Zoological Journal of theLinneanSociety(1995), 114: 91102.

Onychophora: past andpresent. EditedbyM. H. Walker andD. B. Norman

A llo zy m e e v i d e n ce fo r e x te n si v e a n d a n ci e n t
ra d i a ti o n s i n A u stra li a n O n y ch o p h o ra
D. A. BRI SCOE AND N. N. TAI T
School of Biological Sciences, MacquarieUniversity, Sydney, N.S.W. 2109, Australia
Allozyme electrophoresis has been employed to examine genetic differentiation among eight
described species, and representatives of an additional 15 taxa, of Australian peripatopsid
Onychophora. The data reveal extremely high genetic differentiation among the described
species and among the other taxa, each of which warrants specic recognition. Rapid protein
evolution cannot account for the large genetic distances and it is proposed that these are a
consequence of ancient divergence times. A method is presented for extracting phylogenetic
information from allozyme data sets which are not amenable to conventional analysis.
ADDI TI ONAL KEY WORDS:Peripatopsidae Australian fauna species radiations
genetic distances.
CONTENTS
I ntroduction . . . . . . . . . . . . . . . . . . . 91
Material and methods . . . . . . . . . . . . . . . . . 92
Results . . . . . . . . . . . . . . . . . . . . 94
Genetic differentiation among populations . . . . . . . . . . . 94
Sympatry . . . . . . . . . . . . . . . . . . . 95
I sland populations . . . . . . . . . . . . . . . . 96
Discussion . . . . . . . . . . . . . . . . . . . 96
Allozyme species and biological species . . . . . . . . . . . 99
High levelsof genetic differentiation . . . . . . . . . . . . 99
Phylogenetic reconstruction . . . . . . . . . . . . . . 100
Acknowledgements . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . 101
I NTRODUCTI ON
The technique of allozyme electrophoresis has permitted the detection of
cryptic species within groups of organisms as diverse as bacteria (Selander
et al., 1986), cellular slime moulds (Briscoe et al., 1987) and mammals
(Dickman et al., 1988). I t is of particular value in separating taxa of small
or soft-bodied organisms where clear morphological characters may be difcult
to discriminate, and in identifying sibling species. I n this paper, we present
results obtained from using this technique as part of a wider study of the
evolution of the Onychophora in Australia.
91
00244082/ 95/ 050091+12 $08.00/ 0 1995 The Linnean Society of London
92 D. A. BRI SCOE AND N. N. TAI T
The phylum Onychophora is noted for its overall conservatism of body
form, both in terms of the apparently low level of morphological differentiation
among extant taxa, and the striking similarity between present-day and fossil
specimens (Thompson & Jones, 1980; Heyler & Poplin, 1988). This
conservatism may reect strong stabilizing selection imposed by the
microhabitat in which they are found. Onychophorans are highly susceptible
to desiccation (Dodds & Ewer, 1952) and restricted to humid microhabitats
such as soil, leaf litter and, in the case of most of the Australian forms we
have studied, the interstices of rotten logs. They are patchily distributed
throughout the forests of eastern Australia, Tasmania and southwest Western
Australia and occur very locally in South Australia (Ruhberg, 1985).
I n contrast to morphological conservatism, the natural history of
onychophorans may predispose them to local genetic differentiation. Small
population sizes, patchy distribution, and low vagility imposed by strict
microhabitat requirements, are all conditions known to promote allopatric or
parapatric speciation.
When we commenced our studies, eight species in six genera of
Peripatopsidae were recognized in Australia, approximately 8% of the world
onychophoran fauna (Ruhberg, 1985). Our interest was stimulated by our
discovery of a new species, Cephalofovea tomahmontis (Ruhberg et al., 1988),
sympatric with Euperipatoides leuckartii (Saenger) at Mt Tomah in the Blue
Mountains of New South Wales. As part of the delineation of C. tomahmontis,
we compared it with E. leuckartii using allozyme electrophoresis. The two
species differed at 17 of the 21 loci screened (see Reid et al., 1995: p. 119).
Furthermore, the differences in electrophoretic mobilities were sufciently
great to indicate that more than one charged amino acid substitution had
accumulated in many of the proteins and, hence, that divergence of the
lineages was not a recent event. This paper reports an extension of these
ndings to a survey of all named, and 15 unnamed, taxa. The data indicate
that the Australian fauna is far more diverse than was previously recognized
and that some of the radiations are of considerable antiquity.
MATERI ALS AND METHODS
Specimens were hand collected at the localities given in Table 1. Sample
sizes are included in Table 3. The Mt Lewis and Mt Allyn (taxon J of Tait
& Briscoe, 1990) forms were sorted from rainforest leaf litter and the Gibralter
Range and Sydney specimens were obtained from crevices in soil. All other
forms were found in, or under, rotting logs. Sample sizes for many of the
forms are restricted by their low population densities. Capture rates of
approximately one specimen per collector per day are typical. I n some cases,
animals were anaesthetised (see Reid et al., 1995: p. 117), and parts removed
for morphological examination prior to storage of the body at 80C. Most
specimen were frozen whole. Homogenates were prepared by grinding the
bodies with ne ground glass in a small mortar and pestle at a ratio of 1: 3
(w:v) in cold homogenizing solution (0.1 mg NADP ml
1
; 0.1%
b-mercaptoethanol, plus a few crystals of bromophenol blue in distilled
water). The homogenate was divided into 20 ml aliquots in microhaematocrit
tubes and stored at 80C. Cellogel (Chemetron, Milan) electrophoresis was
93 RADI ATI ONS I N AUSTRALI AN ONYCHOPHORA
TABLE 1. Collection localities for eight named and fteen undescribed taxa of
Onychophora from Australia

