BwlogicalJournal ofthe Linnean Society (1996), 58: 197-219. With 6 figures
Phylogenetic evolution and biogeography of
Southeast Asian shrews (genus Crociduru:
Soricidae)
MANUEL RUED1
Imtitut de <oologie et d'Ecologie Animale, Bdtiment de Bwlogk?, I015 Luusanne, Sm't.wland
and Museum of Virkbrate <oology, Univenip of Calgomiu, Berkly, C4 94720, USA.
Received I0 March 1995, accepted fw publication 14 August 1995
Genetic variation of 20 species of shrews from the Malay peninsula, Sumatra, Java, Borneo, the
Philippinesand Sulawesiwas assessed by allozyme electrophoresiis at 32 loci. According to Mantel's tests,
the genetic differentiation of these species of shrews is not a function of the geographic distance
separating them (r = 0.09,NS), but is correlated to the water depth surrounding the islands where they
live (r = 0.49,P < 0.01). The results are just the reverse if the correlations are computed for the Sunda
Shelf taxa only. In this case, the sampled populations show an isolation-by-distance relationship
(r = 0.32; P < 0.01), while no significantcorrelation with water depth was detected (r = 0.20; P = 0.07).
Qualitative predictions based on eustatic sea level variation and water depth were formulated as a model
of historic connections between the islands. This palaeogeographic model was tested through Brooks
Parsimony Analysis. The assumption of a simple vicariant evolution of the shrews was rejected, but
several concordant patterns indicate that the phylogeny of these mammals was indeed shaped by these
events. Homoplasies demonstrated that the SE Asian species of Crocidura include composite
zoogeographic histories. Sulawesi, for example, supports at least six species, five of which are closely
related, while the last one, C. n w e s , is more closely related to a Bornean taxon. This pattern was
interpreted as the result of a fmt wave of colonizers which subsequently radiated, followed by a more
recent, second colonization event from Borneo. The overall small genetic distance found within the
= 0.151 f 0.041)suggests that the radiation was not
assemblage of the five old endemics (DN
accompanied by extensive differentiation, although from a karyological point of view, they exhibit
unusual variations when compared to other Indomalayan Crdura. By contrast, the four species found
on Sumatra are more differentiated (DN= 0.221 f 0.063) and never form sister-group relationships in
any phylogenetic reconstruction; each one is more closely related to different taxa living outside
Sumatra. This suggests that they are probably remnants of an important centre of dispersal for the entire
Malay Archipelago. The standard genetic distance averaged among all Southeast Asian species
(DN= 0.235 f 0.094)is about half that measured within Palearctic or African taxa. Such an overall
lower mean level of genetic variability is consistent with the hypothesis of a relatively recent colonization
of the Malay Archipelago by shrews of the genus Crocidura.
01996 The Linncan Society of London
ADDITIONAL KEY WORDS:-Crocidurinae
Indomalayan Region.
00244066/96/060197
t 23
$18.00/0
-
197
allozymes
-
phylogeny
-
zoogeography
-
631996 The Linnean Society of London
I98
M. RUED1
CONTENTS
Introduction . . . . . . . . . . . . . . .
Material and methods . . . . . . . . . . . .
Animals and sampling . . . . . . . . . .
Electrophoresis . . . . . . . . . . . . .
Phylogenetic reconstruction . . . . . . .
Zoogeographic analyses . . . . . . . . . .
Results . . . . . . . . . . . . . . . . .
Geneticdifferentiation . . . . . . . . . .
Phylogenetic reconstruction . . . . . . .
Mantel’s tests . . . . . . . . . . . . .
Brooks Parsimony Analysis . . . . . . . . .
Discussion . . . . . . . . . . . . . . . .
Genetic differentiation and taxonomy . . . .
Phylogeny.. . . . . . . . . . . . .
Zoogeography within the Malay Archipelago . .
Acknowledgements . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . .
Appendix . . . . . . . . . . . . . . . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
.
.
.
.
198
199
199
199
201
202
203
203
206
207
208
213
213
213
215
216
217
219
INTRODUCTION
Fragmentation of the distributional range of a species presents an opportunity for
genetic differentiation and is traditionally thought to be the dominant mode of
speciation (Bush, 1975; Mayr, 1982). Causes of this fragmentation may be through
range retraction, local extinctions or vicariant events (Frey, 1992 and 1993).
Pleistocene variations in climate have shaped not only range shifts of organisms
following habitat changes (Vbra, 1992), but also have produced physical barriers
such as glaciers or marine transgressions. These changes suggested the existence of
refugia in presently continuous habitats such as in the Amazon basin (Lynch, 1988)
or the North American continent (Ellsworth at al., 1994).Although these refugia are
often invoked to be the causal origin of sibling sister species or of closely related
groups, evidence for refugia is still much debated, partly because their localization is
based on indirect palynological or glaciological reconstruction (Bermingham et al.,
1992; Zink, 1994). A way to test the existence of such refugia and to evaluate their
role in speciation events is a carefd analysis of phylogenetic patterns of co-occurring
species groups (Myers & Giller, 1988).
In this context, the Malay Archipelago lying between the Asian and Australian
continents has many ideal topological features to test the historical impact of
vicariance in speciation events. Unlike true oceanic islands which were never
connected to continental land masses (e.g. Hawaii or the Galapagos), islands of the
Sunda Shelfwere periodically connected during low sea levels (Fig. 1).At these times,
Borneo, Sumatra, Java, the Malay peninsula as well as smaller intervening islands
formed a single large peninsula dissected by big river systems (Heaney, 1991).
Alternatively, during interglacial periods the water previously accumulated in ice
caps filled the oceans and progressively raised the sea level to give the appearance of
the present-day islands (Fig. 1). Moreover, detailed knowledge of the sea level
variations as deduced from various geophysical sources (see e.g. Bartlein & Prentice,
1989) and records of the water depths separating the islands allows documentation
of the precise timing and duration of these isolation/connection episodes (Heaney,
1984; Melnick & a d d , 1985; Peterson & Heaney, 1993). From this precise record,
SE ASIAN SHREWS
199
two predictions about species differentiation and phylogenetic relationships may be
formulated: (1) the genetic differentiation between island populations of any
previously continuously distributed species should be proportional to the isolation
time of these islands, and (2) ifvicariant speciation is the dominant mode of species
formation, their phylogenetic history should be concordant with the chronology of
the vicariance events, at least on continental islands (Brooks, 1990; Frey, 1993).
In the following analyses, the first prediction of whether there is any sigtllficant
correlation between genetic distance and isolation time will be evaluated by Mantel's
test (Manly, 1991). The second hypothesis of congruent phylogenetic and
palaeogeographic evolution will be addressed by Brooks Parsimony Analysis (Brooks,
1990). The general goal of these analyses is to establish historical genetic patterns of
geographic differentiation in Southeast Asian shrews so that it can be compared with
those of other Indomalayan species.
MATERIAL AND METHODS
Animalr and sampliq
Shrews of the genus Crocidura are small terrestrial mammals distributed throughout
the Old World, from Africa and Eurasia eastwards to Japan and to the Moluccas
(Corbet & Hill, 1992). Karyological and genetic data suggest that the origin of the
genus is in the Afirotropical Region (Maddalena, 1990; Ruedi & Vogel, 1995), where
most of the about 150 species of Crocidura are found (Hutterer, 1993). According to
our present knowledge, the Malay Archipelago is inhabited by 27 species (Ruedi,
1995). The genus probably entered into this archipelago quite recently, perhaps in
late Pleistocene, but no fossil record of shrews have been discovered in the
archipelago and this estimation is based on genetic data alone (Ruedi, 1994).
In order to obtain an adequate geographic representation of the Malay shrews, at
least two different sites per major island were sampled (Fig. 1). The taxonomy of
these shrews is problematic (Corbet & Hill, 1992; Hutterer, 1993); sampling sites
were therefore selected as closest to type localities of previously described taxa. The
nomenclature of the shrews from the Philippines follows Heaney & Ruedi (1994),
that from the Sunda Shelf and Sulawesi follows Rue& (1995). Specimens were
captured with Longworth live-traps baited with prawn paste or sardines in oil (Ruedi
et al., 1990).After classical measurements, specimens were killed, skull extracted and
skin mounted on hard paper or stored in alcohol. Species sampled, localities, sample
sizes and original collection number are given in the Appendix. These 142 shrews
represent 20 of the 27 species of Crocidura presently recognized in the Malay
Archipelago (Ruedi, 1995).Ten specimens of the African species Crocidura olivien' and
seven Suncus dayi from India were also analysed. According to genetic and
chromosomal data (Maddalena, 1990; Maddalena & Ruedi, 1994; Rued & Vogel,
1995; Jenkins, Ruedi & Catzeflis, in press), these two species are distantly related
outgroups to other Euroasiatic species of Crociduru and will serve to root the trees.
Heart, liver and kidney of each animal were maintained frozen in the field in
SE ASIAN SHREWS
20 1
on a Tris-citrate pH 8.0 buffer (Ayala et al., 1972). In order to yield comparative
results with previous studies on Eurasian shrews, allozyme variation were scored by
side-by-side comparison to the same standard, C.oliuieri (Maddalena, 1990; Ruedi et
al., 1993). Alleles were designated according to their relative mobility compared to
the most frequent allele found in C. olivier.
PLylogenetic reconstmction
Individual genotypes were transformed into allelic frequencies and genetic
distances by using the BIOSYS-1 program version 1.7 (Swofford & Selander, 1989).
To find the presumed true phylogeny from allozyme data, there is no standard
approach (Swofford & Olsen, 1990);therefore, both phenetic and cladistic treatment
of electrophoretic results were performed. In both approaches, the trees were rooted
with C.olivien' and S.dayi as outgroups.
