APPLICATIONS
OF ELECTROPHORETIC
AVIAN
DATA
IN
SYSTEMATICS
RONALD
H. MATSON
Departmentof Biology,Universityof California,LosAngeles,California90024USA
ABSTR^CT.--This
paper is a survey of applicationsof electrophoretictechniquesin ornithology,with an emphasison post-1970publications.The majorityof electrophoreticstudies
of birdshave beenlimited in a variety of ways.Many havedealt with "domesticated"
species
or have been limited to the examination of blood and/or egg-white proteins. Problems in
comparingresultsfrom different studieshave arisenbecauseof: (1) dissimilarelectrophoretic
techniques;(2) varying numbersof taxa;(3) nonstandardizedenzymeand locusnomenclature; and, especially,(4) different methodsof data analysis.Thesemethodologicalproblems
mustbe addressedin orderto broadenthe utility of electrophoretic
datain aviansystematics.
I suggestthat the enzyme namesrecognizedby the International Union of Biochemistrybe
usedexclusivelyand that a standardizedlocusnomenclature,comparablewith that usedin
other vertebrateclasses,be developed.The predominatinguse of allozyme characterscan
be supplementedby "isozymecharacters"(e.g. different numbersof genes,heteropolymer
assembly,and regulationof expressionsensu
Buth in press),which possiblycouldbe applied
to a determinationof systematicrelationshipsof higher-leveltaxonomicranks.Allozyme
and/or isozymedata should be retained in particulateform (i.e. not summarizedas genetic
distances).The use of outgroupsto assignevolutionarydirection is encouraged.Received
2
May 1983,accepted
19 April 1984.
THEevolutionaryrelationshipsamong birds
have long been a matter of interest.Among
chemicaltechniquesto systematicanalyses,a
with results obtained through other biochemical techniques.
Electrophoresis
is a useful tool in examining
a variety of genetic and developmental phenomena.In avian biology, it has been used to
obtain quantitative estimatesof genetic variation in natural populations, to study the patterns of gene activation, and to examine the
genetic control of various proteins. Another
important applicationof thesedata hasbeen as
a systematictool. It was noted quite early that
biochemicaldatawould be useful in systematic
studies(e.g. McCabeand Deutsch1952,Sibley
1960, Gysels 1963), but "... even by 1970, a
major review of electrophoretic literature in-
new suite of characters became available.
cluded
vertebrates, birds seem to constitute a rather
homogeneousassemblage,a situation that has
contributedto the difficulty of establishingdetailed systematicrelationships.In attempting
to determinetheserelationships,avian systematistshave relied upon a number of taxonomic
characteristics.
Most have usedmorphological
features (e.g. intestinal convolutions, muscu-
lature,nasalbones,and palatetype), although
some have used ecological,ethological, geographical,or physiological
attributes(Van Tyne
and Berger 1976). With the application of bioTech-
almost
no discussion
of uses of electro-
niques that have used biochemical characters phoretic data in systematics,other than for deand that have been applied in ornithological scription and identification of species"(Avise
studiesinclude DNA-DNA hybridization (Sibley and Ahlquist 1980),immunoelectrophoresis (Ryttman et al. 1980), micro-complement
fixation (Ho et al. 1976), and electrophoresis
(studiesreviewedherein). Historically,electrophoresishas been the most important in terms
of numbersof studiesusing biochemicaltechniques to solve problems in avian biology.
1974:465). Even now, there are relatively few
comprehensivestudiesusing this data base to
elucidatesystematicrelationshipsamongbirds,
particularly at higher taxonomicranks (but see
Sibley 1970 and Sibley and Ahlquist 1972 for
exceptions).
Electrophoreticdataprovide alternative suites
of charactersthat can be used to discern sysElectrophoresiscontinues to be a cost-effective tematic relationshipsamong birds. Initially, it
method of collectinglarge amountsof data and was thought that biochemical(molecular)charproviding alternative data setsfor comparison acterswere more conservativethan other types
717
The Auk
101: 717-729.
October
1984
718
RONALD
H. MATSON
of charactersand therefore had greater utility.
That is, it was thought that these characters
would reflect the evolutionary history of the
group under investigation, despite possible
[Auk, Vol. 101
If past studiesare examined, however, prob-
1960% the number of electrophoretic studies
increaseddramatically(Fig. 1). This increasecan
be attributed partially to the developmentof
starch-gelsupporting medium, combined with
the use of histochemicalstainsto locatethe positions of enzymatic proteins (Avise 1974).
During the 1970% further improvements in
biochemical techniques and new methods of
data analysisresulted in an increasedapplication of electrophoresisin ornithology. I will
present a synopsisof these studiesafter arbitrarily dividing them into three overlapping
categories:biochemical genetics, population
genetics,and systematics.
