Vol. 7. pp. 38^3

Asiatic Herpetological Research

1997

Phylogeny of Chinese Oreolalax and the Use of Functional Outgroups to SelectAmong Multiple Equally Parsimonious TreesJlNZHONG FU AND ROBERT W. MURPHY
Department of Ichthyology and Herpetology, Royal Ontario Museum, 100 Queen's Park. Toronto, Out. M5S 2C6,
Canada and Department of Zoology, University of Toronto
Abstract. -Re-analysis of a published data set from Xu et al. (1992) reveals that their proposed phylogenetic tree
is notthe most parsimonious explanation. Ten shorter, most parsimonious solutions are derived from these data.

The progressive functional outgroup approach found a single tree from among the ten to be the best explanationof the data. Such an approach may prove to be a powerful method for dealing with multiple equally parsimonious
trees.

Key words: Amphibia. Anura. Oreolalax. China. Phylogeny, Functional outgroups

phylogeny of Oreolalax using data provided by Xu etal. (1992) and Wu et al. (1993).

Our initial examination of Xu et al. (1992) showed
several problems in their analysis potentially invali-

dating their conclusions. Because of this, we first pursued a re-evaluation of their data, and subsequently

incorporated the new species into our analysis toobtain a more defensible phylogenetic estimation for
the genus Oreolalax.

Materials and Methods
The data set from Xu at al.
tial

Figure 1. Phylogenetic Cladogram of Oreolalax pro-

posed by Xu et al. (1992).

(

992 was used in our iniOur phylogenetic analysis
1)analysis (Appendix
was undertaken using Hennig86 (version
II).

and PAUP version 3.1.1. Swofford(

Introduction
Although considerable interest has been shown in theevolution of pelobatid frogs relatively recently, thephylogeny of many pelobatids remains largely enigmatic. Xu et al. ( 1992) proposed a phylogeny for four-

This genus ofpelobatid frogs is endemic to China and most of theteen species of Oreolalax (Fig.

1.5: Farris1988). Random Cladistics (version 2.0: Siddall 1994)1).

15 described species occur at high elevations (1500 to

3300m). Xu et al. used 29 morphological characters intheir analysis (Appendix I), and these were gathered
for the 14 species known at the time of their analysis.

Subsequent to the proposed phylogeny. a new species,
O. multipunctatus, was described (Wu et al.. 1993).
However, the phylogenetic relationships of this new
species were not considered in the description; its
position among the other species remained uncertain.

Consequently, we undertook a re-examination of the1993 ). Branch andbound algorithm (Hendy and Penny 1982) as provided
in these software packages was used for all tree
searching. The resulting trees were rooted initially bya hypothetical ancestor scored plesiomorphic for all
characters based on the hypotheses of Xu et al.

(1992). Using Random Cladistics. a permutation tailprobability test (PTP; Faith and Cranston 1991) wasused for assessing the presence of pattern in the data
set. which

we assume to represent phylogenetic struc-

Bootstrap values (BS: Felsenstein 1985) wereused for assessing the relative strength of support for

ture.

monophyletic groups. Examination of character state
distributions on trees was accomplished using Mac-

Clade (version 3.04; Maddison and Maddison 1992).
estimations of phylogenetic analysis wereaccomplished using the unaltered data of Xu et al.
Initial(1992). Three methods were exploited for dealing

with multiple most parsimonious trees (MPTs): con-Vol. 7. p. 39

Asiatic Herpetological Research

1997

Figure 3. Three MPTs resulting from successive
approximations weighting. The unstable position of O.

omeimontis and O. popei is responsible for the threetrees.

fewer than 7000 equally or more parsimonious solutions having 61

steps;

we terminated the program

after 30 minutes analysis.

MPTs resulting

A strict consensus tree, Adams consensus tree

from the original analysis with 7 unresolved nodes. B:
50% majority rule consensus tree with 2 unresolved(Adams 1972), and 50% majority consensus tree weregenerated from the ten MPTs respectively. The strict
consensus tree (Fig. 2a) showed low resolving power.

Figure 2. A: Strict consensus tree of 1

nodes: 6=0.6; 9=0.9; 1 =1 .0. C: Adams consensus treewith 4 unresolved nodes.

Only seven of 13 nodes were resolved demonstratingmuch conflict among various patterns of relationships.
The Adams consensus tree (Fig. 2b) gave a slightly
sensus evaluation, successive approximations weighting

(SAW;

Fams

1969.

1988),

andbetter resolution with four nodes unresolved. Finally,functional

outgroup approach (FOG) modified from Watrous andWheeler (1981). Following resolution of the tree forthe initial 14 species treated by Xu et al., the positionof Oreolalax multipunctatus relative to the other species was estimated.