Latitude Longitude
Locality (S) (E) Taxon*

Mt Lewis, Qld. 1635? 14517? Austroperipatus paradoxus

Mt Tomah, N.S.W. 3333? 15025? Euperipatoides leuckartii
Cephalofovea tomahmontis
Undescribed sp.
Carey Gully, S.A. 3458? 13847? Mantonipatus persiculus
Mundaring, W.A. 3157? 11610? Occiperipatoides gilesii
Ferguson R., W.A. 3323? 11545? Occiperipatoides occidentalis
Mt Macedon, Vic. 3723? 14435? Ooperipatus oviparus
Ooperipatellus insignis
Mt Allyn, N.S.W. 3208? 15126? J, undescribed sp.
Gloucester Falls, N.S.W. 3206? 15136? E
Brown Mtn., N.S.W. 3636? 14923? H
Tinderry Mts, N.S.W. 3544? 14916? G
Gibralter Ra., N.S.W. 2936? 15211? undescribed sp.
Sydney, N.S.W. 3349? 15101? undescribed sp.
Mt Dromedary, N.S.W. 3618? 15002? undescribed sp.
Armidale, N.S.W. 3031? 15140? 2 undescribed spp.
Henrietta Ck, Qld. 1736? 14545? C, undescribed sp.
Dunk I s., Qld. 1757? 14609? undescribed sp.
Wide Bay, Qld. 2552? 15307? 2 undescribed spp.
Fraser I s., Qld. 2505? 15313? as for Wide Bay