Biochemical
genetics.--Many
studieshavedealt
with avian biochemicalgenetics,and the resultshave supplied empirical supportfor certain assumptionsnecessaryin the application
of electrophoreticdata to systematics.For example, it is usually assumed that the gene
products examined via electrophoresis (i.e.
electromorphs)are controlled by codominant
alleles at a single structuralgene locus,an assumption confirmed by numerous studies.
lems can be evaluated and mistakes rectified so
Variation
that the full potential of this techniquecan be
realized. In this paper, I will discussproblematic areas identified in past electrophoretic
studiesand make suggestions
to insurethat future investigatorswill be better able to apply
this techniqueto problemsof avian systematics.Barrowclough
(1983)and Corbin(1983)have
recently reviewed ornithological papers em-
controlled by two codominant alleles at an autosomal locus in the Black-tailed Gull (Larus
ployingelectrophoresis
(andotherbiochemical
techniques)in microevolutionaryand certain
systematic
studies.This review will be limited
to papersin which electrophoreticdata have
cations. I will further restrict my comments to
(Przytulski and Csuka 1980) and albumin in
Willow Ptarmigan (LagopusI. scoticus,
Henderson 1976a)were also found to be controlled by
codominantexpressionof alleles at a single locus. As with those using egg-white proteins,
many of the investigatorsemploying blood as
a tissue source have determined the genetic
paperspublishedbetween1970and 1984;for a
control of the various loci. Variation
review of earlier papersseeSibley et al. (1974).
esteraseshas been found to be controlled by
adaptivechanges
or convergences
(Sibley1965).
Avise (1974) stated that electrophoreticcharacters were more precise and objective than
othertypesof characters.
Althoughit hasbeen
shown that these are not necessarilyvalid attributes of biochemical characters (Selander
1971,Wiley 1981),thesecharactersare still useful in systematicstudies.Some advantagesto
the useof electrophoreticdatainclude high inheritanceof characters,the expressionof which
is usually not subjectedto environmental effects, and codominant phenotypic expression
(Whitt 1983), which permits an accurate estimation of the allelic and genotypic composition of the sample.
Although it was initially believed that electrophoretic techniqueswould resolve many
systematic
problems,a pert•salof the avianliterature indicates that this has not been the case.
been applied to avian systematicsor in which
such data have contributed to systematicappli-
REVIEW
The work of Landsteiner et al. (1938), in
which the egg albumin of six specieswas com-
has been found
to be
crassirostris,Kimura 1972), the Black-crowned
Night-Heron (Nycticoraxnycticorax,
Kimura and
Isogai 1973),the JapaneseQuail (Coturnixjaponica, Kimura et al. 1977), and in domesticated
ducks(Anasplatyrhynchos,
Przytulski and Csuka 1980). Pre-conalbumin in domesticatedducks
codominant
HISTORICAL
in conalbumin
alleles
at autosomal
in several
loci
in
the
Ringed Turtle-Dove (Streptopelia
risoria,Bohem
and Irwin 1971), the chicken (Gallusgallus,Tanabeand Ise 1972),the BlueGrouse(Dendragapus
obscurus,
Redfield 1973a), and in the Willow
pared, was the first reported application of Ptarmigan (Henderson 1976b).In other tissues,
electrophoretictechniquesto an avian study. the following systemswere found to be exDuring the next two decades,there were at least pressedby codominant alleles: alkaline phostwo dozen electrophoretic studies of various
avian proteins, the majority of which were
aimed at determining the electrophoreticconstituentsof blood or egg-white proteins.In the
phatase (Maeda et al. 1972), adenosine deaminase (Grunder and Hollands 1977), albumin
(Lucotteet al. 1978), and transferrin (Montag
and Dahlgren 1973,Przytulskiand Csuka1979).
October1984]
15
AvianElectrophoresis
719
• Moving
Boundary
• Polyacrylamide
k• Paper
• Agar
IJlCellulose
J• Starch
40
45
50
55
60
65
70
75
80
Year of Study
Fig. 1. Histogramdepictingthe changesin supportmediausedin avian electrophoretic
studiesconducted
from 1938 to 1984. This graph is basedupon the examinationof 238 papers;258 studiesare indicated,
however,becausesomeinvestigatorsemployedmore than one medium in a singlestudy.
