Tenrec Phylogeny and the Noninvasive Extraction of Nuclear DNA

Abstract

Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses and disturbance of natural populations.

Full text

Syst. Biol. 55(2):181–194, 2006
Copyright
c
 Society of Systematic Biologists
ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150500433649
Tenrec Phylogeny and the Noninvasive Extraction of Nuclear DNA
ROBERT J. ASHER,
1
AND MICHAEL HOFREITER
2
1
Museum f
¨
ur Naturkunde, Invalidenstraße 43, D-10115 Berlin, Germany; E-mail: robert.asher@museum.hu-berlin.de
2
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
Abstract.—Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living
and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum
specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships
proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences
of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly
of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods
favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island
paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to
rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses
and disturbance of natural populations. [Afrotheria; DNA; fossils; Madagascar; museums; phylogeny; Tenrecs.]
With the exception of a single genus of shrew (Sun-
cus), insectivoran-grade mammals from Madagascar
are members of the family Tenrecidae (Eisenberg and
Gould, 1970; Olson and Goodman, 2003). This group
of placental mammals consists of eight genera endemic
to Madagascar and two from equatorial Africa and
is remarkably diverse, occupying terrestrial, semiarbo-
real, fossorial, and semiaquatic niches. Other Malagasy
groups are similarly diverse; previous morphological in-
vestigations of its primates (Cartmill, 1975; Yoder, 1992),
carnivorans (Veron, 1995), and rodents (Ellerman, 1940,
1941), as well as its tenrecs (Butler, 1984; Asher, 1999,
2000), have indicated multiple sister-group relationships
with mainland taxa within each group.
Given the absence of modern taxa from the Mala-
gasy Cretaceous and the isolation of Madagascar from
other landmasses over the past ca. 80 to 90 million years
(Krause, 2003), dispersal has become the primary hy-
pothesis for explaining the arrival of many of Madagas-
car’s inhabitants (cf. Raxworthy et al., 2002; Zakharov
et al., 2004). Phylogeny can further illustrate the biogeo-
graphic history of a given group. Monophyly (Fig. 1A)
and paraphyly (Fig. 1B) of island taxa are compatible
with a single dispersal event leading to island coloniza-
tion, whereas polyphyly (Fig. 1C) implies multiple colo-
nization events.
The aforementioned morphological studies noting the
diversity of Malagasy mammalian groups have to vary-
ing degrees implied island polyphyly (Fig. 1C); i.e., that
each of the modern groups has undergone multiple dis-
persal events across water barriers in order to colo-
nize the island. In contrast, recent molecular phyloge-
nies of terrestrial Malagasy mammals have supported
island monophyly (Fig. 1A) for living primates (Yoder
et al., 1996), carnivorans (Flynn et al., 2005), tenrecs (Ol-
son and Goodman, 2003), and possibly rodents (Jansa
and Weksler, 2004; Steppan et al., 2004; see discussion
below).
Many tenrecid species are rare and/or endangered
(Vogel, 1983; Benstead and Olson, 2003) and are diffi-
cult to obtain for research purposes. For example, the
semiaquatic Limnogale mergulus is known from barely
over a dozen museum specimens in Europe and North
America. Destructive sampling of such material (e.g., for
DNA sequencing) is generally not possible. Because it
is so difficult to obtain tissues, most molecular studies
sampling this group (e.g., Emerson et al., 1999; Mouchaty
et al., 2000; Douady et al., 2002; Malia et al., 2002) have
included between one and five of the over two dozen
species. Olson and Goodman (2003) described a much
better sample and were the first to publish a study with
representatives of all living tenrecid genera, including
sequences from one nuclear (vWF) and three mitochon-
drial (12S, tRNA-Valine, ND2) genes. However, as of this
writing (August 2005), their DNA sequences and align-
ments are unavailable from public sources (e.g., Gen-
Bank). No published study has yet cladistically analyzed
the three recognized fossil tenrecids, Erythrozootes, Pro-
tenrec, and Parageogale (Butler, 1984; McKenna and Bell,
1997). Jacobs et al. (1987) named a fourth fossil genus,
Ndamathaia. However, we follow Morales et al. (2000) in
regarding this taxon as a non-tenrecid.
In this article, we provide new DNA sequence data
from the nuclear growth hormone receptor (GHR) gene
using a noninvasive procedure applied to museum speci-
mens. We also include a morphological data set, enabling
us to sample all recognized living and extinct tenrecid
genera. To reconstruct phylogenetic trees, we apply both
maximum parsimony (MP) and a Markov k (Mk) model
(Lewis, 2001) in a Bayesian framework (Nylander et al.,
2004). Using these data we estimate the fit of living and
fossil tenrecs to phylogenetic and biogeographic patterns
proposed for other Malagasy groups.
M
ATERIALS AND METHODS
The Noninvasive Extraction Method
We obtained between 756 and 855 base pairs from
exon 10 of the growth hormone receptor (GHR) gene
from crania accessioned at the Zoologisches Museum
Berlin (ZMB), Harvard Museum of Comparative Zool-
ogy (MCZ), and the Department of Ecology and Evo-
lution, University of Lausanne (IZEA). Specifically, we
used skulls of Hemicentetes semispinosus (ZMB 71599),
181