* Letters refer to the taxon designations used by Tait & Briscoe (1990).
conducted at 4C using the gel buffer of Spencer, Hopkinson & Harris (1964)
for presoaking the strips (10 cm17 cm) and in the electrode vessels.
Preliminary trials were conducted to ensure that the electromorphs recorded
were not artifacts. Comparisons were made of fresh versus frozen, male
versus female, new born versus adult and gut versus body wall. Some
systems, including esterases, phosphatases, peptidases and general proteins,
yielded ambiguous data and were discarded. Run durations, at 15 V cm
1
,
for each reliable enzyme are given in Table 2. Eighteen enzymes representing
21 gene loci were visualized using the histochemical staining methods of
Richardson, Baverstock & Adams (1986). Staining for AAT, I DH and MDH
produced two independent zones of enzymatic activity. The gene loci encoding
these enzymes are distinguished by the sufxes A and C for the more
anodally and cathodally migrating zones respectively. When loading the gels,
a reference sample derived from a mass homogenate of 30 E. leuckartii from
Mt Tomah, N.S.W., was interspersed ve times among the other samples
across the gel. Following staining, the migration of each electromorph was
measured with dial callipers and subsequently identied by its mobility
relative to the reference sample. This facilitated comparisons between runs
and allowed the method of data analysis proposed later in this paper. As
large numbers of alternative mobility states were observed among our
specimens, line-up gels were frequently required to conrm mobility
differences of 2 mm.
RESULTS
The variation observed in the zymograms following histochemical staining
is consistent with segregation, or xation of alternative alleles, at the protein-
94 D. A. BRI SCOE AND N. N. TAI T
TABLE 2. Enzymes examined by cellulose acetate electrophoresis of onychophoran tissue
homogenates

Run time
Enzyme E.C. Number Abbreviation Loci (Mins)

Aspartate aminotransferase 2.6.1.1 Aat 2 90

Aconitase 4.2.1.3 Acon 1 75
Adenylate kinase 2.7.4.3 Ak 1 60
Aldolase 4.1.2.13 Aldol 1 60
Enolase 4.2.1.11 Enol 1 105
Fructose 1,6 Diphosphatase 3.1.3.11 Fdp 1 60
Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 Gapd 1 60
Glycerol-3-phosphate dehydrogenase 1.1.1.8 aGpd 1 60
Glucose phosphate isomerase 5.3.1.9 Pgi 1 90
Hexokinase 2.7.1.1 Hk 1 60
I socitrate dehydrogenase 1.1.1.42 Idh 2 60
Malate dehydrogenase 1.1.1.37 Mdh 2 75
Mannose phosphate isomerase 5.3.1.8 Mpi 1 60
Phosphoglycerate mutase 2.7.5.3 Pgam 1 60
6-Phosphogluconate dehydrogenase 1.1.1.44 6Pgd 1 60
Phosphoglycerate kinase 2.7.2.3 Pgk 1 60
Phosphoglucomutase 5.4.2.2 Pgm 1 120
Pyruvate kinase 2.7.1.40 Pk 1 60