The general pattern of glucose-6-phosphatedehydrogenasein birds and other vertebrateswas
describedby Nobrega et aL (1970). Shiraishi
and Hirai (1983) have determined that man-
nosephosphateisomeraseis controlled by autosomal
codominant
alleles
in
the
Helmeted
Guineafowl (Numida meleagris).Many of these
investigators have employed mating experi-
ments to determine the genetic basis of the
electromorphpatterns obtained (e.g. Maeda et
al. 1972, Tanabe and Ise 1972, Przytulski and
Csuka 1979, Shiraishi and Hirai
1983). Al-
though the vast majority of these studiesdemonstrate that these patterns resulted from the
expressionof codominant alleles, some did not
[e.g. esterase-4in chickens (Tanabe and Ise
1972), prealbumin in domesticated ducks
(Przytulski and Csuka 1979)]. For the purposes
of datainterpretation,however, it is reasonable
to assume that electromorphs represent prod-
was studiedby Shaugnessy(1970a).Brushand
Scott (1972) examined developmental changes
in geneexpression
at severallociin Red-winged
Blackbirds(Agelaiusphoeniceus).
The ontogeny
and activation of gene loci encoding for alcohol dehydrogenase(Le Vine and Haley 1975),
6-phosphogluconate
dehydrogenaseand glucose-6-phosphatedehydrogenase(Leung and
Haley 1974), and lactate dehydrogenase,fructose 1,6-diphosphatase,"malic enzyme," and
aspartateaminotransferase(Meyerhof and Haley 1975, 1976) have been studied in Coturnix
japonicaand Coturnixx Gallushybrids. It was
found that embryos,chicks,and adultsmay differentially expresselectrophoreticallydistinct
forms of gene productsand that enzymes are
activated at different times during ontogeny.
There
were
also indications
that some mater-
nally produced enzymes were stored in eggs
(Meyerhofand Haley 1975).Thesefindingslend
ucts of codominant
alleles.
credence to the conclusionsof Tegelstrom et
Patternsof gene activation have been inves- al. (1980) that, unlesscomparisonsare made betigated as well. The ontogeny of hemoglobins tween comparableontogeneticstages,eggsand
in Macaroni Penguins (Eudypteschrysolophus) embryosare often not the bestsourcesof tissue.
720
RONALDH. MATSON
Due to the high fat contentin theseembryonic
tissues,
resolutionof electromorphs
maybe poor
(Tegelstromet al. 1980).
Population
genetics.--During
the 1970's,a large
numberof workersattemptedto determinethe
amountsof geneticvariationin populationsand
to distinguish between populations and/or
subspecies
(e.g. Brown et al. 1970;Shaugnessy
1970b; Redfield 1973a, b; Corbin et al. 1974;
Morgan et al. 1977a, b; Guttman et al. 1980;
Matson 1980;Parker et al. 1981; Ryttman and
Tegelstrom1981;Barrettand Vyse 1982).These
[Auk, Vol. 101
phology, dominance,and mortality in captive
and wild Juncohyemalis.
Systematics.--Investigators
have usedelectrophoresistechniquesat the interspecificlevel to
study systematicrelationships.Electrophoretic
patterns of egg-white proteins (e.g. Ferguson
1971; Sibley 1973, 1974, 1976; Sibley and Ahlquist 1973)and blood proteins(e.g. Sibleyand
Hendrickson1970,Fergusonand Barnford1973,
Hendrickson
and Yow 1973, Ford et al. 1974,
Harper 1978, Lopes et al. 1979, Mosher et al.
1982) have been used to make interspecific
workersused the productsof relatively large comparisonswithin a number of families. The
data
numbersof loci to computevaluesfor genetic mostdetailedapplicationof electrophoretic
descriptors
suchaspercentage
of polymorphic to avian systematicswas provided by Sibley
loci (P), average heterozygosity (H), and (1970) and Sibley and Ahlquist (1972), who hyWright'sF-statistic(F) (e.g.Manwell and Baker pothesized the relationshipsof 668 speciesof
1975; Lucotte and Kaminski 1976a, b; Fleischer
passerinesand 816 speciesof nonpasserines,
1983; Zink and Winkler 1983). Various distance respectively. These relationships were exand similarity coefficients(e.g. Nei 1972,Rog- pressedas a seriesof probability statements,a
ers 1972) were also calculated. The results of
summary of which can be found in Sibley et
these studies[which have been reviewed by al. (1974). It was Sibley'sstatedgoal to assess
Barrowclough(1983) and Corbin (1983)] sug- degressof "genetic relatedness"among taxa
gestthat, within species,birds are not lacking examinedand to develop a classificationthat
in genetic variation. Genetic distance mea- would reflect degrees of genetic difference
sures, however, are generally lower among demonstratedbetween the taxa (Sibley 1970).