In the phenetic approach, a matrix of Nei's unbiased (DN;Nei, 1978)and Rogers'
Rogers, 1972)was computed, but because the former distance
genetic distances (DR;
is not a metric (Rogers, 1986)only the latter will be used in subsequent analyses. The
genetic relationships of the taxa were reconstructed with the distance-Wagner
procedure (Farris, 1972). This is because such tree-building method is not
constrained with equality rate of evolution along the branches and because it uses a
parsimony criterion to find the best tree (Rogers, 1986).These two conditions are not
met in the usually performed UPGMA dendrograms (Sneath & Sokal, 1973) and
may lead to erroneous phylogenetic interpretations if the taxa evolved through
unequal rates of genetic differentiation (Swofford & Olsen, 1990).Wagner trees were
produced by BIOSYS- 1 with the multiple addition criterion and with a maximum of
20 subtrees remained at each step of the growing network (the default setting of five
subtrees often failed to find the shortest tree). These settings should insure that the
most parsimonious tree under the optimality criterion will be chosen (Swofford &
Selander, 1989).The Wagner-tree procedure is, however, sensitive to the nature and
number of taxa included in the original matrix. We therefore tested the repeatability
of the results by a jackknife method of resampling over the taxa (Lanyon, 1985) to
i d e n w portions of the tree which are weakly supported and those which depend on
the taxa included. Only nodes supported in more than 80% of jackknifed replicates
were considered to have relevant phylogenetic information.
In the cladistic approach, the alleles were scored as present or absent regardless of
their frequencies. Although this coding scheme has many drawbacks (Buth, 1984;
Carpenter et al., 1993), the unequal sample sizes, the high number of taxa and loci
included, and the relatively high level of polymorphism found among SE Asian
shrews (Ruedi et al., 1993) precluded a locus-as-character approach. The presence/
absence matrix of alleles was submitted to a phylogenetic analysis using parsimony
(PAUP program version 3.1.1, Swofford, 1990). Characters were treated as
unordered and the most parsimonious solution was found through an heuristic
search. Because the alleles are not independent characters, the repeatability of the
results was not tested by classical bootstrap resampling (Felsenstein, 1985).
M. RUED1
202
Genetic differentiation between the Malay shrews may be separated into two
major components, one purely geographic and the other phylogenetic. The
geographic component may be either simply due to isolation-by-distance or due to
the more complex effect of allopatric differentiation when previously contiguous
populations became isolated on different islands (Peterson & Heaney, 1993). We
investigated these two geographic effects with Mantel’s test (Manly, 1991) using the
‘ R program package developed by Legendre & Vaudor (1991).The significance of
the correlation coefficient measured between the standardised matrices was
evaluated by 1000 randomizations. If more than 50 correlations within these
randomized matrices were higher than that calculated from the original ones, then
the correlation was interpreted as non-significant at the 0.05 level. The isolation-bydistance effect within the Malay shrews was evaluated from the matrix of Rogers’
genetic distance and a matrix of cord distance between the sampling sites (polar coordinates given in the Appendix). As the time of the last isolation for any Sunda
islands is determined by the depths of its surrounding waters (Edwards, 1993;
Heaney, 1984; Melnick & Kidd, 1985), the second effect on genetic differentiation
was calculated between the Rogers’ genetic distance matrix and a matrix of
minimum sea depth separating any two sampling sites (Fig. 1). For example,
minimum sea depth between Java and the Malay peninsula is the value found
between Sumatra and Java (61 m) because the shortest way to reach Java from the
Malay peninsula passes through Sumatra; if the taxa compared were sampled on the
same island, this value was Om. Minimum water depths were estimated on
Admiralty Charts (No 1066, 1312, 1358, 2056 and 2637) and from the literature
(Tija, 1980; Heaney, 1984 and 1986).
Finally, a Brooks Parsimony Analysis (Brooks, 1990) was performed in order to
evaluate the historical involvement of the islands in the evolution of the Malay
shrews. Under the assumption of a purely vicariant evolution, this analysis tests the
correspondence between the phylogenetic relationships of the shrews and a
palaeogeographic model (area cladogram) representing the chronology of the last
separation of the islands (Fig. 2). For the same reason as mentioned above, the
historical connections among these islands are related to the water depth
surrounding them; the palaeogeographic model was therefore simply represented by
P.MALAYSIA
SUMATRA
--_
--_
JAVA
I
PHILIPPINES
SULAWESI
I
I
I
I
I
1
BORNEO
Figure 2. Paheogeographic model showing the presumed historic separation of the Sunda islands
according to the depth of their surrounding waters. It was reconstructed by the UPGMA applied to the
log water depths. As Sulawesi and the Philippines (other than Palawan) are oceanic islands, they had no
dry-land connections with continental masses.
SE ASIAN SHREWS
203
an UPGMA cluster of the minimum water depth between the islands. For the shrews
sampled on oceanic islands like Sulawesi or the Philippines, thii minimum sea depth
just means that no connection with other land masses existed at all.
RESULTS
Of the 32 protein loci essayed, eight were invariant across all SE Asian taxa
analysed. These monomorphic loci are AK-2, GOT-2, LDH-1, LDH-2, MDH-1,
PROT, SOD-3 and XDH. The 24 remaining loci showed different levels of
variation, the most diverse being ADA (up to nine different alleles consistently
revealed) and SOD-1 (seven alleles). For space efficiency, results of allozyme
variation are presented by island in three assemblages (Tables 1, 2 and 3). Data for
the two outgroup species (C. olivien' and 5'. dayz) may be found elsewhere (Jenkinset al.,
in press). Within each island, any species recognized by morphological characters
(Ruedi, 1995) is usually defined by at least one fixed allelic variant. This is in
agreement with the expectation that sympatric or parapatric populations should
belong to different species if they do not mix their gene pools (Mayr & Ashlock,
1991). Exceptions to this observation are C. malayana from the Malay peninsula
(Table l), C. beccarii from Sumatra (Table 2) and C. elongutu from Sulawesi (Table 3).
In these cases, alleles at each locus are shared among species living on the same
region, but painvise comparisons show that each taxon has different arrays of fixed
alleles at many loci and are thus genetically independent. C. 0. orientalis from western
Java and C. 0. lawuana from eastern Java (Table 2) are also characterized by three
diagnostic loci. As the two subspecies are represented by 19 and 10 animals
respectively, these differences are unlikely to be due to sampling errors, but suggest
that they have evolved independently and should warrant specific rank. A similar
case is found between the northern and central subspecies of C. n w p e s from Sulawesi
which are also well differentiated at the subspecific level (Table 3). The three
subspecies of C.foetida from Borneo show similar discrete variations, but as two of
them are represented by single specimens, no definitive conclusion on gene flow can
be drawn. The same is true for the three allospecies sampled on the Philippines (C.
grgyi, C. mindorus and C. beatus; Table 1). Until more specimens and more localities are
sampled, a conservative taxonomy is adopted (Heaney & Ruedi, 1994).
The Nei's unbiased genetic distances computed from the allozyme variations
(Table 4) give a similar pattern of differentiation, with intra-specific comparisons
being usually under DN = 0.1 and inter-specific above this value. This is consistent
with previous estimations made on shrews of the genus Crocziiura from the West
Palearctic and African regions (Catalan et al., 1988; Catzeflis, 1983; Maddalena,
1990). A notable exception is the very low genetic distance found between largely
allopatric species. C. foetida dorim from Borneo and C. n. n w p e s from Sulawesi.
Although they differ markedly at several loci (ALB,SOD-1 and MPI; see Tables 1
and 3) the genetic distance separating them (DN = 0.052) is very low for interspecific
standards. As both have a fairly representative sample size (n = 7), this similarity is
probably not due to sampling errors, but should reflect a recent common
ancestry.