182 SYSTEMATIC BIOLOGY VOL. 55
FIGURE 1. Biogeographic implications of (A) monophyly, consis-
tent with a single dispersal event to colonize Madagascar (cf. Eisenberg,
1975; Olson and Goodman, 2003); (B) paraphyly, consistent with a sin-
gle dispersal event coupled with limited back-migration from Mada-
gascar to Africa (cf. Butler, 1985); and (C) polyphyly, consistent with
multiple dispersal events between Africa and Madagascar (cf. Asher
2000: fig. R1-12). Dotted lines in B and C indicate uncertainty in the
positions of Erythrozootes and Protenrec.
FIGURE 2. Lateral view of crania in Micropotamogale (top, IZEA
4975), Limnogale (middle, ZMB 35258), and Setifer (bottom, ZMB 44586),
used for noninvasive extraction of nuclear GHR sequences. Images
were taken after DNA extraction. Boxes highlight patent lacrimal fora-
men in Setifer, and absence thereof in Micropotamogale and Limnogale.
Limnogale mergulus (ZMB 35258; Fig. 2), Potamogale velox
(ZMB 46588), Setifer setosus (ZMB 44586; Fig. 2), Geogale
aurita (MCZ 45044), and Micropotamogale lamottei (IZEA
4975; Fig. 2). New sequences were aligned with previ-
ously published GHR sequences (Malia et al., 2002; Ad-
kins et al., 2001; Pantel et al., 2000; van Garderen et al.,
1999; Zogopoulos et al., 1999; Wang et al., 1995; Adams
et al., 1990; Baumbach et al., 1989; Smith et al., 1989; Le-
ung et al., 1987). Table 1 shows GenBank accession num-
bers for extant taxa, including DQ202287 to DQ202292,
for our new sequences.
Expanding upon the method of Rohland et al. (2004)
for mitochondrial DNA, we obtained nuclear GHR se-
quences from museum crania, leaving the treated speci-
mens completely intact. We incubated either lower jaws
or rostra in 20 mL of a buffer containing 5 M guanidinium
isothiocyanate, 50 mM Tris, pH 8.0, 25 mM NaCl, 1.3%
Triton-X, 20 mM EDTA, and 50 mM DTT. To minimize
the possibility of damage, we incubated the specimens
at room temperature and rotated them in near-vertical
tubes that permitted flow of the buffer but kept speci-
mens stationary. DNA was then eluted from the buffer
and the specimens washed and dried as described in
Rohland et al. (2004). The DNA was eluted in a final vol-
ume of 200 µL1×TE. PCR amplification was done using
2 units of Taq Gold and 60 cycles under the conditions de-
scribed in Hofreiter et al. (2002). Depending on the taxon,
we used seven to nine primer pairs to amplify GHR se-
quences (Tables 2, 3). When possible, we designed at least
one primer per primer pair that selected against human
GHR sequence to avoid amplification of contaminating