conspecific
bird populationsthan amongpop- It was assumedthat the possessionof similar
ulations of other vertebrates (e.g. Barrow- patternsby taxa resultedfrom the genetic reclough and Corbin 1978, Barrowcloughet al. latednessof the speciesexamined.Variation in
1981, Barrowclough1983).
number and mobility of proteins was considElectrophoreticdata have been used by oth- ered to be taxonomicallysignificant (Sibley
ers to investigate additional population-level 1970), and taxa that exhibited similar electrophenomena.For example,Martin and Selander phoretic patterns were deemed to be more
(1975), Corbin et al. (1979), Barrowclough closely related than taxa with different pat(1980), Braun (1981), Johnson and Zink (1983), terns.In thesestudies,relatively few characters
and Braun et al. (1984) have studied hybridiza- were used(usually only general proteins),and
tion between speciesor integradationbetween there usually was no attempt to analyze data
subspecies.In general, it appearsthat electro- quantitatively.
phoretic data are useful in separatingparental
The number of systematicstudiesincreased
and hybrid individuals. Electrophoreticdata with the advent of widespread allozyme elechave also been used by Johnsonand Brown trophoresisin the latter part of the 1970'sand
(1980) to investigatethe breeding structureof into the 1980's.This correspondedwith the shift
Grey-crownedBabblers(Pomastostotnus
tempo- away from usingegg whites and/or blood as a
ralis). Sherman (1981) encouragedthe use of tissuesourceand toward the incorporation of
electrophoresisto investigate problems con- a variety of organs.Refinementsin histochemcerning mating systemsand genealogy. Not- ical staining proceduresmade it possibleto astebohm and Selander (1972), Handford and
say more enzymes, thereby increasing the
Nottebohm (1976), and Baker (1974, 1975) used number of possiblecharactersin a study. Conelectrophoretic
datain investigations
of the role currently, a major change in the treatment of
of songdialectsin reducinggeneflow between electrophoreticdata occurred with the intropopulations of Zonotrichiacapensis
and Z. leu- ductionof measuresto quantify the geneticdifcophrys,respectively. Results of these studies ferencesbetween populations.Nei (1972), Roghave been equivocal.Baker and Fox (1978) ex- ers (1972),and othersdevelopedmathematical
amined relationshipsbetweengenotypes,mor- formulae that summarize allelic frequencies
October1984]
AvianElectrophoresis
721
TABLE
1. Electrophoretic
studiesin which systematic
relationships
amongaviantaxaare depictedby means
of clustering algorithms.
Study
Orientation
Smith and Zimmerman (1976)
Distance
Coefficient
•
Codingb
Clusteringc
Rogers(S)
NA
Nei (I)
NA
WPGMA
WPGMA
Jaccard
Rogers(D)
NA
NA
Average linking
DistanceWagner
Morgan et al. (1977b)
Barrowcloughand Corbin (1978)
Distance
Distance
Lopeset al. (1979)
Distance
--
NA
Pairingaffinity
Avise et al. (1980a)
Distance
Nei (I)
NA
UPGMA
Character
NA
+ /-
Bonde
Distance
Nei (D)
NA
Avise et al. (1980b)
UPGMA
FM
Distance Wagner
Avise et al. (1980c)
Yang and Patton(1981)
Distance
Distance
Nei (D)
Rogers(S)
Rogers(D)
NA
NA
NA
Avise et al. (1982)
Mosher et al. (1982)
Zink (1982)
Distance
Distance
Distance
Nei (D)
Jaccard
Nei (D)
NA
NA
NA
UPGMA
UPGMA
FM
Distance Wagner
UPGMA
Average linking
UPGMA
Rogers(D)
WPGMA
Rogers(D)
Distance Wagner
UPGMA
FM
Gutierrez et al. (1983)
Distance
NA
WPGMA
FM
Johnsonand Zink (1983)
Distance
Rogers(D)
NA
Distance Wagner
UPGMA
WPGMA
FM
Distance Wagner
aNei (I) = geneticidentity; Nei (D) = geneticdistance(Nei 1972).Rogers(S) = geneticsimilarity; Rogers
(D) = genetic distance(Rogers1972).
bNA = Not Applicable;+/- = scoredas presence/absence
of alleles.