M. RUED1
204
TABLE1. Allele frequencies at 16 polymorphic protein loci among C d u m
taxa from
Peninsular Malaysia (P Mal), Borneo and the Philippines (Philip).At eight additional loci, this
subset of taxa were fixed for allele 100 at ADH, CK-1, IDH-2, MDH-2, PGI and SOD-2, for allele
150 at IDH-1, and for allele 80 at PA. An asterisk mentions the allele(s) found in one or both of
the outgroups (C. oliviffi and S. days. Abbreviation of the taxa uses the first three
letters of the species or subspecies name
C.&l C . d l C . d C . m g C.&fbc C,&hel C.fdor C.min C.gra
PMal PMal PMal PMal Borneo Borneo Borneo Philip Philip
n-9
n-6
n-5
n-6
n-1
n-1
n-7
n-1
n-9
LOCUS
Ada
Ah-1
Alb
a-2
Est
Got-1
GPd
Gapd
Hbb
Hh-1
Hk-2
Me
Mpi
Gpgd
pgm
SOAl
150
138
119
*I00
112
*lo0
*I00
94
91
88
84
*lo0
61
*112
*lo0
82
200
"100
153
*lo0
70
123
115
"I00
88
163
110
*100
100
77
45
135
120
111
"137
*100
180
140
*lo0
150
117
*100
78
"100
94
84
77
66
0.111
0.889
0.111
0.889
1.ooo
1.Ooo
0.083
0.833
0.083
0.083
0.417
0.500
0.300
0.500
0.200
1.OOO
1.OOO
1.Ooo
1.Ooo
1.Ooo
0.667
0.333
0.400
1.000
0.500
1
1.Ooo
.ooo
0.071
0.643
0.286
1.000
0.167
0.833
n-I
1.000
0.625
0.375
1.000
1.000
1.Ooo
1.000
1.Ooo
0.056
0.278
0.667
1.Ooo
0.600
0.500
1.ooo
1.000
1.Ooo
0.800
0.200
1.OOO
1.000
1.000
1.OOO
1.OOO
1.ooo
1.000
1.om
0.857
0.143
1.ooo
1.000
1.000
1.000
1.ooo
1.000
1.ooo
1.ow
1.Ooo
1.Ooo
1.000
1.000
1.Ooo
1.Ooo
1.OOO
1.000
1.ooo
1.000
1.OOO
1.Ooo
1.ooo
1.Ooo
1.ooo
1.000
1.ooo
1.ooo
1.OOO
1.ooo
1.Ooo
1.OOo
1.OOO
1.Ooo
1.om
1.Ooo
1.Ooo
1.ooo
1.OOO
1.000
1.ooo
1.000
1.Ooo
1.Ooo
0.111
0.889
1.ooo
0.500
0.500
C.bea
Philip
1.000
1.Ooo
0.111
0.333
0.111
0.889 1.000 1.000 0.667 1.000 1.000 0.889 1.OOo 1.ooo 1.ooo
0.417 0.500 0.800 1.000 1.om 1.000 1.000 1.ooo
1.000 0.583 0.500 0.500
1.ooo
0.083 0.250
1.ooo
0.917 0.750 1.ooo 1.000 1.ooo 1.ooo 1.000 1.000 1.ooo
1.om 0.800 0.700 0.417
0.500
0.200 0.300 0.583 1.ooo 1.Ooo 1.ooo 0.500 1.ooo 1.ooo
0.333 0.200 0.583 1.om 1.Ooo 1.000 1.ooo
1.000 1.ooo
0.667 0.800 0.41 7
1.000
0.278 0.083
0.071
0.722 0.917 1.000 1.000 1.ooo 1.ooo 0.929 1.ooo 0.61 1 1.ooo
0.111
0.278
0.944 0.083
0.300
0.059 0.500 0.500 1.ooo 1.om
1.000 0.700 1.000 0.056
0.417 0.250
0.250
0.556 1.ooo
0.389
SE ASIAN SHREWS
205
TABLE
2. Allele frequencies at 20 polymorphic protein loci among Crocidura taxa from Sumatra
(Sumat) and Java. At four additional loci, this subset of taxa were fixed for allele 100
at GOT-1, HBB, and PGI, for allele 80 at PA. See Table 1 for other conventions
C.@r
Sumat
n-1
LOCUS
150
138
129
119
*loo
*83
*64
32
Adh
135
*lo0
91
Ak-1 *lo0
67
Ah
*I00
94
91
88
CK-1
103
*I00
CK-2 *I00
61
Est
127
117
*112
*I00
Gpd
*lo0
88
60
G e d 123
115
HA-1
163
C.kc
Sumat
n-4
C.hut
Sumat
n-3
C.@
Sumat
n-17
*loo
Idh-1
Idh-2
100
77
70
*I50
115
110
*loo
Mdh-2 *lo0
66
Me
147
135
111
Mpi
174
*137
*loo
6Pgd 218
180
I40
*I 00
Pgm
150
117
Sod-I *I 00
94
84
77
Sod-2 '1 00
80
C . h
Java
n-6
C.o.Iaw
Java
n-10
C.0.m'
1.000
1.ow
Java
n-19
0.500
Ada
HA-2
C . m
Java
n-1
1.000
0.167
0.250
0.125
0.500
0.125
1.ooo
1.ooo
0.167
0.667
0.500
1.Ooo
0.667
0.250
0.089
1.ooo
1.ooo
0.941
0.059
1.ooo
1.ooo
1.000
1.000
0.667
1.Ooo
1.Ooo
1.000
1.000
1.ooo
1.ooo
1.000
1.om
1.000
1.om
1.om
1.ooo
1.ooo
1.Ooo
1.ooo
0.079
0.921
1.ooo
1.000
1.ooo
0.917
1.ooo
1.000
1.ooo
1.om
1.ooo
1.ooo
1.000
1.000
1.000
0.333
1.000
1.ooo
1.ow
1.000
1.000
1.ooo
1.ooo
1.ooo
1.ooo
1.000
1.000
0.333
1.ooo
1.Ooo
1.000
1.000
1
.om
1.000
1.ooo
1.000
1.000
1.000
1.ooo
1.ooo
1.ooo
1.ooo
1.000
1.ooo
1 .ooo
1 .ooo
1.ooo
0.765
0.235
0.059
0.941
0.059
0.941
1.000
1.ooo
1.ooo
1.000
1.000
1.000
1.ooo
1.ooo
1.ooo
1.000
1.ooo
1.000
1.000
0.029
0.971
0.912
0.088
1.000
1.Ooo
1.000
1.000
0.400
0.600
1.ooo
1.ooo
1.ooo
1.000
0.059
0.941
1.ooo
0.250
0.667
0.750
0.333
1 .ooo
1.ooo
1 .ooo
1.ooo
1.ooo
1.000
1.000
1.ooo
1 .ooo
1.ooo
0.083
1.000
1.ooo
1.000
1.ooo
0.667
1.ooo
1.000
0.029
0.971
1.000
1.ooo
1.ooo
1.ooo
0.450
0.550
0.361
0.333
0.306
1.000
0.083
0.917
1.ooo
0.050
0.950
1.ooo
1.ooo
0.050
0.950
1.ooo
1.ooo
1.ooo
1.000
1.ooo
1.000
1.000
1.ooo
1.ooo
1.ooo
1.ooo
1.om
1.ooo
1.000
1.ooo
1.000
1.ooo
1.ooo
1.ooo
1 .ooo
1.000
M.RUED1
206
The shortest tree found in the distance-Wagner analysis for the 25 SE Asian
populations of shrews investigated is presented in Figure 3. It has a total length of
2.516 and is a fair representation of the original distance matrix with a cophenetic
correlation coefficient of 0.88. Populations or subspecies of the same species
consistently clustered together, a result which is in good agreement with the
presumed monophyly of the taxa (Ruedi, 1994). Several poorly sampled taxa
(including C. mindorus, C.J&etidu and C.f:kehbit)have very short branches (less than
TABLE
3. Allele frequencies at 14 polymorphic protein loci among Cnn-idurutaxa from northern
Sulawesi (N Sulaw) and central Sulawesi (C Sulaw).At ten additional loci, this subset of taxa
were fixed for allele 100 at ADH, AK-1, CK-1, GOT-1, GPD, HBB, IDH-2, MDH-2, and
SOD-2, for allele 150 at IDH-1, and for allele 80 at PA. See Table 1 for other conventions
C.n.nigC.n.lip
NSulaw
CSulaw
n-7
n-15
LOCUS
Ado
Alb
ck-2
Est
G6-Pd
Hk-1
Hk-2
Me
Mpi
Pa
6-pgd
pgi
PiP
sod-1
138
119
106
*lo0
91
88
*I00
61
117
*112
123
115
110
*lo0
109
100
77
135
111
74
187
*137
*lo0
a100
*80
38
180
140
*lo0
62
200
*lo0
150
117
78
94
84
80
77
73
66
C.h
CSulaw
n-4
C.eb
CSulaw
n=2
C.rh
CSulaw
n-3
C.lca
CSulaw
n-3
1.000
0.500
0.500
1.ooo
1.000
1.000
1.Ooo
1.ooo
1.000
1.Ooo
1.000
1.ooo
1.ooo
1.000
1.OOO
1.ooo
1.Ooo
1.ooo
1.Ooo
1.Ooo
1.000
1.Ooo
1.ooo
1.Ooo
1.Ooo
1.ooo
1.ooo
0.167
0.833
1.Ooo
1.ooo
1.000
1.ooo
0.167
0.833
1.000
0.333
1.ooo
1.ooo
0.643
0.357
1.Ooo
1.Ooo
0.800
0.200
0.071
0.929
0.200
0.800
0.333
0.667
1.ooo
0.067
0.933
1.ooo
1.000
0.800
0.200
0.357
0.769
0.231
1.ooo
1.ooo
1.ooo
0.967
0.033
1.ooo
0.500
0.500
1.000
1.ooo
1.000
1.ooo
1.Ooo
1.000
1.000
1.ooo
1.om
1.ooo
1.ooo
1.om
1.ooo
1.Ooo
1.ooo
0.833
1.000
1.Ooo
1.Ooo
0.300
0.700
1.Ooo
1.Ooo
1.ooo
0.167
0.833
1.Ooo
1.ooo
0.667
1.000
1.ooo
1.ooo
0.643
1.000
C.mw
CSulaw
n-6
1.ow
1.ooo
1.ooo
1.000
0.075
0.250
0.500
1.ooo
1.ooo
0.167
1.000
1.ooo
1.om
0.500
1.om
SE ASIAN SHREWS
207
1Yo of the total length of the tree); therefore their precise position in this tree is weakly
supported in resampling tests (Lanyon, 1985).Among the well differentiated species,
C. purudoxuru and C. beccurii were the most unstable as in nearly half replicates, C.
purudoxuru was substituted to the place of C. beccurii in a basal position, while the latter
was placed among the C.n&y$es-C.&etida cluster. This instability is known to occur
with long branches (Felsenstein, 1978). The C. graYi-C. beutus cluster was also
ambiguously joined as the sister taxon of the C. mulayunu-C. negligm cluster. Within
the Sulawesian species, C.lea was placed at a more ancestral position (sister taxon of
the cluster C. mmsm*-C.elongata-C. levicula), and C. rhoditis joined the other Sulawesian
species in about 25% of replicates. The sixth Sulawesian species, C. nigntes, was
consistently placed within the Sunda species in all replicates. The relative position of
C.&l@nosa and C. monticola within the basal clade was not robust to resampling as the
latter species was more closely linked to the other SE Asian species in about 80% of
replicates. As a conclusion to the fist phenetic approach, only boldfaced parts of the
tree in Figure 3 are sufficiently robust to warrant significant phylogenetic
information.