2006 ASHER AND HOFREITER—TENREC PHYLOGENY 183
T
ABLE
1. Taxon sample and accession numbers of taxa used in our
sample of nuclear GHR sequences. Boldface indicates new GHR se-
quences; daggers indicate extinct taxa. For nomenclature we follow
Nowak (1999) and Asher (2005).
Supra-generic Accession
High-level clade clade Genus number
Didelphimorphia Didelphidae Monodelphis AF238491
Artiodactyla Bovidae Bos X70041
Capridae Ovis M82912
Suidae Sus X54429
Carnivora Canidae Canis AF133835
Ursidae Ursus AF392879
Chiroptera Phyllostomidae Artibeus AF392895
Pteropodidae Pteropus AF392893
Vespertilionidae Myotis AF392894
Hyracoidea Procaviidae Procavia AF392896
Lipotyphla Erinaceidae Echinosorex AF392887
Erinaceus AF392882
Soricidae Blarina AF392880
Crocidura AF392884
Sorex AF392881
Suncus AF392888
Talpidae Parascalops AF392883
Macroscelidea Macroscelididae Elephantulus AF392876
Perissodactyla Equidae Equus AF392878
Primates Cercopithecidae Macaca U84589
Papio AF150751
Hominidae Homo X06562
Proboscidea Elephantidae Elephas AF332013
Loxodonta AF332012
Rodentia Geomyidae Geomys AF332028
Muridae Mus M33324
Rattus X16726
Scandentia Tupaiidae Tupaia AF540643
Sirenia Trichechidae Trichechus AF392891
Tenrecoidea Chrysochloridae Chrysospalax AF392877
Geogalinae †Parageogale —
Geogale DQ202287
Oryzorictinae Limnogale DQ202289
Microgale AF392885
Oryzorictes AF392886
Potamogalinae Micropotamogale DQ202290
Potamogale DQ202291
Protenrecinae †Erythrozootes —
†Protenrec —
Tenrecinae Echinops AF392889
Hemicentetes DQ202288
Setifer DQ202292
Tenrec AF392890
Tubulidentata Orycteropodidae Orycteropus AF392892
Xenarthra Myrmecophagidae Myrmecophaga AF392875
T
ABLE
3. Primers used to obtain tenrec GHR sequences.
Primer fragment Sequence
F1g G AAT TCAACAATGATGACT CTT GG
R1g GAATGT CAG GTT CATAAC AAC TGG TAC
R1gap AT CAT CAT CCT TTG CCC CA
F2 GCT TCTAAS CAT TGA CCT GC
F2a CTAAGC ATT GAC TYD CAAAAATCA CT
R2 TG GTC AAG GCA CAA GAG ATC TA
F2g G ATC RGA CAC AGA CAG RCT TCTAA
R2g TTGATT CTT CTG GTC AAG GCA C
R2.2s right GGT CAA GGC ACAAGA GAT CA
F3 GTGACATGT GTGATG GTACCT CAG AGG TG
R3 A KGA GCT GAC TCA GAY CCA
F3g ACA RAG GTT RAAAGG GGAAG
R3g TG GGC ATAAAA GTC GAT GTT TG
F4 TAG CTTACT GTC TMYWGAYRC TG
R4 CGG GGAAAG GAC CAC ACT C
R4gap1 GG GAC ATC CCT GCT TTAAG
F4gapg CAG GTAAGC GAGATTACA CCA G
F4.1g CAATAC CAC TTC TTAATG GTG GAT C
R4.1s right CA CTG GAATAT CCC TGC TTTAAG
F5 GAC TTT TAT GCC CAG GTAAGC GAG
F5g GC AGG GAG TGT GGT CCT TTC
R5g CT GTG GTG ATG TAA CTG TCT TCC TG
R5gap TGG TAA GGC TTT CTG TGG TGA
F6 A CCT GGC CAA GCC AAC TTCA
R6 GC ATC TCG GAG CTV GGK GCT
F6g AC TTC TGT GAG GCA GAT GCC A
R6g TGGACT ATATGGATG GAG GTATAG TCT G
F6gap CAG ATG CCAAAAAGT GCATTG
R6gap AGC TCG GGG CTC CTT CTG
F7 AGAAAG CCT TAC CAC TAC TGC TGT
R7 T GTT CAG TTG GTC TGT GCT CAC
F7gap CA CCA CAG AAA GCC TTACCA CTA
F7gap CA CCA CAG AAA GCC TTACCA CTA
R7 short a T GTT CAG TTG GTC TGT GCT C
human DNA, ubiquitous in the environment (Hofreiter
et al., 2001). Amplification of human sequences occurred
regularly when it was not possible to select against hu-
man DNA, showing that not only mitochondrial but also
nuclear human DNA is an abundant contaminant. Due
to the variability of the GHR sequences, different primer
pairs were used for the different species for some of the
amplified fragments (Table 3). Amplification products
were cloned using the TOPO TA cloning kit (Invitrogen,
The Netherlands) and multiple clones sequenced.
TABLE 2. Primer pairs for the amplification of the seven fragments used to determine GHR sequences in the six tenrecid species. Primer
sequences are listed in Table 3. The length of the products is given in base pairs, including primers. n.p.: no product obtained.
1234567
Potamogale
velox
n.p. F2a/R2g 201 F3/R3 200 F4.1g/R4.1s 175
F4gapG/R4gap1 73
F5g/R5g 223 F6g/R6g 205 F7gap/R7shorta 195
Limnogale
mergulus
F1g/R1g 181 F2/R2 199
F2a/R2g 201
F3g/R3g 225 F4/R4 217 F5/R5gap 264 F6g/R6g 205
F6/R6 206
F7/R7 189
Geogale aurita F1g/R1g 181 F2/R2 199
F2a/R2g 201
F3g/R3g 225 F4/R4 217 F5/R5gap 264 F6g/R6g 205 F7gap/R7 195
Micropotamogale
lamottei
n.p. F2g/R2 209 F3g/R3g 225 F4.1g/R4.1s 175
F4gapG/R4gap1 73
F5g/R5g 223 F6g/R6g 205 F7gap/R7 195
Hemicentetes
semispinosus
F1g/R1gap 147 F2/R2 199 F3/R3 200 F4/R4 217 F5/R5gap 264
F5g/R5g 223
F6gap/R6gap
150
F7/R7 189
Setifer setosus F1g/R1gap 147 F2/R2 199 F3/R3 200 F4/R4 217 F5/R5gap 264
F5g/R5g 223
F6gap/R6gap
150
F7/R7 189