Results

the

50% majority consensus tree (Fig. 2c) left only

two nodes unresolved. In all analyses, O. rhodostigmatiis always appeared at the base of the ten MPTs.

As another means of selecting a preferred tree
from among the ten MPTs, we used SAW. Following
the successive re-weighting of characters based on the
RI.

three

trees

were

found.

Whenthecharacterweights were re-set to 1, each tree had a length of 58

Our PTP test showed the data set of Xu et al. has pat-steps, and thus all fall within the suite of initial MPTs.

which is significantly different from random. A
PTP value of p<0.001 was obtained based on 999 ran-Among the three trees, the position of O. omeimontis

terndomized data sets. The minimum length tree derived
from the randomized data was 64 steps whereas theminimum length tree from the original data was 58
steps.

We interpret this pattern to reflect the presenceof phylogenetic structure.

and O. popei were unstable, accounting for threealternative trees, and two other nodes were resolved aspolytomys (Fig. 3).

We had concern that the plesiomorphic states may
not have been correctly identified as no outgroup wasused in the analysis, although the ancestral states the-Ten MPTs of 58 steps were found, each with a=consistency index (CI; Kluge and Farris 1969) of CI
0.50, and a retention index (RI; Farris 1989) of RI =oretically represent the conditions observed in Lepto-

0.59. When the data are mapped onto the tree of Xu et

more likely the polarization of character states will be

was observed to have a length of 61 steps(CI = 0.47; RI = 0.54). Using PAUP, we found noaccurately determined, we used a functional outgroupal.. their tree

brachium. Given

this,

and the observation

that,

in

general, the closer the outgroup is to the ingroup the(FOG) approach (Watrous and Wheeler 1981)

to

1997

Asiatic Herpetological ResearchVol. 7. p. 40

Figure 4. Strict consensus tree of 2 MPTs resulting

Figurefrom using Oreoialax rhodostigmatus as functional out-

as functional outgroup.

5.

Resulting tree using Oreoialax lichuanensis

group.

examine the data. Because O. rhodostigmatus always
appeared at the base of the ten MPTs. this species was

We excluded the hypotheticalused as our FOG.

and has a high value of 0.634. The weakest support

from BS is the O. popei and O. omeimontis clade thathas a value as low as 0.06

ancestor from our secondary and subsequent analyses.

Our FOG approach revealed two equally parsimo-

Finally,

the

data

for

1

.

Oreoialax multipunctatus

were combined with those of Xu et al. (1992: Appen-

From the characters available for all ingroupnious trees, each with 53 steps (=58 steps with HA; CIdix= 0.54, RI = 0.60). Neither tree was the same astaxa, O. multipunctatus was resolved as either the sis-

SAW evaluation but both were

11).

among our original ten MPTs. The strict consensus

group of O. schmidti or O. puxiongensi, both ofwhich were most frequently resolved as sister taxa.

tree shows two unresolved nodes (Fig. 4). For resolv-

The missing data were responsible for the ambiguity.

ambiguous nodes, a closer FOG, Oreoialax
lichuanensis, was selected. Taxa below the FOG (O.
rhodostigmatus. O. popei and O. omeimontis) wereDiscussion

resolved from ourtering theexcluded from this analysis. A single

MPT with 37=

steps was found (=58 steps with the deleted taxa; CI
0.67, RI = 0.70) (. 5). It is one of the ten MPTs fromthe original analysis.

Examination of character state

Phylogeny of Oreoialax
Oreoialax rhodostigmatus was consistently resolved
as the sister of all other species in the genus. Threecharacters (8, 10, 18;

distributions on this tree showed no potential for fur-other species.

ther resolution to the relationships of O. chuanbeien-

the

sis,

O.

liangheiensis

and

more

terminal

taxa.

Combining the results of our progressive FOG analysis, a single cladogram emerged as our preferred tree
(Fig. 6).

Thus, our tree

is

left

with one unresolved

polytomy.

Appendix I) separate it fromA BS value of 0.634 was calculated fornode excluding O. rhodostigmatus. The rugosus

group of Xu et al. (1992) also appears to be monophyletic and received a relatively high BS value(=0.732). Zhao and Adler (1993) regarded O. puxion-

gensis as a synonym of O. schmidti. Here, the highest
BS value observed from the data, BS = 0.840, shows

1994)

that these two taxa are very similar. Although further

was used to assess strength of support of monophyletic groups. The BS value based on 999 randomized
data sets was mapped on the resulting tree (Fig. 6).

detailed work is needed to determine the status of O.

puxiongensis. because O. multipunctatus is resolved
as the sister species of either O. puxiongensis or O.