The presence/absence coding scheme of polymorphic alleles yielded 54
phylogenetically informative characters. The heuristic search of the shortest tree
produced 16 equally parsimonious solutions, all requiring 170 steps with a low
consistency index of 0.318. The strict consensus tree resulting from this cladistic
analysis is presented in Figure 4. Again, conspecific taxa cluster together in all 16
trees. Interestingly, most ambiguous nodes identified in the Wagner tree of Figure 3
are also unresolved in the cladistic approach, as exemplified by some Sulawesian taxa
(C. rhoditis and C. lea), some Philippines species (C. grayi-c. beatus clade) and by C.
beccurii from Sumatra. In turn, C. purudoxuru is consistently placed as a sister species
of a whole cluster of Sunda species, a position which was found in about half of the
jackknifed simulations of the distance-Wagner approach. Forcing C. purudoxuru into
the same position as in Figure 3 produces eight additional steps in the tree; this
position is therefore less likely. The position of C. hutanis relative to the clade C.
oriatalis-C. brunneu is different in the phenetic and cladistic approaches; if C. hutanis is
placed as the sister-species of the latter clade, the tree is only two steps longer than
the most parsimonious solution of Figure 4.The same cost to the overall length of the
tree is found if C. nignpes is joined to C.foetida as in Figure 3. Finally, forcing C. lepidura
to be the sister species of the remaining five Sulawesian taxa has a minimal one step
cost.
In the final best estimates of the true phylogeny (Fig. 5) we retained all nodes
which were supported by either or both trees. In the three cases mentioned above the
topologies were contradictory but within one or two steps of the optimal cladistic
solution; we favoured the phenetic result. When the placement of a particular clade
or taxon was not consistently supported by the phenetic nor by the cladistic analyses,
it was left as an unresolved polytomy.
Mantel’s tests
Among the 25 populations sampled, the Mantel’s test between genetic and
geographic distances showed no significant effect of isolation-by-distance (r = 0.08;
NS), the correlation between genetic distance and minimum inter-island water depth
M.RUED1
208
is moderate (r = 0.29; P = 0.001). The phylogenetic reconstruction was unambiguous in placing the primarily continental C.*l&nosa and the widespread Sunda C.
monticola well apart from the other Malay shrews (Fig. 5). It is thus very likely that
these two species experienced different zoogeographic histories when compared to
the other Malay shrews. Excluding them from the original matrix does not increase
the relationship between genetic and geographic distances (r = 0.09; NS), but
substantially increases the correlation between genetic distance and island isolation
(r = 0.46; P = 0.001). The influence of the nine species sampled on oceanic islands
was however determinant for these trends, especially those from Sulawesi. If the
same correlation tests are restricted to the Sunda Shelfpopulations only, then the
isolation-by-distance relationships has a better support to explain the genetic
variance (r = 0.32; P = 0.005) than the water depth (r = 0.20; NS).
Brooks Parsimony Ana&-b
The genetically derived phylogeny of Figure 5 served as the basis for the present
Brooks Parsimony Analysis; the same figure indicates the areas in which species
occur according to a recent revision (Ruedi, 1995). The geographical units
considered here were Peninsular Malaysia, Sumatra, Java, Borneo, Sulawesi and the
Philippines. The palaeogeographic model giving the chronology of connections
between these areas was deduced from an UPGMA clustering of minimum water
TABLE
4. Matrix of genetic distances among 25 SE Asian shrew populations and two outgroups
(C. oliviai and S. &yS. Above diagonale Nei’s (1978) unbiased estimate and below diagonale
Rogers’ (1972)
Taxon
1 C. olivieti
2 S. duyi
3 C.fuliginaca
4 C. makayanal
5 C. m a h a d
6 C.neglip
7 C . 8 foGfidu
8 C . 8 helabit
9 C. mindorus
10 C . f d m i a e
11 C grayi
12 C. beatzcs
13 C. paradonura
14 C. beccani
15 C. hutanis
16 C. nwnticola
17 C. lepdura
18 C. o. h n a
19 C. brunnca
20 C. o. orientalis
21 C. n. lipam
22 C. n. nigrpes
23 C. elongat0
24 C. rhoditis
25 C.lea
26 C. lcuicuka
27 C. museti
1
3
2
-
-
.372
.424
.405
.403
.467
.439
4
5
--
6
7
-
8
9
-
10
11
12
-
.447 .528 .500 .495 .627 .568 .584 .531 ,522 .529 .539
-
.343
.340
.341
.410
.382
.448 .391
.410 375
.423 371
.425 371
.425 .391
.459 .359
.391 .344
.444 .411
.349 .299
.403 .373
,451 .448
.476 .398
.448 .414
.445 .376
.435 .382
.390 .366
.387 .359
.404 .346
.416 367
.366 398
.401 .384
- 296
.293 .303 .048
.362 .125
.326 .206
.303 .208
345 .158
.340 .174
363 .178
.392 .199
358 .228
.314 .158
.408 .217
.249 ,214
.352 .214
,416 .259
.386 .215
.320 .240
.349 .206
.367 .220
.367 .267
353 .226
.303 .232
360 .269
392 .304
.385 ,498 .470
302 .410 362
.Ooo .072 .180
- .072 .177
.096
.134 .207 .133 .223 .164 .063
.185 .124 .121
.181 .120 .085
.177 .163 .139
.202 .193 .179
,225 .188 .199
,162 .215 .127
.204 .189 .160
.219 .291 .293
.208 .278 .253
.263 .243 .209
.222 .209 .180
.258 .205 .126
.205 .186 .146
.211 .177 .134
.260 .322 .307
.212 .268 .252
,227 .287 ,315
.245 .305 .273
.302 .336 .315
.486
,340
,179
.190
.145
.049
-
.078
.074
.140
.188
.250
.lo5
.170
.297
.230
.249
.157
.146
.147
.123
.328
.260
.354
.281
.323
.064
-
.439
.429
.142
.125
.128
.122
.123
.lo6
.083
.133
.141
.215
.142
.139
.261
.183
.202
.142
.162
.134
.lo6
.261
.203
.276
.266
.276
.113
.153
.199
.146
.125
.253
.206
.211
.135
.182
.118
.086
.283
.220
.313
.240
.279
-
.486
.479
.116
.155
,154
.154
.170
.108
.114
.023
.088
.215
.135
.124
.270
.251
.233
.179
.233
.122
,143
.258
.192
.305
.238
.251
.219
.167
.134
.277
.234
.215
.185
.209
.182
.166
.285
,159
.292
.281
,260
.452
.397
.ll8
.147
.lo6
.110
.073
-
.445
.381
.138
.141
.077
.067
.050
.035
-
SE ASIAN SHREWS
209
depths. The resulting tree (Fig. 2) places Sulawesi and the Philippines as the first
areas to split off the Sunda Shelf. For the time scale encompassed here (Pleistocene
to recent), this is consistent with geological data because, ifwe except Palawan, these
true oceanic islands were probably never connected to the continental shelf (AudleyCharles, 1987) and should support the most divergent fauna (Musser, 1987; VaneWright, 1991). Because Java is delimited by deep ancient river systems ("$a, 1980;
Heaney, 1991 and Fig. l), it was the first of the Sunda islands considered here to be
isolated from the rest of the shelf, followed by Borneo. Peninsular Malaysia and
Sumatra are presently separated by the narrow strait of Malacca which has a
minimum depth of 21 m (Fig. 1); these areas were thus only recently isolated from
each other (Tjia, 1980) and are accordingly placed close together in this
palaeogeographic reconstruction (Fig. 2).