½UPGMA = unweighted pair-group method with arithmetic averages;WPGMA = weighted pair-group
method with arithmeticaverages(Sneathand Sokal 1973);FM = Fitch and Margoliash(1967);DistanceWagner = Farris (1972); for other methods,refer to the specificstudy.
across loci as a measure
of the amount
of dif-
ference or similarity among taxa. Biologicalsignificance was ascribed to electrophoretic data
under the assumption that these distances(as
measuredby Nei's coefficient)actually reflected the number of codon differencesper locus
ers' coefficientsof genetic similarity. Using
these coefficients, they constructed phenogramsdepicting relationshipsof generawithin
the family. Similar typesof data were collected
and analyzed by means of phenetic and distance-oriented cladistic methods. A Wagner
network produced by Barrowcloughand Corbin (1978) depicted relationshipsamong three
genera of parulid warblers. The relationships
of turdids, emberizids, parulids, mimids, and
Sneath and Sokal 1973) and, to a much lesser vireonids were examined by Avise and coldegree, methods of constructing phylogenetic leagues(Avise et al. 1980a,b, c, 1982).Yang and
trees such as the Fitch-Margoliash method Patton (1981) discussedthe relationships of 11
(Fitch and Margoliash 1967) and the distance speciesof Gal•pagos finches, whereas Gutierrez et al. (1983) examined 10 speciesof galliWagner procedure (Farris 1972) (Table 1).
In severalstudies,clusteringtechniqueshave form birds. Zink (1982) comparedmorphologbeen employed.Smith and Zimmerman (1976) ical and genic variation in an attempt to
examined six icterid genera and, from allelic describethe relationshipsamong four emberifrequency data, calculatedboth Nei's and Rog- zid species.Finally, Johnsonand Zink (1983)
(Nei 1972). The distance data could be clus-
tered to produce dendrogramsdepicting relationships.The mostpopular methodsusedwere
the UPGMA phenetic clustering technique (see
722
RONALD
H. MATSON
[Auk, Vol. 101
cladisticallyand phenetically examined elec- of separateloci) is made, then it is possibleto
trophoretic data from four speciesof sapsuck- obtain additional information from electrophoers in the genusSphyrapicus.
retic data (e.g. heteropolymerformation,numTrees based on electrophoretic data, when
ber of structuralgenescontrolling multilocus
compared with trees constructed from other systems,tissue-specificexpression)for use in
data bases(e.g. morphology, behavior, ecolo- higher-level taxonomicstudies(Buth 1981, in
gy), have varied in agreement. In studies by press).As noted by Whitt (1983:20), the "tissue
Smith and Zimmerman (1976) and Yang and and developmental patterns of isozyme locus
Patton (1981), biochemicalresultswere concor- expressionare often characteristicof a species,
dant with those based on more traditional
data
genus, or higher taxon ..." (emphasis mine).
sets. In other situations, however (e.g. Zink
Making this distinction between allozyme and
1982, Gutierrez et al. 1983, Johnson and Zink
isozyme data has been useful in solving sys1983), the systematicrelationships from elec- tematic problems in fishes (Buth et al. 1980)
trophoretic data did not necessarilyagree with
and lizards (Murphy et al. 1983). Recognition
thosepreviouslyreported.In mostof the above of this distinction may increase the range of
studies, it was found that both phenetic and applications of electrophoretic data in avian
cladistictypes of data analysisyielded similar systematics.How the distinction between alpatterns of relationships in the taxa examined. lozyme and isozyme characterscan be made is
Most authors seemedto agree that the cladistic explained below. Other problems have also
method of data analysis was, at least theoretilimited the application of this technique as a
cally, the method of choice. Although these systematictool. These include problems with
methods represent improvements over those the number of taxa examined, electrophoretic
used in the past, there are further modifica- techniquesemployed, tissuesused, nomenclations that will increasethe utility of these data ture used, and methods of data analysis.
Taxa.--Of 225 avian electrophoreticpapers
in the study of evolutionary relationships.
These suggestedimprovements will be dis- published since 1938, 105 (47%) concentrated
cussed below.
on nine taxa (Meleagrisgallopavo,
Gallusgallus,
Columba
livia,Streptopelia
spp.,Coturnixjaponica,
Chrysolophus
spp., Phasianuscolchicus,Cairina
OBSERVATIONS AND CONCLUSIONS
moschata,Anas platyrhynchos)
as the primary
As evidenced by the foregoing discussion, subjectsof investigation.These"domesticated"
electrophoresishas had a wide range of appli- specieshave worked well for thoseinvestigatcations in ornithology. The use of this tech- ing problemsconcerningthe modesof inherinique has been limited as a tool in systematics, tanceof a particulargeneproductor the chemhowever. It is generally acceptedthat electro- ical structureof a particular protein. With the
phoreticdata are useful at or below the generic increasedinterest in population geneticsand
level (e.g. Feduccia 1970) but of questionable evolution,however, it becamenecessaryto exutility in solving higher-level systematicprob- amine natural populations.Yet, even though
lems (Prager et al. 1976, Bushand Kitto 1978). Sibley (1970) and Sibley and Ahlquist (1972)
As noted by Buth (in press),the basisof this examinedegg-whiteproteinsfrom 1,484species
limitation is that only allozymes[i.e. alterna- of birds and Kuroda et al. (1982) and Kakizawa
tive forms of an enzyme producedby different et al. (1982)examinedmalatedehydrogenase
in
alleles at a locus(Prakashet al. 1969)]were ana- 285 species,the number of bird speciesthat
lyzed in thesestudies.With higher-level taxo- have been examinedfor allozymevariationrenomic categories,allozyme charactersbegin to mains proportionately small. Since 1970, 141
demonstratedivergence at all loci. This even- speciesrepresenting 7 orders and 20 families
tually renders the taxa totally different and of birds (Morony et al. 1975,A.O.U. 1983)have
leaves nothing shared among the taxa with been the subjectsof electrophoreticstudiesinwhich to assessrelationships.Therefore, these volving 10 or more loci. If this technique is to
data have been applied only to lower taxonom- continue to have an impact on avian systemic levels, an unnecessarylimitation on the use atics,it must be applied to natural populations
of "electrophoreticdata." If the distinction be- from a wide variety of taxa.