Three nodes were shown to be well supported by BS

schmidti,that have values over 0.5. The O. schmidti and O. pu.x-

would also require synonymization of O. multipuncta-

Bootstrap (BS; Felsenstein 1985: Siddall

iongensis clade has a highest value of 0.840. while theO. jingdongensis, O.rugosus and O. granulosus clade

has the second highest value of 0.732. The ingroupmember excluding O. rhodostigmatus from the clade

synonymization of theselattertwo taxa

tus if a holophyletic classification is to be maintained.

Thus, we recommend recognition of all three species
until far more detailed evaluations can be made.

Vol. 7, p. 41

Asiatic Herpetological Research

1997

Functional Outgroup Method

Our progressivefunctional

outgroup methodmaymultiprove to be a powerful method for dealing withwhen levels
ple equally parsimonious trees, especiallyof homoplasy are relatively great, and the outgroup is
so distant from the in-group that mispolarization of
the character statesiscommonplace.

When diver-togences are great, and characters are constrainedalternative
states,fewamay
homoplasy
possibleonly

be so rampant that there is very little resolving power
to the data when a taxonomic outgroup is used to

431

0.634

polarizecharacter states.

Selectinga closersistergroup, as in the use of FOGs, may prove particularly
Figure 6. Phylogeny of Oreolalax as resolved by using
our progressive functional outgroup analysis. One
node remains unresolved. Bootstrap values are pro-vided for each node.

beneficial for reducing this problem. Thus, the use of

FOGs might possibly result in the defensible selectionof a single most parsimonious tree when, otherwise, asuite of equally parsimonious trees would be found.
The application of consensus tree methods has notbeen recommended as a way of selecting among

Although O. popei and O. omeimontis are grouped
together on our preferred tree (ure 6). the BS analysis

stanceequally parsimonious solutions, although thisWilkinson
has not been universally accepted (e.g.,shows a very low value of 0.06 1. They shared five1994). Our trial of this approach with the Oreolalax

characters (8, 10, 18. 21, 29). Optimizing these characters on the preferred tree shows that character 8, 10

data did not demonstrate high resolving power. The
strict consensus tree seeks the unambiguous nodes

and 18 are shared by all ingroup members excluding
O. rhodostigmatus, with 1 or 2 independent reversals.
Characters 21 and 29 are shared by all ingroup mem-

more than 2 independent reversals. These
two taxa do not share any uniquely derived characters.

bers with

We believe that this association should be considered
tenuous because of the low BS value, and the resolution of a different set of relationships for thesetaxa in ourtwo

SAW evaluation (Fig. 3). More work isneeded to determine the relationships of these species
with the others.

num-

appearing on all MPTs. It often results in large
ber of unresolved nodes. A single taxon with ambigua

ous relationships may cause the whole consensus tree
to be a total "bush". The Adams consensus method

improves this problem, but in both our case andinothers, it still frequently resolves ambiguous nodes. In

our case,strictand Adams consensus

left

6 and 5

nodes unresolved respectively. Although the majority
consensus method often gives a more complete
solution, the percentage is arbitrary and it is doubtful

rulethat all nodes reflect phylogeny.

The relationships of O. chuanbeiensis, O. liang-

Successive

approximations

weighting(Farrisheiensis and the rugosus group are unresolved. There

1969) often reduces the number of cladograms to be

are no unambiguous characters to support any resolu-

considered by restricting attention to the most consis-

tion of these three taxa.

More characters are required

for the resolution of this polytomy.

Most species of Oreolalax are distributed along

tent characters,

and hence the "best" evidence (Car-nodespenter 1988). Consequently, some ambiguousthat are supported by the "best" evidence may get

ambiguous nodes not sup-the east edge of the Qinghai-Tibet Plateau. Oreolalax

resolved. However, therhodostigmatus occurs in the far east of the QinghaiTibet Plateau and is almost allopatric in distribution

willbyremain unresolved. In our trials, SAW resolved three

from all other species. The rugosus group occurs at
highest elevations. 2100-3300m. This suggests

equally parsimonious solutions. The ambiguous position of O. popei and O. omeimontis is responsible forcommon ancestor was adapted for the rigors

these three trees. Character 28, which clusters thesethethat theirportedthesuiteof consistent charactersFOG

was notof alpine life. The speciation within the rugosus group

two taxa together undertheappears to be closely associated with the uplift ofincluded in the "best" evidence; It received a weight-

Qinghai-Tibet Plateau.

ing value ofanalysis,from the SAW analysis.

our case, the high resolving power of FOG
analysis results from the changing of the characterIn

state polarizations.

Some symplesiomorphies which

Asiatic Herpetological ResearchVol. 7. p. 42

are useless in providing evidence of common ancestry

6. Post-otic projection of squamosal either present (0),became synapomorphies after re-polarization. However, we believe that the revised data result in a better

solution. For example, the polarity of the states ofor absent

FOG,

the polarities of thestatesof Character 24).