To reconstruct the 'biological area cladogram', the 20 current species and 14
ancestral taxonomic units identified in the tree in Figure 5 were coded as
recommended by Brooks (1990) in order to produce a matrix relating geographic
areas and phylogenetic patterns. The branch-and-bound search of the most
parsimonious solution yielded one tree of 32 steps with a rescaled consistency index
(see Swofford, 1990) of 0.47. This tree is not shown but is almost unresolved with all
areas exceptJava and Sumatra constituting a polytomy. Such a lack of resolution is
expected not only because of the few phylogenetically informative characters used,
but also may happen when the Brooks Parsimony Analysis includes widespread taxa
such as C. monticolu or includes areas such as Peninsular Malaysia or Sulawesi which
support both derived and basal species (see Fig. 5); such species may have
experienced dflerent zoogeographic histories. Following Brooks (1990), we repeated
-
-
18
.486
.446
.400
.175
.165
.286
.267
.241
.176
.193
.241
.246
.309
.158
.206
.301
.584
.583
.519
.247
.243
.242
.224
.280
.212
.220
.233
.237
.237
.276
.135
.404
.200
16
-
.479
.403
.348
.124
.119
.203
.115
.093
.125
.123
.lo5
.158
.274
.252
.218
.246
.283
,215
,185
* 240
.220
.228
.305
.308
.229
.281
.354
.565
.518
.496
.191
.165
.168
.146
.162
.117
.lo2
.080
.119
.227
.149
.167
.262
.183
.262
,179
.205
.197
.167
.253
,197
,281
.220
.260
.418
.350
.263
.202
.203
.314
.340
.344
.293
.270
.292
322
.280
.292
.336
.294 .219 .280 .152 .336 .211
.lo7 .274 .192
.176 .325 .237
.170 .275 .255
.164 .270 .178
.287 .292 .204
.200 .270 .139
.293 .225 .167
.238 .277 .172
293 .319 .242
14
~
.613
.435
.421
.217
.201
.183
.211
.288
.239
.202
.218
.247
-
-
-
17
15
13
20
-
.634
.500
.469
.190
.190
.198
.185
.162
.136
.117
.165
.197
.197
.170
.086
312
.182
.118
.122
.lo8
.241
.249
.317
,268
.244
.296
349
-
.578
.521
,361
.214
.236
.187
.114
.148
.159
.173
.230
.225
.265
.201
.159
382
.236
.lo3
.141
.147
.194
.166
345
.282
.289
.279
351
-
-
-
19
-
21
22
23
-
24
25
26
27
-
.584
.458
.420
.180
.174
.155
.130
.135
.120
.093
.loo
.175
.221
.182
.143
.299
.254
.253
.183
.256
.243
.236
332
.269
.276
.328
346
-
.564
.471
.426
.190
.181
.145
.116
.lo1
.087
.052
.lo1
.156
.234
.137
.138
.297
.161
.260
.149
.242
.077
.112
.236
.233
.314
.272
.261
.472
.425
.421
.250
.232
.334
.330
.369
.271
.298
.262
300
323
.251
.289
.315
.173
.333
.383
.360
.235
.224
.245
.173
,290
.232
.239
.475
.434
.410
.214
.189
.289
.275
.291
.215
.225
.168
.166
364
.189
.196
.305
.125
.294
.317
.296
.237
.164
.134
.495
.412
338
.207
.194
323
.366
.429
.307
.357
.333
.336
.252
.302
325
.244
.151
.261
326
305
348
319
.087
.195
.528
.453
.429
.276
.234
.336
.314
330
.301
.263
.243
.330
330
.233
.261
322
.167
.341
.320
.389
.297
.243
.lo2
.172
.137
.434
.495
.484
322
312
.384
.365
.380
.306
.310
.267
292
.428
.272
.323
.373
.250
.418
.418
.411
.275
.239
.120
.175
.211
.173
-
-
-
-
.163 .128 .190 .129 .167 .135 .140 .177 .198 .167
-
M. RUED1
210
this analysis considering these areas as composite: C. &l@nosa from Peninsular
Malaysia was transferred to a new area called ‘Continent’, C. monticola to a new
‘Sunda’ area and C. nigripes to ‘New Sulawesi’. The two equally parsimonious
biological area cladograms resulting from this matrix needed 34 steps and had a
rescaled consistency index of 0.86. One of the two solutions (Fig. 6) corresponds very
closely to the hypothesized palaeogeographic model of Figure 2 and therefore partly
supports the idea of a vicariant evolution of shrews in this region (the alternative tree
links Sumatra with Java but is otherwise identical to Fig. 6). Inconsistencies with the
palaeogeographic model are the positions of Borneo, New Sulawesi and the
Philippines. These homoplasies in Brooks Parsimony Analysis are interpreted as
colonization events which are independent of the history of vicariance events
,
and
predicted by the model (Brooks, 1990). These cases involve C.n ~ p e s C.foetida
C. mindow (species 8, 7 and 6 respectively) which all possess a common, direct
ancestor (taxon 22) which was more widespread (Fig. 6). Interestingly, the other two
closely related shrews of the Philippines (C.grayi and C. beah; species 9 and 10 in Fig.
6) evolved from a common ancestor which was also endemic to t h i s archipelago, but
as the position of the Philippines contradicts the palaeogeographic model, it is also
C.fuliginosa
C. monticola
7
C. pamdoxura
c. mlayafla1
c.maluyana3
c.
C. negligens
mindom
C. f.foetida
C. f. hlabit
C. f. doriae
-
e
C. n. nigripes
C. n lipara
C. gmyi
C. b e a u
1
C. beccarii
-.I
C. lepidum
C. rhoditis
SE ASIAN SHREWS
21 1
likely to have reached this archipelago independently of sea level changes. The
common ancestor to the two species occurring in Peninsular Malaysia (C. malayana
and C. negl&ens) is also found on the same area; both extant species thus probably
speciated in situ. The same case is found on Java and Sulawesi where the endemic
species (C. brunnea-C. orientalis, and C.rhodih to C.mmseri clade respectively) are also
the result of a local radiation. The situation of the shrews sampled on Sumatra is
more complex. The phylogenetic tree of Figure 5 indicated that the four species were
not closely related to each other but shared sister-group relationships with many
other taxa, including the old endemics of Sulawesi or ofJava. For example, the most
recent ancestor of C.hutanis (taxon 30) appears both on Java and Sumatra (Fig. 6);
according to the palaeogeographic model, this should result from an active dispersal
of the ancestor 30 from Java to Sumatra. Another example is taxon 34 which is the
common ancestor of the old endemics of Sulawesi and of the Sumatran C.lepidura.
Similarly, it should have dispersed from Sumatra to Sulawesi or was more
widespread but became extinct (or unsampled) in the other parts of the Malay
Archipelago. These are only tentative conclusions as the major shifts in vegetation on
c.malrrycur01
C. rnahyana 3
C. nsgligsnr
c.nlindom
c. f.foeti&
C. f; kslabU
C. f.dodas
- -
C. n. lipam
c.gnryi
C. b e d
C. bee&
C. hutan&
C. bmnnsa
C. 0. orlsntaus
M. RUED1
212
1- C.fuUgimsa P. Malaysia
2-C.mont&ola Sunda
3- C.paradorrua
4- C. mahyana
- 2 1 K s m C.negligcns
2 2 1
6= C.rnMorus
7- C.f o e ssp
-
25
-27-
Sumatra
P. Malaysia
P. Malaysia
Philippines
Borneo
8- C. nigripes ssp
9- c.gmyi
--28K10
C.=
beatus
24
11. C. b e c c d
Sulawesi
Philippines
Philippines
Sumatra
12-C.hutMlS
Sumatra
13. C.brunnea
Java
14- C.orlentalls ssp Java
23
I
-15-
C.kpidurrr
Sumatra
16 C. rhoditis
17- C.&a
C. levicuh
19. C. elongah
201 C.mussed
Sulawesi
Sulawesi
Sulawesi
Sulawesi
Sulawesi
Figure 5. Hypothesized phylogeny derived from the genetic variations of the Malay shrews. This tree
retains only those nodes which were supported by the phenetic and/or by the cladistic approaches. The
i
k
r was ambiguous in both reconstructions,but according to karyological investigations
position of C. M
(Rued & Vogel, 1995), it is more closely related to the other old endemics of Sulawesi than to C. h@ikra.
To the right of the species names appears their distribution range as understood in a recent revision
(Rued, 1995). The numbers on the branches refer to terminal (1-20) and to hypothesized ancestral taxa
(21-34) used in the Brooks Parsimony Analysis.
Figure 6. Biological area cladogram derived from a Brooks Parsimony Analysis of Malay shrews. To
obtain this cladogram, C.&liginaCa (sp l), C. modcola (sp 2) and C. [email protected] (sp 8) were treated separately
from other co-occurringspecies (see text). This tree is one of the two solutionsrequiring 34 steps and with
a rescaled consistency index of 0.86. The other solution unites Sumatra with Java, but according to the
palaeogeographicmodel of Fig. 2, it is a less likely configuration. Bold numbers 1 to 20 represent extant
species (see Fig. 5 for caption), while italic numbers 21-34 are hypothetical ancestral taxa.
SE ASIAN SHREWS
213
the Sunda Shelf which occurred during the Pleistocene (Heaney, 1991) may have
changed substantially the distribution of some taxa.
DISCUSSION
Genetic dfltentiation and taxonomy
As a natural test to the biological species concept, taxa should conserve discrete
gene pools when they occur in the same area (Map & Ashlock, 1991). Lack of gene
flow as evidenced by the existence of discriminant alleles at one or several loci was
demonstrated in all pairwise comparisons of sympatric taxa of SE Asian Crocidura. As
an example, in central Sulawesi we trapped during the same session up to five species
in an area of less than 5 ha of mossy forest (Ruedi, 1995). Four of them (C. n. lipara,
C. rhodih, C. laricula and C. mussen) are characterized by one to four diagnostic loci
(Table 3), the only exception being the morphologically highly divergent C. elongata
(Musser, 1987). The overall genetic independence required by the biological species
concept is therefore verified at least for the sympatric or parapatric species of shrews
analysed here. However, species status is more problematical for allopatric taxa, i.e.
found on Merent islands. Since actual gene flow is prevented by physical barriers,
the biological species concept is not applicable. The potential of two taxa to
interbreed is then a matter of speculation of the future biological interactions (if any)
of these taxa (O'Hara, 1993). Hence the definitive taxonomic treatment of some
closely related allopatric taxa (e.g. C. gryi/C. b e a h or C.jietida/C. mindow) cannot
be solved by our genetic results. We therefore adopt a conservative attitude to favour
taxonomic stability which relies on a traditional, morphological approach (Heaney &
Ruedi, 1994; Ruedi, 1995).
When compared to African or to Palearctic Crocidura, the overall genetic distance
found among SE Asian shrews is much reduced. Excluding all intra-specific
comparisons which would reduce artificially this value, the mean Nei's genetic
distance is 0.235 f 0.094 (range 0.052-0.519) for the 20 Indomalayan species. It is
therefore about half the value calculated for 15 African (0.453 f 0.183, range
0.074-1.007; Maddalena, 1990) or for six Palearctic species (0.391 k 0.1 18, range
0.038-0.545; Maddalena, 1990). If we rule out the possibility of a slower allelic
change in this group, this result suggests that SE Asian shrews are, on average, of
more recent origin than the Old World Crocidura, or that they are issued from only
part of the broader phylogenetic diversity found elsewhere. This interpretation is
consistent with chromosomal evidence which suggests an African origin for the
genus, followed by a colonization of the west Palearctic region, and finally of the
Indomalayan region (Ruedi 8z Vogel, 1995; Ruedi et al., 1993).