tween allozymesand isozymes(i.e. alternative
Techniques.--A
majorproblemin comparing
formsof an enzymethat are the geneproducts the results of these various studies is due to the
October1984]
AvianElectrophoresis
fact that a number of different electrophoretic
techniqueshave been used.For example,of 125
post-1970papersexamined,81 (65%)have used
starch-gel electrophoresis(including the majority of systematicstudies). The remaining
studiesemployed polyacrylamide(21%), agar
(2%), cellulose (1%), or paper (1%), or some
combination of these (10%) as the support medium. Although the relative advantagesand
disadvantagesof each technique are reported
elsewhere (Brewer 1970, Smith 1976, Ferguson
1980), it is important to realize that these different techniques produce different results,
which are not necessarilycomparable.For example, acrylamide disc electrophoresis "does
not permit careful comparative studies or the
detection of subtle differences in migration
which may arisefrom geneticvariation or other sources" (Brewer 1970: 48). Furthermore,
caution must be observedeven whem comparing resultsobtained with the sametechnique.
Brush(1979) used cellulose-acetate
electrophoresis to examine egg-white proteins from 63
speciesof birds representing seven orders. He
found that different electrophoreticconditions
may affect results and warned that this should
be accountedfor during comparisonsof results
from different studies. Aquadro and Avise
(1982) reexaminedsix enzymesin nine species
of thrushes and their relatives
in order to assess
the effectof varying electrophoreticconditions
on the resultsobtained.They usednine buffers
with varying ionic concentrations,pH values,
and running times. Although their taxonomic
conclusionsremained essentiallythe same,they
were able to resolve eight additional electrophoretic variants. When compared with the
work of Tegelstrom et al. (1980), Zink and
Winkler (1983) sharpened resolution and detected more variation in the gulls they examined by using alternativebuffer systems.These
examples serve to emphasize the need for
screeningmultiple buffer systemsfor a determination of optimal electrophoreticconditions
in order
to resolve
the maximum
amount
of
variation.
Tissues.--Thedistinction between allozyme
and isozyme characters has not been recognized in avian systematic analyses. Distinguishing between allozymesand isozymeshas
been complicatedbecauseof the paucity of tissues examined in past studies. In some studies
conducted in the 1970's, egg white and blood
(i.e. hemoglobin, plasmaand serum) were used
723
as a tissue source. Blood was a favorite
tissue
for electrophoreticwork, becauseit was readily
obtainable, and its collection did not necessi-
tate sacrificeof the specimen.Other tissueswere
also examined. Brush (1976) and Knox (1980)
had some limited successin obtaining taxonomically useful charactersin feather proteins.
Frenkel and Gillespie (1979) usedbeak proteins
to determine taxonomic relationshipsand concluded that, even with some limitations, elec-
trophoresisof beak proteins provided more information than electrophoresis of feather
proteins. In most studies, the general protein
stainsemployed could detect the presenceof
6-12 gene products (e.g. lysozyme, ovoalbumin, transfertin), but only the overall pattern
(of differences in mobilities and numbers of
proteins) was consideredto be of systematic
importance.With the advent of allozymeelectrophoresis,the numberof charactersavailable
for study increased.Yet, recent studies have
also been limited
in numbers
of tissues exam-
ined. The four most commonly examined tissuesare heart, kidney, liver, and muscle, and
in many studiesonly one or two of these are
examined (e.g. Tegelstrom et al. 1980). Although examinationof only one or two tissues
may be necessaryor even desirablein some
instances,suchrestrictionsmay result in potential loss of characters.For example, Marsden
and May (1984)found that featherpulp yielded
moregene productsthan blood but fewer than
internal organs.Whitt (1983:28) hasstated"...