encoding loci, within or among populations of sexually reproducing, diploid

organisms. Presumed heterozygotes at the Ak, Mpi, Pgk and Pgmloci exhibited
two zones of activity while those for Aat, aGpd, Idh, Pgam, 6Pgd and Pgi
displayed three zones with the intermediate band most intensely stained.
These patterns are characteristic of the known monomeric and dimeric
structures of these proteins. The electromorph states observed for each
enzyme in each population are given in Table 3.
Observed intrapopulation polymorphism is low. I n the two species for
which we have sufciently large sample sizes to allow reasonable estimates,
E. leuckartii and C. tomahmontis, observed heterozygosity is 0.005 and 0.057
respectively.
Genetic differentiation among populations
I n contrast to the low level of intrapopulation polymorphism, variation
among populations is remarkably high. For ease of interpretation we illustrate
this differentiation as matrices of percentage xed gene differences. I n any
pairwise comparison of populations, a gene locus is dened as displaying a
xed difference if the populations do not share any common mobility state
for the enzyme coded for by the locus.
As a benchmark we have examined variation among specimens collected
at, or near, the type localities of seven named species, and morphologically
referable to those species (Table 4). The number of shared mobility states
in pairwise comparisons among these species varies from six to one, giving
xed gene differences of 7095% (Table 4). Thus, our initial observation of
extensive differentiation between E. leuckartii and C. tomahmontis is not atypical
of Australian onychophorans.
Variation among other populations is, surprisingly, almost as great as that
95 RADI ATI ONS I N AUSTRALI AN ONYCHOPHORA
for the named species. Extensive sampling throughout Australia has revealed
a large number (100) of genetically distinct populations (Briscoe & Tait,
unpublished). Table 5 presents a subset of the data illustrating heterogeneity
among populations sampled within the State of New South Wales. On
morphological criteria, none of these populations are referable to E. leuckartii,
Ooperipatus oviparus (Dendy) or C. tomahmontis, the only described taxa from
the State. The populations included in Table 5 represent a variety of
geographical distributions of allozyme-genetic forms. The Mt Allyn and
Tinderry forms appear to be restricted to several geographically adjacent
sites in the Barrington Tops area (220 km north of Sydney), and the Tinderry
Ranges (40 km south of Canberra) respectively. I n contrast, the Gibralter
Range population and the Mt Dromedary sample are apparently genetically
related, respectively, to complexes extending through northern New South
Walessouthern Queensland and southeastern New South Wales. Similarly,
the Brown Mountain specimens form part of a complex extending from
southeastern New South Wales into Victoria. The Armidale 1 and 2 samples
are closely related to populations distributed, respectively, to the west and
east of Armidale city, where they are sympatric. Samples from suburban
Sydney are anomalous. They are apparently related to the Gibralter Range
complex, 470 km to the north, in terms of their allozyme proles (Tables 3
and 5), in the structure of the heads of males, and in their microhabitat
within soil crevices.
Thus, while the data presented are selected, they are representative of our
full data set, and their purpose in this report is to illustrate the extent of
allozyme differentiation among populations. Examples of ner-scale
differentiation are given in accompanying papers (Tait & Briscoe, 1995, Reid
et al., 1995).
With the exception of the Sydney-Gibralter Range comparison, each of
the populations described in Table 5 differs from all others at 1420 (67
95% xed gene difference) of the enzyme loci assayed. This level of genetic
differentiation is not compatible with conspecicity of populations of sexually
reproducing, diploid organisms, even for geographically isolated populations
(Richardson et al., 1986).
Sympatry
I n several localities, we have identied sympatry of two or more species
or putative species. At Mt Tomah, E. leuckartii, C. tomahmontis and a third,
undescribed, species can be collected from the same rotten log. Despite the
intimacy of this sympatry, the three forms retain gene pools differing by 81
86% xed gene difference (Table 3). A similar situation exists in the
Barrington Tops area (Mt Allyn and Gloucester localities) where a further
three species are sympatric but differ from each other at 7176% xed gene
difference (Table 3).
Island populations
To assess the rate at which amino acid substitutions may accumulate in
onychophoran proteins, we have examined two situations where we have
96 D. A. BRI SCOE AND N. N. TAI T
TABLE 3. Allozyme mobility states observed for eight populations of described Australian
peripatopsid Onychophora and 15 undescribed taxa from 13 localities. nSample size

n Enol Aat
a
Aat
c
Ak 6Pgd Pk Pgk Aldol

Mt Lewis A. paradoxus 2 7 4 3 12 5 4 8 10:11

Mt Tomah E. leuckartii 30 8 8 2 10 3 2 8 5
C. tomahmontis 8 14 12 5 5 6 2 10 9
undescribed sp. 2 4 13 1 6 6 4 5 3
Carey Gully M. persiculus 2 10 9 6 8 6 2 8 6
Mundaring O. gilesii 8 13 7 2 5 6 8 10 6
Ferguson R. O. occidentalis 4 5 6 3 10 6 9 11 11
Mt Macedon O. oviparus 3 1 11 1 11 6 1 2:4 2
O. insignis 2 15 8 3 1:2 10 6 6 2
Mt Allyn undescribed sp. 1 5 8 1 3:5 3 4 4 1:3 11
undescribed sp. 2 4 2 2 3 6 7 2:3 6:8 7
Gloucester undescribed sp. 4 6 8 3 5 3 2 9 4
Falls
Brown Mtn. undescribed sp. 6 11 5 1 6 1 3 10 5
Tinderry Mts undescribed sp. 8 4 10 6 4 5:7 1 6 2
Gibralter Ra. undescribed sp. 7 8 8 3 7 2 4 7:8 8
Sydney undescribed sp. 3 8 8 3 7 1 4 6:7 8
Mt Dromedary undescribed sp. 3 8 8 2 6 8 1:2 8 5
Armidale undescribed sp. 1 9 8 8 2 6 2 2 10 1
undescribed sp. 2 8 14 12 2 10 9 2 10:11 5
Henrietta Ck undescribed sp. 1 2 9 5 4 7 5 4 5 8
undescribed sp. 2 2 3 4 2 11 5 4 4:6 8
Dunk I s. undescribed sp. 2 9 5 4 7 5 4 5 8
Wide Bay undescribed sp. 1 1 12 6 2 9 3 7 7 8
undescribed sp. 2 1 9 5 3 6 1 5 6 12
Fraser I s. undescribed sp. 1 1 12 6 2 9 3 7 7 8
undescribed sp. 2 2 9 3:5 3 6 1 5 6 12