The historical connections of islands of the Malay Archipelago have shaped the
phylogenetic relationships of shrews living in this area as evidenced by the Brooks
Parsimony Analysis. One important feature of the phylogenetic tree presented in
Figure 5 is the basal position of two taxa, C.&l$jnosa and C. monticolu. This result is
independent of the method used to reconstruct the tree (either phenetic or cladistic).
214
M. RUED1
C. CfiLliginosa is a continental species which only marginally enters the Malay
Archipelago. This view is supported by morphological (Ruedi, 1995), karyological
(Ruedi et al., 1990; Ruedi & Vogel, 1995)and by the present phylogenetic results and
contrasts with the former belief that it is a common species widespread from the
Himalayas to the entire Sunda Shelf and adjacent islands (Corbet & Hill, 1992;
Medway, 1977;Jenkins, 1982). Our own revision of the Philippine shrews (Heaney
& Ruedi, 1994) was confused by this odd taxonomic concept. Other taxa distantly
related to C.@liginosa are in fact living within the Malay Archipelago.
The second basal Malay species of Figure 5 is C. monticolu, represented by one
individual from Java (see Appendix). This sample is clearly insufficient to support or
refute the idea that it is a widespread taxon occurring over the entire Sunda Shelf
and adjacent islands (Jenkins, 1982). However, if this is true, then C. monticola would
be the only widespread Malay shrew. This unique pattern and the case of C.@liginosu
were responsible for a failure to detect consistent zoogeographic information from a
first Brooks Parsimony Analysis on the raw data. When both taxa were treated apart,
the results were much more concordant with the predictions drawn from the
palaeogeographic model (Fig. 2).
Another important result appearing in all phylogenetic analyses is the monophyletic assemblage comprised of the old endemics of Sulawesi plus one species from
Sumatra (Fig. 5). The monophyly of the Sulawesian species within this clade is more
weakly supported, but a recent karyological survey (Ruedi & Vogel, 1995)strengthen
this view as, unlike all other Malay shrews which have a diploid number of 38
(including C. lepiduru), these Sulawesian taxa have 30 to 34 chromosomes. Despite
this unusual karyologic variation, the radiation of the old endemics of Sulawesi was
not accompanied by extensive genetic differentiation as measured by the allozyme
data (mean D N = 0.151 f 0.041). According to the continental origin of the genus
Crocidura and to the interpretation of the biological area cladogram, the ancestral
taxon 34 in Figures 5 and 6 dispersed from Sumatra to Sulawesi by ovenvater
colonization, but apparently left no descendent in between. In fact, C. ba1umui.v is an
endemic species from the higher mountains of northern Borneo which could not be
sampled for this study. As C. balm.& is morphologically barely distinguishable from
C. lepidura (Ruedi, 1995),it may be a close phylogenetic relative to this species. It thus
could represent a remnant of ancestral taxon 34, and thus represent the hypothetical
ancestral link between Sumatra, Borneo and Sulawesi.
C. n@pes is another species endemic to Sulawesi but it is only distantly related to
the old endemics living on this island (Figs 3, 4 and 5). It is not only separated by a
large genetic distance ON= 0.258 f 0.053), but has a distinct karyotype (2n = 38)
which is most similar to other Sunda species (Ruedi & Vogel, 1995). Therefore, the
interpretation of the biological area cladogram of Figure 6 indicates that C. nipipes
is a recent colonizer to Sulawesi which arrived from Borneo where its closest relative
C.&etida is widely distributed (Ruedi, 1995).
The same C.&etida is also closely related to C. mindorus which represents one of the
two species groups endemic to the Philippines (Heaney & Ruedi, 1994). In fact, one
species group is represented by a suite of allospecies spreading from Palawan (C.
puluwanasir) through the Philippines (C. mindorus),Negros (C. negr'na) and Mindanao
(C. grandis). It is not yet clear if this species group entered the Philippines from
northern Borneo and Palawan or from a southern route via the Sulu islands to
Mindanao. Comparative samples from Mindanao are still lacking in our genetic
analyses to answer t h i s question. The second species group is represented by C. gryi
SE ASIAN SHREWS
215
and C. beatus and, according to the phylogenetic tree (Fig. 5), is more distantly related
to the other species. The biological area cladogram of Figure 6 suggests that they
speciated in situ but their common ancestor (taxon 28) dispersed from the Sunda
Shelf at an earlier time; this ancestral taxon left apparently no other descendants. A
last species, C. athuata, has been recorded from the extreme northern Philippines.
From our previous view (Heaney & Ruedi, 1994), C. attenuatu most likely is a recent
introduction from Taiwan and did not enter the Malay Archipelago elsewhere.
The central role played by the four sampled Sumatran Crocidura species in the
phylogenetic tree is striking (Fig. 5). Unlike the relationships predicted by the
palaeogeographic model (Fig. 2) there is no direct link between species living in
Peninsular Malaysia and Sumatra. Rather, taxa from Sumatra are associated with
those of Java (common ancestor of C. hutanis), Sulawesi (C.lepidura) or with most
Sunda species (C. paradomra), but no special link with Peninsular Malaysia was
evidenced. One interpretation is that Sumatra functioned as a major source area for
dispersal over the rest of the Sunda Shelf, or was a refugium where descendants of
early ancestors survived to the present. Alternatively, this may reflect the existence of
missing taxa (either unsampled or extinct) or of uncertainties concerning the
hypothesised true phylogeny of Figure 5. For example, the position of C. beccarii was
highly dependent on the method of reconstruction used (compare Figs 3 and 4) and
of the other taxa included in the analysis (poor jackknife support); its phylogenetic
position is therefore still provisional.
Ambiguous or poorly resolved parts of the phylogenetic tree in Figure 5 may be
due not only to lack of resolution of the allozyme technique, but may represent true
polytomies (Hoelzer & Melnick, 1994; Purvis & Garland, 1993). These ‘hard’
polytomies reflect nearly simultaneous speciation events. In the case of C. nipipes-C.
malayana clade of Figure 5, both lack of resolution and nearly simultaneous origin
may be valid interpretations of their seemingly uniform level of differentiation. At
certain taxonomic scale, the influence of small sample size may be a serious problem
for the evaluation of genetic distances (Archie, Simons & Martin, 1989). Indeed,
when closely related species are compared, they are likely to differ in allele
frequencies subjected to sampling or stochastic errors, while this problem is less
severe when the species differ by presence or absence of fixed allelic variants.
<oogeography within the Malay Arch$elago
Shrews from continental islands show positive isolation-by-distance relationships
(r = 0.32; P = 0.005), but no simple clinal arrangement of progressively differentiated species was evidenced within the Malay shrews as a whole. Indeed, Mantel’s
tests failed to detect any significant correlation between genetic and geographic
distances, even if outsiders such as C. &l@nosa and C. monticola are excluded. By
contrast, the relationships between island isolation as measured by the depth of its
surrounding water, and genetic distance was moderate but significant (r = 0.29;
P = 0.001). Although local demographic hazards or small sample size may affect
estimation of genetic differentiation, there is no reason to believe that this ‘noise’ will
affect all sampled populations in the same direction to produce these significant
results. The magnitude of the correlation is however modest (r = 0.49 if C.&l@nosa
and C. monticola are excluded) and explains only a small part of the genetic
variance.
216
M. RUED1
This may be due to several limitations of the palaeogeographic model proposed in
Figure 2. For example, rampant dispersal during periods of dry land connections
may have obscured part of the sequence of speciation; as a result, phylogenetically
distantly related species may coexist on the same island and confound the correlation
with island isolation even if these taxa ultimately resulted from vicariant speciation.
As suggested by the biological area cladogram (Fig. 6), there is strong evidence of
both vicariance and dispersal having had an impact on the existing patterns, but
neither is sdlicient by itself to explain all the patterns.
The palaeogeographic model presented in Figure 2 accounts for the vicariance
order of areas only during one cycle of glaciation-deglaciation.However, any group
of organisms which colonized the Malay Archipelago before the last glaciation must
have experienced more than one cycle. This limitation is not necessarily problematic.
In the case of no speciation following the separation of the islands, and subsequent
extensive gene flow between formerly isolated populations, the genetic clock is reset
to zero and no coevolution between species and areas is expected. If speciation
follows one cycle of island connection/separation, the trace of this vicariance event
is imprinted in the genes of the resulting species. Any subsequent colonization or
redistribution of the taxa would confound the correspondence between phylogeny
and history of island separation. It is only if exchanges between islands are limited
that the corresponding trace of this speciation should be detectable in their
phylogenetic history.
Another problem of the proposed model is the non-linear distribution of water
depths versus time of isolation. For the f i s t 120m of water depth, this relationship
is nearly linear (see e.g. Bartlein & Prentice, 1989) and should account fairly well the
chronology of separation of continental islands. But the fluctuations of sea levels
during the late Pleistocene probably did not exceed much this value (Heaney, 1991).
Therefore beyond this critical value, no dry land connection is expected at all, and
shrews from Sulawesi and the Philippines, for example, must have reached these
oceanic islands by overwater colonization. Although these events may have been
promoted by low sea levels which expanded exposed land masses, they were
independent of any vicariant event.