the tissuepatternsof expressionof a given set
of isozymes can differ from one taxonomic
group to another.... In suchcases,thesedifferent tissueexpressions
can be usedto understand the evolutionary and systematicrelationships among speciesand higher taxa." Tissue-
specificexpressionhas been employed as an
isozymecharacterby workersusinglactatedehydrogenase
of fishes(Shakleeand Whitt 1981),
creatinekinaseof amphibiansand reptiles(Buth
et al. in press), and NADP-dependent malate
dehydrogenase("malic enzyme") of larids and
passerids(Matson unpubl. data). If isozyme
charactersare to be resolved and applied to systematic studies,more tissuesmust be routinely
examined.
Increasingthe number of tissuessurveyed
will alsoaid in determiningthe homologiesof
loci between
taxa. "Because of the substantial
differencesamongisozymes,and the relatively
conserved tissue-specificexpressions,we can
724
RONALD
H. MATSON
readily determine orthologousisozymesin different species,i.e., which isozyme loci of one
speciescorrespondsto which isozyme loci of
another species..." (Whitt 1983:20). Knowing
locus homology is essentialto the establishment
of a standardized
nomenclature
and
to
the determination of polarity.
Nomenclature.--Comparisonsof results are
complicatedby the lack of a standardizedsystem of enzyme and locus nomenclature in avian studies. Independent investigators have
often developed their own systemsof nomenclature. This practice has resulted in a multiplicity of namesfor homologousenzymesand
genes in different species.As noted by Buth
(1983: 394): "... many biologists fail to keep
abreast of ... changes in enzyme nomenclature, many are unaware of the multiplicity of
names,their synonymsor even published recommendations
for their
use!...
The real need
[Auk,Vol. 101
retic paper to present results that had been
analyzed cladistically was that of Barrowclough and Corbin (1978). Since then, cladistic
methodshave been employed more often (Table 1).
Even with this shift in systematicapproaches
to the use of cladisticmethods,problems still
remain with the treatment of data. In most systematic studies, the loci were not treated as
characters, but, rather, electromorphs were
converted into the allelic frequenciesused to
compute various distance coefficients.The dis-
tancecoefficientswere then analyzed cladistically. Farris (1981), however, raisedseriousobjections to the use of any sort of distance data
for determination of phylogenetic relationships of taxa (cf. Felsenstein 1984). Farris ar-
guedthat the useof Nei's distanceis not valid,
becausethis measure does not satisfy the triangle inequality. [The triangle inequality states
for improvement lies in the area of information that, when the distances between three taxa
content, i.e., improving the designations for (A,B,C) are compared,the following matheidentifying relationshipsamongloci... somore maticalrelationshipmusthold:distance(A,C) -<
accuratestatementsregarding homologiescan distance(A,B) + distance(B,C).] Nonmetricity
be made." To avoid confusion,enzyme names makes it impossible to interpret the network
shouldbe thoserecognizedby the Internation- branchlengthsin a phylogeneticallymeaningal Union of Biochemistry(1979) and should be ful way. For distance coefficientsthat are metaccompaniedby the EnzymeCommission(E.C.) ric (e.g. Rogers' distance), the results are no
number (a practice that is on the increase). better but for different reasons. On a tree conButh's (1983) suggestionsfor stabilizing locus structed using any distance coefficient,Farris
that the common
ancestor
of
nomenclature in the ichthyological literature demonstrated
can and should be applied to avian studiesas three taxa cannot exist on any branch that conwell. These simple steps would facilitate the nectsany two of the three taxa. This is clearly
comparisonof results from different studies.
impossible,and it lead Farris (1981: 18) to conSystematicmethods.--The most serious diffi- clude that metrics" .. no more yield physicalculty in comparing the resultsof previous avi- ly interpretablebranch lengths than do nonan studies is the result of different philoso- metrics .... " He suggested analyzing
phies used in making systematicdecisions. electrophoretic data by using the electroPhenetic techniques have been most common- morphsdirectly and discardingthe use of frely used for analysisof electrophoreticdata, al- quencydata.Farris(1981:22) further statedthat
thoughmorerecentstudieshaveemployedcla- ".. there is not much comparative informadistic methods. Cladistic analyses assess
relationships that are based on the possession
of synapomorphic(shared, evolutionarily derived) characters rather than on overall similarity (as in phenetic analyses).The possession
of synapomorphiescharacterizesholophyletic
groups (sensuHolmes 1980) that are thought to
tion in the frequencies beyond simple presences..." and that "direct phylogenetic analysis of alleles as characters,moreover, avoids
the information loss that attends reducing
character data to distances."