obtained samples from mainland localities and adjacent offshore islands (Table
3). At Henrietta Creek, on the north Queensland mainland, two undescribed
species exist in sympatry. Specimens from nearby Dunk I sland differ from
one of these species at only one of the 21 loci. Wide Bay on the southern
Queensland coast also supports two sympatric species, both of which are
also sympatric on the adjacent Fraser I sland. The disjunct populations differ
at only two and zero enzyme loci.
DI SCUSSI ON
The allozyme data reveal an extensive diversity of previously unrecognized
putative species of peripatopsids in eastern Australia. Here we argue that the
high levels of genetic divergence among these forms indicate that they are
not the products of recent evolutionary events.
Our argument is, however, entirely dependent upon our interpretation that
differences in electrophoretic mobility of homologous enzymes reect
underlying variation in structural genes. We have no evidence to reject this
generally-held assumption. First, we have not used gene-enzyme systems
which yielded patterns which were ambiguous with respect to sex, tissue
97 RADI ATI ONS I N AUSTRALI AN ONYCHOPHORA
TABLE 3continued.

Idh
a
Mpi Acon Pgm Pgam Mdh
a
Pgi aGpd Fdp Gapd Hk Mdh
c
Idh
c

4 3:7 4 5:7 4:7 6 9 9 6 4 8 1 2

5 7 8 10 7 3 10 4 4 3 6 1 4
3 5 3 8 9 5 12:14 1 3 6 2 1 4
1 2:5 2 8 3 3 1 5 4 4 13 1 5
3 3 5 11:13 3:5 7 8 4 5 4 2 6
7 2 2 5 7 1 2:5 4 7 9 9 7 1
7 6 2 4 1 10 2 10:11 1 6 1 5 1
2:3 3:5 5 13:15 5 6 4 3 1 1 3 1 2
6 11 5 2 8 9 1:3 8 8 8 14 7 6
5 5 4 7 4 3:8 9:13 9 3 10 10 1 1
5 5 8 8 2 3 10:13 4 3 4 2 5 3
5 5 4 9 8 3:8 11 5 2 2 11 1 4
3 1 6 1:3 6 11 6 2 2 1 4 1 5
2 9 4 3 4 3 15:16 7 2 4 7 1 4
3:5 10 6 13 1 10 8:11 5 5 4 5 1 5
3 10 6 6 6 10 6 9 2 4 8 1 1
5:7 7 1 11:14 6 3 9:13 2 4 3 11 1 4
5 3 8 7 7 2 11 6 4 5 3 6 1
5 2 8 6 2 4 13 6 2 7 12 4 4
5 8 2:7 5:6 7 12 5 4 2 5 8 3 1
5 3 7 5:7 4 6 7 9 5 1 8 1 1
5 7 7 5:6 7 12 5 4 2 5 8 3 1
3 3:5 6 16 10 6 6 9 4 6 1 1
3 10 4 12 1 6 6 5:9 4 4 1 1
3 5 4 16 10 6 6 5 4 6 1 1
3 9:10 4 10:12 1 6 6 5:9 4 4 1 1