Although shrews are small terrestrial mammals with high energy requirements
(Genoud, 1988), they have surprisingly good colonizing abilities (Heaney & Ruedi,
1994) and are found from the sea level to the top of the highest mountains. Such
vagility, and the presumed recent origin of the genus Crociduru in the Malay
Archipelago, explain part of the disagreement with predicted patterns. As an
independent test and in order to compare if the patterns observed among shrews are
common to terrestrial mammals, other taxa of different age and ecology should be
analysed as well.
ACKNOWLEDGEMENTS
This study was carried out as part of a Ph.D thesis under the supervision of Prof.
P. Vogel. Special thanks are addressed to M. Chapuisat, D. Iskandar, G. Dandliker,
T. and C. Maddalena, F. Simon, and A. Rue& for their help in the field work.
Authorizations for trapping in the different national parks were obtained from Prof.
H. S. Yong and the leaders of the Sarawak and Sabah National Parks (Malaysia),the
Indonesian Department of Forestry (PHPA), the Indonesian Institute of Sciences
SE ASIAN SHREWS
217
(LIPI) and the Institute of Technology of Bandung (Indonesia). Tissues from the
Philippines shrews were kindly donated by L.R. Heaney at the Field Museum of
Natural History (Chicago).Laboratory assistance was provided by N. Di Marco.J. L.
Patton and P. Vogel offered valuable comments on early versions of the manuscript.
Costs of the field work were partly supported by the following Swiss institutions:
AcadCmie suisse des Sciences naturelles, Basler Stiftung fur wissenschaftliche
Forschung, the Georgine Claraz fundation, the SociCtt Acadkmique vaudoise and
the Institut de Zoologie et d’Ecologie Animale at the University of Lausanne.
REFERENCES
ArchieJW, S h o n C, Morda A. 1989. Small sample size does decrease the stability of dendrograms calculated
from allozyme-frequencydata. h l u r i o n 43: 678-683.
Audley-Charles MC. 1987. Dispersal of Gondwanaland relevance to evolution of the angiosperms. In:
Whitemore TC, ed. Bwgeop$hual Euolurion ofh Mahy Archipekgo. Oxford Clarendon Press, 5-25.
Ayah F, PowellJR, Tracey ML, Mourano CA, Perer-Splu 8. 1972. Enzyme variability in the Drosqbhla
zudlistoni group. IV. Genic variation in natural population of Dracophila willistuni. Genetits 70: 1 13-1 39.
Btutlein PJ, Prentice IC. 1989. Orbital variations,climate and paleoecology. T i in Ecobgy and Evolution 4:
195-199.
Be&+
E, Rohwer 8, Freeman 8, Wood C. 1992. Vicariance biogeography in the Pleistocene and
speciation in North American wood warblers: A test of Mengel’s model. fiocdings of hNiationalA c h y ofS&nct,
USA 89: 6624-6628.
Brewer CJ. 1970. A n introduction to i r o p technics. New York Academic Press.
Brooks DR. 1990. Parsimony analysis in historical biogeography and coevolution: methodological and theoretical
update. SystnatiC <oobgy 39: 14-30.
Bush CL. 1975. Models of animal speciation. A d Revim ofEcology and SySlrmalicJ 6: 339-364.
Buth DC. 1984. The application of electrophoretic data in systematic studies.AnnualRcvinu of&ology and &stmatics
15: 501-522.
Carpenter JM,Strassmpnn JE, Turilh.d 8, Hufiea CR, S o h CR, Cervo R. 1993. Phylogenetic
relationshipsamong paper wasp social parasites and their hosts (Hymenoptera: Vespidae, Polistinae). Claditits 9:
12S146.
CatalanJ, Poitevin F, Foru R, Cuerp.imov 8, Croset H. 1988. Biologie hvolutive des populations ouesteurophennes de crocidures (Mammalia, Insectivora). III. Structure ghnhtique des populations continentales et
insulaires de Crocidura ~ s s u l a( H e m , 1780) et de Crocidura suaveoh (PaUas, 1811). Mmnmalia 52: 387-400.
Catzetlis F. 1983. Relations gknttiques entre trois espCces du genre C d u r a (Soricidae, Mammalia) en Europe.
Mammalia 47: 229-236.
Corbet CB, Hill JE. 1992. fi manunah of h Indmkayan rgion: a y s d revim. Natural History Museum
Publications. Oxford Oxford University Press.
Edwards SV. 1993. Mitochondrial gene geneaology and gene flow among island and mainland populations of a
sedentary songbird, the grey-crowned babbler (htos&mw&tn@&). Euolurirm 47: 1 118-1 137.
EUworth DL, Honeycutt RL, S h y
BiclrhpmJW, Klinwtra WD. 1994. Historical biogeography and
contemporary patterns of mitochondrial DNA variation in White-tailed deer from the Southeastern United
States. Euolurion 48: 1 12-1 2 I .
F d s JS. 1972. Estimating phylogenetic trees from distance matrices. AmniGM N&r&t106: 645468.
Felsenstein J. 1978. Cases in which parsimony and compatibility methods will be positively misleading. SySrcmatiC
<oology 27: 401L4.10.
Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. EvoluLion 39:
w,
783-791.
Frey JK. 1992. Response of a mammalian faunal element to climatic changes. Joumal ofMmnma&gy734 43-50.
Frey JK. 1993. Modes of peripheral isolate formation and speciation. SyStcmnriC Biologv 42: 373-381.
Cenoud M. 1988. Energetic strategies of shrews: ecological constraints and evolutionary implications. Mmnmal
RGvinu 18: 173-193.
Harris H, Hopkinson DA. 1976. Handbook ofclcch.ophmcsir in human genetics. Amsterdam: North-Holland
Publication Co.
Heaney LR. 1984. Mammalian species richness on islands of the Sunda Shelf, Southeast Asia. Occologia 61:
11-17.
Heaney LR. 1986. Biogeography of mammals in SE Mia: estimates of rates of colonization, extinction and
speciation. BwlogicalJoumal ofh Linncrm soCic3,28: 127-165.
Heaney LR. 1991. A synopsis of climatic and vegetational change in Southeast Asia. ClimclriG Change 19:
53-61.
218
M. RUED1
Hepney LR, R d M. 1994. A preliminary analysis of biogeography and phylogeny of Crodwa from the
Philippines. SpGEial Atblication oftht Camcgic Museum ofN&al Histoy 18: 357-377.
Hoelzel GA, Melaick DJ.1994. Patterns of speciation and limits to phylogenetic resolution. Trends in Ecology and
Euolurion 9: 104-107.
Hutterer R. 1993. Order Insectivora. Ln: Wilson DE, Reeder DM, eds. Mammol speck of& WwU.A tuxmomic and
geographic r&rcnce. Washington: Smithsonian Institution Press, 69-130.
Jcnulu PD. 1982. A discussion of the Malayan and Indondesian shrews of the genus C d u r a (Insectivora:
Soricidae). &dgishe Mc&lingen, Lcidm 56: 267-279.
JcnuluPD,Ruedi M, Catzefli. FM. in press. A biochemical and morphological investigation of Suncuc h y i
(Dobson, 1888) and discussion of relationships in Suncur Hemprich & Ehrenberg, 1899, Cm&
Wagler, 1832,
and SyliuirorGx Thomas, 1904 (Insectivora: Soricidae). Bonner zwhgirche &&I@.
h y o n SM.1985. Detecting internal inconsistencies in distance DNA. syslrmotic +hgy 34: 3 9 7 4 3 .
Legendre P, Vaudor A. 1991. L+l
R - h h s e m u l h t i o n n e l h , anahse sp&. MontrCak DCpartement
de sciences biologiques, UnivenitC de MontrCal.
L p c h J D . 1988. Refugia. In: Myen AA, Gder PS, eds. Analytical BWgeogr@hy: An integrated approach to tht srudy of
animal muiphnt dishibuhnr. London: Chapman & Hall,31 1-342.
Maddalean T. 1990. Systematics and biogeography of Mrotropical and Palaearctic shrews of the genus Cduura
(Insectivora:Soricidae): An electrophoretic approach. In:Peters G, Hutterer R, eds. Ve&br& in tht f~opics.Bonn:
Museum Alexander Koenig publication, 297-308.
Maddalean T, Rue& M. 1994. Chromosomal evolution in the genus Crocidura (Soricidae, Insectivora). Special
A b l i c h n ofL Cam& Mweum OfNiral Histoy 18: 335-344.
Manly BFJ. 1991. Ranabmk&m and Monte C a ~ hm
c
W in biology. London: Chapman & Hall.
Mayr E. 1982. Speciation and macroevolution. Evolution 36: 1119-1 132.
Mayr E, Amhlock PD. 1991. FbncipICs of@shn& Zoohgy. New York McGraw-Hill Int.
Medwoy L. 1977. Mammals of Borneo. Field keys and an annotated checklist. Monograph ofthe Mhyan Branch of
L RpdAsiotic SoGiCfy 7: 1-172.
M e l a i d DJ,Kidd KO,1985. Genetic and evolutionaryrelationships among Asian macaques. Inhational3ournal
OfRimatoh~6: 123-160.
Musscr GG. 1987. The Mammals of Sulawesi. In: Whitemore TC, ed. Bwgeographical Evolution ofthe Malay
Arch@hgo. Oxford: Clarendon Press, 73-91.
Myers AA, Ciller PS. 1988. Analytical B~gtwgraphy:An intcgrafcd appmuh to tht srudy ofanimal and p h t dishibutionr.
London: Chapman & Hall.
Nei M.1978. Estimation of average heterozygosity and genetic distance from a smallnumber of individuals. Genetics
89: 583-590.
O'Hara RJ. 1993. Systematic generalization, historical fate, and the species problem. S y s d Bwhgy 42:
231-246.