8.

Body length of males either greater than 65 mm (0),or less then 56

mm

(

1

(

1

).

).

Opening of Eustachian tube either large
small 1 ).

9.

or

(0),

(

10. Tympanic status either obvious (0), or hiddenchanged such that a synapomorphy united O. schmidti
and O. puxiongensis with O. major.

1

Ilium either short (0), or longas the FOG. Unlike in our initial analysis, a derived

O. popei. O. omeimontis and O. liangbeiensis. Similarly, after using O. lichuanensis as our(

7.

Character 28, changes after using O. rhodostigmatusstate united

1997

(1).
1

1.

Dorsal skin either smooth (0). or warty and granular ( 1 ).

Evolution of the Characters
12.

Among the 29 characters used by Xu et al., 10 con-

Skin groove along posterior area of the dorsum
either absent (0), or present).
(

tributed no information to the phylogeny. Characters
7. 11.

1

2. 22

have autapomorphic states and hence are1

13. Belly skin either smooth (0), or granular ( 1 ).

Colour pattern on belly either presentorphylogenetically uninformative. The derived states of
1. 13. 20. 23. and 24 were resolved as inde-

14.

pendent "gains" (parallelism). Character 28 was interpreted to be two independent reversals. Conversely.

15. Triangular pattern between eyes either absent (0),absentcharacters

19 characters showed clear evolutionary trends whenmapped on our preferred

tree.

16.

However, althoughto1,characters 17,

25, 29 show a progression from state 1 to 0. These latter three characters are

ships of the taxa.

(0).

).

(

).

1

Toes either webbed (0), or either not webbed or
only a trace

(

1

).

17. Vocal sac either present (0), or absent ( 1 ).
18.

Spines in lip margins either absent (0), or present(1).

undoubtedly mispolarized by

Xu et al. if our tree reflects the genealogical relation-1

or presentcharacters 2, 3, 4. 5. 6. 8. 9. 10. 14. 15. 16. 18. 19, 21,
26, and 27 evolved from state(

19.

Spinal patches on the chest either small (0), or
large

(

1

).

20. Spines on the chest either small (0). or large

Acknowledgments
This work is supported by Natural Sciences and Engineering Research Council of Canada grant No. A3 148
to RWM, and No. OGP46464 to K. Coates. Amy

Lathrop generously assisted with the preparation of
the figures.

Appendix

4.
5.

Spoon-like cartilage either present(0), orabsentColumella either long (0). or reduced as a small
(

1

1.

23. Spinal patches on the inner side of the lower arm
(

1

).

).

or either small, diameter<1.3 mm, or absent

(

1

).

26. Labial tooth formula of tadpole either 1:7-7/1:7-7

or more (0), or 1 :6-6/l :6-6 or less ( 1 ).
27. Papillae of tadpole nostril more than one circle
clear (0), or only half circle clear ( 1 ).
28. Number of eggs laid either more than 280 (0), or
less than 220 ( 1 ).

( 1

Tympanic annulus either large. diameter>1.9 mm
(1).

1

).

Tympanum either present (0). or absent(0),(absent (0). or present ( 1 ).

Quadratojugal either long, overlapping with max-

projection

the upper arm either absent (0), or present

25. Linea musculinae either absent (0), or present

(1).
3.

(1).

24. Spinal patches on the back of the upper arm eitherI

illa (O). or short, not overlapping the maxilla.

2.

).

1

22. Spinal patches on the ventral surface at the base ofeither absent (0). or present

The Characters used by Xu et al (1992)
1.

(

21. Nuptial spines on fingers either small (0), or large

29. Egg pigment, either present (0), or absent

(

1 ).

1997

Asiatic Herpetological Research

Apendix

Vol. 7. p. 43

II

Coded Data set from Xu et al. (1992). A hypothetical ancestor (HA) was added for analysis.

HA

00000 00000 00000 00000 00000 0000

O. popei

10000 001 II 0000001 Kil 10000 0001<>

00001 001

chuanbeiensis

1

I

00010 01 100 00010 101

I

O. major

00000 00011 001 10 01

O. schmidti

10000 001 1 1 0001

O. omeimontis

00000 00101 00001 10100 10101 0001

O. puxiongensis

00000 00111 00111 11000 11001 1110

O. lichuanensis

oooooooioioooooonoi loin mi

0. rhodostigmatus

00000 01000 00000 01000 10001 001

O. liangbeiensis

00001 00111 00010 01 100 00000 1101

O. pingii

oooooooin lioioonoo loon mi

O. ntgosus

01111 10111 00000 01100 00000 11-0

O. xiangchengensis

01111 ooill 0000001110 00000 1110

O.jingdongensis

0101

O granulosus

01

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