The body of the table represents the allozyme mobility state for each population and enzyme.
Mobility 1 represents the least anodally migrating electromorph. Rare electromorphs which were found
in only one population have been omitted. The symbol indicates that activity for this enzyme in
this sample was too low for accurate identication.
type, age or tissue storage time. Second, where apparent heterozygotes were
observed, the zymograms were concordant with the known quaternary
structures of the proteins. Third, where intrapopulation polymorphism was
observed, we have identied both apparent homozygotes and heterozygotes.
TABLE 4. Matrix of percent xed gene differences among
seven species of Australian peripatopsids based on 21
allozyme loci

E.l. M.p. O.g. O.o. A.p. O.ov. O.i.

E. leuckartii 0
M. persiculus* 85 0
O. gilesii 86 90 0
O. occidentalis 95 95 81 0
A. paradoxus 81 90 90 90 0
O. oviparus 95 70 95 90 81 0
O. insignis 95 95 95 95 95 90 0

* No activity for aGPD was detected in M. persiculus, therefore

comparisons involving this species are based on 20 loci.
98 D. A. BRI SCOE AND N. N. TAI T
TABLE 5. Matrix of percent xed gene differences among eight
populations of undescribed peripatopsids from New South Wales,
Australia, based on 21 allozyme loci

M.A. B.M. T.R. G.R. S. M.D. A.1 A.2

Mt Allyn 0
Brown Mtn. 95 0
Tinderry Ra. 86 86 0
Gibralter Ra. 76 81 90 0
Sydney 71 67 81 38 0
Mt Dromedary 76 76 81 76 81 0
Armidale 1 81 90 100 76 86 67 0
Armidale 2 90 86 90 95 90 71 76 0