Peterson AT, HePney LR. 1993. Genetic differentiation in Philippine bats of the genera GJn0ptm.s and
Haphychis. BwhgicalJoutnal o f h Litman So&& 49: 203-218.
Purvis A, Garland TJr. 1993. Polytomies in comparative analyses of continuous characten. Systmutic Bwhgy 42:
569-575.
RogersJS. 1972. Measures of genetic similarity and genetic distance. Stdies in Genetics, UnivnSty ofTexas Publications
7214: 145-153.
Rogers JS. 1986. Deriving phylogenetic trees from allele frequencies: A comparison of nine genetic distances.
& s ~ I & @logy 35: 297-310.
Ruedi M. 1994. Zmgbgraphie et taxonomie des CrocidurinCs d'Asie du Sud-Est (Insectivora, Mammalia). Ph.D
dissertation, University of Lausanne, Switzerland.
Ruedi M. 1995. Taxonomic revision of C d u r a shrews from the Sun& Shelfand Sulawesi with description of two
new species (Mammalia: Soricidae). ~oah&alJournal oftht Linnean
115: 21 1-265.
Ruedi M, Maddalean T, Vogel P, Oblro Y. 1993 Systematicand biogeographic relationships of the Japanese
d ofMmntnalogy 74: 535-543.
white-tmthed shrew (Cmdura ~ w n r ) 3.
Ruedi M, Maddalean T, Yong HS, Vogel P. 1990. The Cmdurafirligkosa species complex (Mammalia:
Insectivora)in Penninsular Malaysia: Biological, karyological and genetical evidence. Biochemical h ha tics and
Ecohgy 18: 573-581.
Rue& M,Vogel P. 1995. Chromosomal evolution and zoogeographic origin of southeast Asian shrews (genus
Crodura). Expnimh 51: 174-178.
Selpnder RK, Smith MH,Ynng SY,Johnson WE, Gentry JB. 1971. Biochemical polymorphism and
systematics in the genus P n a y s w . I. Variation in the oldfield mouse ( P n a y s w polionotus). Srudies in GmcriGs W,
l 3 e Uniwsity of fixas Atblicahn 7103 49-90.
Sneath PH,S o u RR. 1973. Numnical faxrmmny. San Francisco: Freeman & Co.
Swofford DL. 1990. PAL?. fiyhgcnetic Analysis Using Pwsimoy, Version 3.0. Computer program distributed by the
Uiois Natural History Survey, Champaign, Illinois.
Swofford DL, Olsen GH. 1990. Phylogeny reconsmction. In: Hillis DM, Moritz C , eds. Mohcular Systoatics.
Sunderland Sinauer, 41 1-501.
Swofford DL,Selmder RB. 1989. BIOSYS-1.7. A computer program for the analysis of allelic variation in
population genetics and biochemical systematics. Illinois NaNaktra.4His@ S q .
SE ASIAN SHREWS
219
Tjia HD. 1980. The Sunda Shelf, Southeast Asia. <&chnjlJirr Ceomwpholo~N.F. 24: 405427.
Vane-Wrifit RI. 1991. Transcending the Wallace Line: do the western edges of the Australian region and the
Australian plate coincide?Awfralian Systrmaric Bohy 4: 183-197.
Vbra ES. 1992. Mammals as a key to evolutionary theory. J o u m l of Mmnmalogv 73: 1-28.
Zinlr RM. 1994. The geography of mitochondrial DNA variation, population structure,hybridization, and species
h i t s in the Fox sparrow (Passarelhi l k u ) . Euolution 48: 96-1 11.
APPENDIX
Species, localities (see also Fig. I), sample sizes and animal numbers for the specimens used in the electrophoretic
study. These specimens are deposited at the museums of natural history in Bogor (Indonesia), in Leiden (Holland),
in Chicago (USA) and in Geneva (Switzerland). IZEA is the acronym of Institut de Zoologie et d'Ecologie Animale,
University of Lausanne, Switzerland.
CrociduraJirliginosa. - Peninsular Malaysia: Tana Ratah at 1500m altitude (locality 1: 4'30'N, 101'25'E),
Cameron Highlands (n = 9; IZEA 3542,3553-5,3610,2621,3747,3752-3). C. mnlaym. - Peninsular Malaysia:
Maxwell's Hill at 1 150 m alt. (loc. 2: 4'57'N, 1OO048'E), Perak (n = 5; IZEA 399 1-5); ulu Gombak at 200 m alt. (loc.
3: 3'21'N, 101'45'E), Selangor (n = 6; IZEA 3550-1, 3620, 3977-9). C. &ar. - Malaysia: Tekek at 10m (loc.
4: 2O54'N, 104'09'E), Tioman Island (n = 6; IZEA 355740, 3563-4). C. focrida. - Borneo: Niah Caves N.P.
headquarters at 150m alt. (loc. 5: 3O54'N, 113O44'E), Sarawak, Malaysia (n = 1; IZEA 3980). C . j kclnbit. - Borneo:
Bareo airstrip at 1050m alt. (loc. 6: 4O00'N, 115'38'E), Sarawak, Malaysia (n = 1; IZEA 3981). C . j d w k . Borneo: Mt Kinabalu N. P. headquarters at 1500m (loc. 7: 6'00'N, 116'37'E), Sabah, Malaysia ( n = 7; IZEA 3950,
3986-90,4625). C. gruy'. - Philippines: Mt Isarog at 1200 m alt. (loc. 8: 13O06'N, 123O43'E), Luzon (n = 9; SMG
2315-16, LRH 4067, 4110, 4137, 4161, 4166, 4172, 4178). C. beatus. - Philippines: Naval at 850m alt. (loc 9
12O22'N, 122'37'E), Biliran island (n = I ; EAR 1484). C. mindow. - Philippines: Mt Guitinguitin at 325 m alt. (loc.
10: I 1°37'N, 124'25'E), Sibuyan island (n = 1; SMG 2878). C. hutanir. - Sumatra: Ketambe at 300m (loc. 11:
3'31'N, 97O46'E), Mt Leuser N. P., Indonesia (n = 3; IZEA 4426, 4431-2). C. pmadomru. - Sumatra: Mt Tujuh at
2200m (loc. 12: lo40'S, 1Ol016'E),Mt Kerinci N.P., Indonesia (n = 1; IZE4 4503). C. beccm?. - Sumatra: Mt
Kerinci and Mt Tujuh at 1710-2000m (loc.12: l040'S, 101°16'E), Indonesia (n = 4; IZEA 4475,4500-1,4506). C.
lepidura. - Sumatra: Mt Kerinci and Mt Tujuh at 1700-2200m (loc. 12: Io40'S, 101'16'E), Indonesia (n = 17;
IZEA 4444-5, 4454-8, 4460, 4462-5, 4467, 4476, 4478, 4480, 4502, 4504). C. monticola. - Java: Mt GedePangerango at 2200 m alt. (loc. 1 3 6'26'S, 106'48'E), near Cibodas, Indonesia (n = 1; IZEA 4580). C. 6. bmnncn. Java: Mt Gede-Pangerango at 1250-1450m alt. (loc. 13: 6O29'S, 106'48'E), near Cibodas, Indonesia (n = 6; IZEA
4542, 4544, 4548-50, 4555). C. o. Wim#alir. - Java: Mt Gede-Pangerango at 1450-2450m alt. (loc. 13: 6'29'S,
106'48'E), near Cibodas, Indonesia (n = 19; IZEA 4551, 4553, 4556-8, 4560, 4562-4, 4566-8, 457&4, 4581,
4583). C. o. h u a n a . -Java: Ran0 Pani at 2000-2200m alt. (loc. 1 4 8O13'S, 113"50'E), Mt Bromo-Semeru N. P.,
Indonesia (n = 10; IZEA 4512-3, 4520, 4522, 4524, 4526-7, 452S30, 4538). C. mussm'. - Sulawesi: Mt
Rorekatimbo at 2200m alt. (loc. 15: lo16'S, 120'15'E), Lore Lindu N.P., Indonesia (n = 6; IZEA 4392, 4395,
4398-9, 4403-4). C. rhoditis. - Sulawesi: Mt Rorekatimbo at 2200m alt. (loc. 15: 1'16'S, 12Oo15'E), Lore Lindo
N. P., Indonesia (n = 3; IZEA 4401, 4406-7). C. leu. - Sulawesi: Mt Rorekatimbo at 2200m alt. (loc. 15: lo16'S,
- Sulawesk Kamarora at 800m
120°15'E), Lore Lindu N. P., Indonesia (n = 3; IZEA 4393-4, 4402). C. &la.
and Danau Tambling at 1700m alt. (loc. 15: lo16'S, 120°15'E), Lore Lindu N. P., Indonesia (n = 4; IZEA 4380,
4385, 4390-1). C. elonguta. - Sulawesi: Kamarora at 800m and Mt Rorekatimbo at 2200m alt. (loc. 15 1'16'S,
120"15'E),Lore Lindu N. P., Indonesia (n = 2; IZEA 4365,4396). C. n@@s liparu - Sulawesi: Kamarora at 800 m,
Danau Tambling at 1700m and Mt Rorekatimbo at 2200m alt. (loc. 15: 1'16'S, 120"15'E), Lore Lindu N.P.,
Indonesia (n = 15; IZEA 4366-7, 4370, 4372, 4374, 4377-8, 4381-4, 4386, 4388-9, 4400). C. n. n@pes. Sulawesi: Toraut at 380m (loc. 16: 0°41'N, 124'08'E), Dumoga Bune. N. P., Indonesia (n = 7; IZEA 4412, 4414,
4417, 44214)