiable, and has nothing to do with time since
divergence from the ancestral stock.
My results serve to illustrate some of the dif-
ficulties of using electrophoretic characters in
phylogenetic analyses, as have been pointed out
by other workers (e.g. Buth 1984, Swofford and
Berlocher 1987, Swofford and Olsen 1990). The
highly polymorphic nature of these characters
makes coding and analysis difficult; effects of
population history can reduce the probability
of correctly reconstructing the history of the
group. Although ! believe that useful and ac-
curate information was retrieved from the data
set, the difficulty of exact placement of several
important populations demands further study
with other types of biochemical characters.
Rates of molecular evoIution.--The analyses of
rates of molecular evolution indicated that at
least three ApheIocoma jay populations have un-
dergone marked accelerations in rate of differ-
entiation relative to their sister populations. Re-
gardless of whether they are placed as basal
splits or with apparently conspecific popula-
tions (Figs. 7B or 7A), these populations have
evolved faster than other populations of Aphelo-
coma jays. Inspection of branch lengths in the
tree (Fig. 8) suggests that heterogeneity of evo-
lutionary rates may not be restricted to just those
three lineages, but that rate heterogeneity on a
finer scale may be common in the genus.
Because all tree-building methods are to some
degree sensitive to rate differences (Swofford
and Olsen 1990), trees estimated are not inde-
pendent of the question of whether rates of
evolution are uniform. When presented with a
case of extreme rate inequality, algorithms for
phylogeny estimation err on the side of reduc-
ing heterogeneity of branch lengths. A search
for rate differences based on trees potentially
affected by such differences is a conservative
test; errors introduced will be of not finding a
difference that exists, rather than of finding
spurious heterogeneity. Studies of variation in
other molecules that will permit critical inde-
pendence of phylogeny estimation and branch-
length estimation are in progress.
More interesting is the degree to which evo-
lutionary rates differ. If ACINS and ANGUE
represent basal splits from the genus, they have
been evolving 20-25% more rapidly than the
rest of the genus. If, as seems more likely, they
were derived from within Scrub Jays and Uni-
colored Jays, their evolutionary rates have been
three to four times greater than those of their
sister lineages. Correlations between rate of dif-
ferentiation and features of population biology
are explored in Peterson (1990, in prep.).
Electrophoretic data for populations of Aphe-
locoma jays clearly violate the assumption of rate
equality. Although studies of several other avi-
an groups have come to the opposite conclusion
(e.g. Bledsoe 1987), my results and the results of
others (e.g. Britten 1986, Springer and Kirsch
1989) indicate that the assumption of rate uni-
formity is not always reasonable. Rate-depen-
dent algorithms and interpretations should be
tested first (e.g. Page 1990) and used cautiously,
if at all, in studies purporting to derive phy-
logenetic or biogeographic information from
molecular data alone.
ACKNOWLEDGMENTS
Many people have lent invaluable assistance to me
during this project. I thank especially Scott M. Lan-
yon, Frank A. Pitelka, and John W. Fitzpatrick for
their generous interest throughout the project. Thanks
to John Bates, Shannon Hackett, Scott Lanyon, and
two anonymous reviewers for helpful comments on
the manuscript. I thank Pamela Austin, John Hall,
Victor Pacheco, Mary Anne Rogers, Douglas Stotz,
Thomas Schulenberg, David Willard, Adolfo Navarro
S., and Patricia Escalante P. for their interest in my
work. Shannon Hackett provided invaluable assis-
tance with testing phylogenetic information in the
cladistic data sets. I thank the Direcci6n de Flora y
Fauna Silvestre, Secretaria de Desarollo Urbano y
Ecologla, of the Mexican government, and the state
governments of Oregon, California, Nevada, Arizona,
Utah, New Mexico, Colorado, and Texas for providing
permits for scientific collecting. The Field Museum
generously provided access to its facilities for bio-
chemical analysis. Financial support was provided by
the National Science Foundation Dissertation Im-
provement Grant Program (BSR-8700850), National
Geographic Society, Field Museum of Natural His-
tory, Chapman Fund (American Museum of Natural
History), Sigma Xi, and the University of Chicago.
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