Fourth, in a few instances, females subsequently found to be apparent

heterozygotes have given birth in captivity. Analysis of their offspring yielded
both heterozygotes and homozygotes. Thus, protein structure and pattern of
inheritance are concordant with zymogram variation reecting segregation,
or xation, of alternative alleles at structural gene loci.
I n our analysis we have reduced the data to matrices of percentage xed
gene differences. This is a conservative estimator of differentiation as two
populations sharing a single mobility for a particular enzyme are thereby
considered to be genetically identical at the underlying locus. We have used
this procedure because estimates of allele frequencies are extremely unreliable
when sample sizes are small, and because, in taxonomic studies, loci displaying
xed differences are the most informative (see Richardson et al., 1986). The
statistical validity of our conclusions can be assessed in two ways. First, the
lower 95% condence interval for an estimate of 60% xed gene difference
(based on 21 loci) is 39%a level of differentiation far greater than normally
encountered among allopatric populations of a single, diploid species. Thus,
populations differing at 60% xed difference are highly unlikely to be
conspecic (Table 5). We should note that, on this criterion, the Gibralter
Range and Sydney samples may tentatively be included within the same
taxon, although further biogeographic and morphological evidence may prove
this view to be overconservative.
A second statistical approach to assessing the validity of our conclusions
is to test the null hypothesis that the specimens we have analysed have been
drawn from a single gene pool. When one individual is sampled from each
of two populations, the probability that they will be homozygous for
alternative alleles at a locus is 2p
2
q
2
, where p and q are the relative
frequencies of the alternative alleles in the hypothetical common gene pool.
This probability has a maximum value of 0.125 when p q0.5. When
an apparent xed gene difference between the populations is based on
sampling two individuals from each population, the probability is reduced to
0.0078 (Sarich, 1977). The probability that two or more individuals, drawn
from each of two populations, will, by chance, be homozygous for alternative
alleles at 13:21 loci is vanishingly small.
99 RADI ATI ONS I N AUSTRALI AN ONYCHOPHORA
Allozyme species and biological species
Our interpretation that the distinct genetic forms represent true biological
species is supported on two grounds. First, and most importantly, sympatric
populations of two or more forms sharing no alleles in common at the
majority of their enzyme loci (Tables 1 and 3) indicate a complete barrier
to gene exchange, the essence of the biological species concept. Where we
have detected sympatry, (Mt Tomah, Armidale, the Barrington Tops area,
Henrietta Creek, Wide Bay and Fraser I sland [Tables 1 and 3]), the
differentiation among coexisting compound genotypes is not signicantly
greater than that found between allopatric putative species. Thus we conclude
that the allopatric forms would be extremely unlikely to interbreed with each
other even if their geographic isolation were broken down. Second, a number
of the taxa initially identied on the basis of allozyme analysis have
subsequently proved to be morphologically distinct. This is most obvious for
C. tomahmontis, and the Mt Allyn, Brown Mountain, Tinderry Range,
Gloucester Falls and Henrietta Creek populations. Each of these exhibits a
distinctive male head structure and corresponds to a taxon of Tait & Briscoe
(1990) (see Table 1). Detailed morphological analysis is currently revealing
differences among some taxa which lack head structures (M. Reid, personal
communication).
High levels of genetic differentiation
Several hypotheses may be advanced to account for high levels of allozyme
differentiation among taxa; sampling artifacts, clonal population structure,
strong natural selection, rapid protein evolution and long evolutionary
separation times. We have argued above that the probability of our data
being artifacts of small sample sizes is extremely low. Clonal population
structure is also an unlikely explanation. With the exception of the Trinidadian
peripatid, Epiperipatus imthurni (Sclater), which is parthenogenetic (Read,
1988), all onychophorans so far studied are sexually-reproducing and dioecious.
Within Australian peripatopsids, all populations we have examined to date
have karyotypes consistent with diploidy, and XY heterogamety in the
majority of males (Rowell et al., 1995: pp. 142143). I n addition the majority
of females we have dissected carry sperm in their spermathecae. At the
allozyme level, the zymograms of putative heterozygotes are concordant with
those of sexually-reproducing diploids. I n summary, we have no data which
support an hypothesis of clonal population structure.
While strong heterogeneous selection may lead to local genetic
differentiation, we discount this hypothesis on two grounds. First, this model
is not compatible with the maintenance of very different compound genotypes
among sympatric species. Second, Kimura (1983) has demonstrated that the
fate of an allele is predominantly inuenced by stochastic processes if the
selection coefcient is much smaller than the reciprocal of twice the effective
population size. Onychophoran populations are generally of small census size
and, through genetic bottlenecks, probably of even smaller effective size.
Therefore, the allozyme variants we observe are most likely to be effectively
selectively neutral.
100 D. A. BRI SCOE AND N. N. TAI T
Rapid genetic differentiation of taxa could also result if mutation rates
were unusually high or if the population structure of onychophorans promoted
xation of newly arisen amino acid substitutions. Concomitants of this
hypothesis are the predictions that there would be considerable transient
polymorphism within populations and:or local differentiation among
populations within widespread species. Our data are inconsistent with the