Some problems with distance data can be
avoidedby the use of character/statedata. This
be descended
from a common
ancestor and
type of datahasan advantageover distancedata
therefore genealogicallyrelated. Although the in that homoplasiousstepsmay be identified,
theoreticaladvantagesof cladisticanalysishave and branch order and length have biological
been well documented(e.g. Wiley 1981), its in- interpretation (Buth in press).Few studentsof
fluence has been slow to affect avian system- birds have attempted to analyze electrophoatics (Cracraft 1981). The first avian electropho- retic data cladistically by means of characters
October1984]
AvianElectrophoresis
and characterstates.In one study (Avise et al.
1980a), alleles were treated as characters, the
presence/absenceof a given allele being considered the characterstate. This "independent
alleles"method of encodingdata, however, has
recentlybeen critically examinedby Mickevich
and Mitter (1981), who suggestedalternative
methodsfor encoding electrophoreticdata. In
the new interpretation, the locus per se is considered
to be the character
and the allelic
com-
position of a locusto be the characterstate.This
recognition of charactersand character states
in electrophoretic data is biologically more
meaningful (after all, some allele mustoccupy
a given locus).Although Zink (1982) and Gutierrez et al. (1983) mention encoding data in
this manner, detailed resultsusing this coding
method have yet to be reported in any avian
study.
Problemsalsoexistin linking charactersinto
an evolutionary sequencewhen two or more
statesare recognized.Although several ordering methods have been developed by Mickevich and Mitter (1981, 1983), this area of systematics remains a problem (Buth in press).
Nevertheless,
use of character-state
data de-
servesconsiderationand may be thought of as
an alternative
to distance
coefficients.
725
man 1971, Buth et al. 1980). The ability of multimeric enzymes to form heteropolymersis assumed to represent the primitive condition
(Buth in press).Thus, while outgroup analysis
is the method of choice in inferring polarity,
alternatives
are available.
As Markert (1983: 16) noted: "All problems
in biology involving genesand enzymesare candidatesfor the application of our techniqueand
our understanding of isozymes. The research
frontier is expanding, not shrinking, and isozymes are certain to make basic contributions
to the solution of problems in many areas of
biology simply becausethey are a fundamental
and pervasivemanifestationof the structureand
function of organisms." Electrophoresishas
played an important role in ornithology during the past 46 yr. If former inadequaciesin
data gatheringand data analysisare addressed,
electrophoresismay prove even more useful in
the future. This can be assuredby sampling a
wider array of taxa and tissuesand by using
isozymeaswell as allozyme data. Investigators
should encode data by using the locus as the
character, retain data in particulate form, and
use shared derived charactersto infer phylogeny. Only then will the full potential of this
technique be realized in avian systematics.
A problem related to the ordering of characterstatesis the establishmentof evolutionary
ACKNOWLEDGMENTS
polarity. Part of the reason that loci were not
treateddirectly ascharacterswasbecausemany
I thank D. G. Buth for his encouragement,support,
investigators thought it difficult to establish and valuablecriticisms.I am grateful to C. F. Bennett,
polarity of biochemical characters(e.g. Brush T. R. Howell, N. K. Johnson and E. C. Olson for read1979). There is no reason why, given a suffi- ing the first draft of this manuscript. I thank C. B.
cient
number
of characters
and more
than
one
Crabtree, R. D. Orton, J. A. Peterson, W. J. Rainboth,
P. B. Samollow, and an anonymousreviewer for their
many helpful comments,which improved the qualinferring evolutionarypolarity would not be ity of this paper. Thanks go to all my family and
applicable in avian electrophoretic studies. friends for their continuedsupport.I am mostgrateBaverstock et al. (1979) have shown that stan- ful to my parents, B. and D. Matson; B. Matson, V.
dard outgroupmethodsare suitablefor estab- Matson, K. Delehanty, and J. Pogrebafor all of their
lishing such polarity. A herpetological study support, encouragement, and financial help. I espeby Murphy et al. (1983) demonstratedthe suc- cially thank C. Staggsfor providing both moral supcessful use of the outgroup method for allo- port and clerical assistance.The figure was prepared
zyme data. If the outgroup method cannot be by M. Kowalczyk.
potential outgroup, that outgroup methods of
applied, other methods for determining polarity exist(Crisciand Stuessy1980,Stevens1980).
For isozymes (but not allozymes), heteropolymer assembly (i.e. the interaction of different
enzyme subunits) in multimeric enzymes can
be used as both an isozyme characterand a
method of determining polarity. The restriction of heteropolymerformation hasbeen used
as a derived characteruniting taxa (e.g. Gor-
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