former prediction. We have attempted to test the latter prediction in two
ways. First, we have sampled six populations throughout the 600 km
2
range of Occiperipatoides gilesii (Bouvier) in the Darling Range of Western
Australia. Despite the disjunct nature of populations of this species (Dakin,
1920), no differentiation was observed (Briscoe & Tait, unpublished). Second,
little differentiation was found between island and adjacent mainland
populations (Table 3). Dunk and Fraser I sland lie only 45 km from the
mainland, but have been isolated for approximately 9000 and 2000 years
respectively. This argument is relatively weak as low levels of migration can
maintain genetic homogeneity among conspecic populations. Nonetheless,
we suggest that migration rates are probably restricted by the strict microhabitat
requirements, and susceptibility to desiccation, of onychophorans.
I n summary, our data are not compatible with hypotheses of sampling,
clonal population structure, heterogeneous selection, or elevated amino-acid
substitution rates. Thus, we accept the simplest hypothesis, that at least some
of the lineages diverged an extremely long time ago.
Data from independent character suites are necessary to corroborate or
reject this hypothesis. I nitial karyological analysis is indicating that Australian
peripatopsids are highly variable (Rowell et al., 1995; pp. 139153). Further,
recent evidence from 12 S ribosomal RNA sequences, which evolve very
slowly, has revealed extensive nucleotide substitution between members of
the Euperipatoides complex from New South Wales and a specimen from
north Queensland (Ballard et al., 1992). Taken together, information from
the nucleotide, protein, karyological, morphological and biogeographical levels
are most compatible with a model of ancient radiations with many lineages
surviving to the present day.
Phylogenetic reconstruction
I n addition to delineating taxa, allozyme analysis can provide data for
establishing phylogenetic relationships. Unfortunately, this is not possible with
our data set using current measures of genetic distance. Fixed gene differences
in excess of 70% cannot be distinguished from zero true genetic identity, as
convergent mutations may result in different alleles coding for proteins with
the same electrophoretic mobilities (Richardson et al., 1986). Thus, all taxa
differing at this, or a greater level, must be considered equally unrelated and
conventional genetic distances contain no phylogenetically useful information.
We have attempted to overcome this difculty by devising a measure (Briscoe
et al., 1987) which extracts more information from allozyme data than the
mere presence or absence of differences in electromorph migration. A similar
measure has been proposed independently by Krimbas & Sourdis (1987).
We have observed that, in polymorphic populations, the segregating
electromorphs are physically close on the gels. At a minimum, the distance
101 RADI ATI ONS I N AUSTRALI AN ONYCHOPHORA
between these electromorphs is the consequence of a single charged amino
acid difference between the proteins. I n contrast, the physical distances
separating electromorphs from different taxa are generally considerably larger
and, presumably, reect several to many charged amino acid substitutions.
The essence of our estimator can best be described by way of an example.
I magine two recently diverged species, A and B. Over time each species
will accumulate amino substitutions at allozyme loci until, at time t, the
species share no common electromorphsa genetic identity of zero. At time
t the B lineage speciates again, amino acid substitution continues and, at
approximately time 2t, three species A, B, C will be evident, each apparently
equally unrelated to the others using conventional genetic distance measures.
However, the number of charged amino acid substitutions differentiating B
from C will be approximately half the number separating A from B and C.
This should be reected in greater average mobility differences separating
the A electromorphs from B and C than the mobility differences separating
B from C electromorphs. Clearly, physiological constraints will impose limits
on the nett positive or negative charge carried by an allozyme molecule.
However, the probability that two recently-diverged taxa will display large
differences in mobility for many independent enzymes or, conversely, that
two distant taxa will display convergence of mobility for many enzymes is
remote. At a practical level we sum, over all the enzymes, the physical
distances separating the electromorphs from each pair of species to generate
a matrix of genetic distance. (Where a population is polymorphic we take
the average migration distance of the segregating allozymes to be representative
of that population.)
We have assessed this absolute migration distance metric using both real
data sets and computer simulations and shown it to be informative (Briscoe
et al. 1987; McKay et al., 1989). I n an accompanying paper (Tait & Briscoe,
1995: pp. 103113) we utilize this estimator, together with biogeographical
evidence, to assess the antiquity of Australasian peripatopsid radiations.
ACKNOWLEDGEMENTS
We gratefully acknowledge the companionship and assistance of Ray
Cameron, Mandy Reid, David Rowell and Robyn Stutchbury during eld
work, and the Wildlife Services of Western Australia, Victoria, New South
Wales and Queensland for permits and local knowledge. Ray Cameron and
Rod Nurthen helped greatly with laboratory analysis. Dr Peter Baverstock
and Professors Desmond Cooper, Dick Frankham and Stephen J. OBrien
gave valuable comments on the absolute migration distance analysis, which
we developed in collaboration with Dr George McKay. Margaret Clarke
kindly prepared the manuscript with efciency and charm. This work was
supported by the Australian Research Council and Macquarie University.
This is paper number 154 of the Research Unit for Biodiversity and
Bioresources, Macquarie University.
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