184 SYSTEMATIC BIOLOGY VOL. 55
The challenges confronting ancient DNA studies
(Hofreiter et al., 2001; Olson and Hassanin, 2003) are
relevant to this work, as we used museum specimens
collected nearly 100 years ago. Hence, we used appro-
priate laboratory techniques at a dedicated ancient DNA
facility at the Max Planck Institut for Evolutionary An-
thropology, Leipzig.
Figure 3 shows sequence overlap between adjacent
fragments for all taxa. Except for Potamogale, which has
a 6-bp gap between fragments 3 and 4, all species have
continuous sequences when the amplified fragments are
FIGURE 3. Schematic view of PCR fragments used to reconstruct
GHR sequences (see also Tables 2 and 3). Dashed lines for Potamogale
and Micropotamogale indicate missing sequences due to nonamplifica-
tion of the first PCR fragment. A 6-bp gap between fragments 3 and 4
in the sequence data for Potamogale is indicated by the box. Deletions
are shown as gaps in the sequence but do not represent missing data.
The length of overlap among fragments is shown to scale.
concatenated after trimming the primers. Table S1 details
sequence overlap across our amplified fragments, all of
which are identical within species, both between differ-
ent fragments and alternative amplicons that span ho-
mologous regions. Moreover, each amplified fragment
excluding primers is unique in our data set, making it
highly unlikely that our sequences are chimaeric (see
Olson and Hassanin, 2003). When compared to the avail-
able sequences in GenBank by Blast searches (Table S2),
all fragments show closest matches to members of the
Afrotheria, and 38 out of 47 fragments are closest to
published GHR sequences of the Tenrecidae. Given the
occasionally short length of the fragments, slightly closer
matches to other members of Afrotheria are not surpris-
ing. Finally, except for two fragments from Setifer setosus
(which match corresponding sequences from Echinops
AF392889), all others differ slightly from previously pub-
lished sequences available in GenBank.
Sequence Alignment
Using MacClade 4.07 (Maddison and Maddison, 2000)
and Clustal X (Thompson et al., 1997), we concatenated
GenBank files, added new sequences, and constructed
alignments that preserved reading frames and con-
tained few indels. GHR shows several conserved regions
that facilitate a priori homology assessment. Neverthe-
less, some ambiguity remains regarding the positions
of certain indels and adjacent nucleotides. Exploration
of alignment ambiguity has occasionally (e.g., Messen-
ger and McGuire, 1998), but not always (e.g., Douady
et al., 2003), led to revised phylogenetic interpretations.
For this reason, we explore a limited number of alter-
native alignments, differences across which are sum-
marized in Table S3. Confidence indices mentioned in
the text, as well as statistical comparisons of alterna-
tive topologies, are based on the first alignment (with
the addition of the morphological partition, as indicated
below), unless stated otherwise. Topological results
were not significantly altered by using the other three
alignments.
Each series of internal (i.e., not leading or trail-
ing), contiguous gap characters was assumed to rep-
resent a single insertion and/or deletion event (indel).
For all of our analyses including sequence data, we
coded indels for each alignment, adding them as bi-
nary characters following the aligned nucleotides. Actual
gap characters interspersed among the aligned nu-
cleotides were treated as missing data. In Bayesian anal-
yses, indels were treated using the binary (restriction
site) model without assuming that all presence/absence
characters have been observed (MrBayes command
“LSET CODING=VARIABLE”). Sequence alignments
and other supplementary data are available online at
http://systematicbiology.org.
Morphological Data Collection
We used an anatomical dataset consisting of 126 char-
acters, 20 of which are from the soft-tissues of the ros-
trum and cranial arterial supply, 46 from the cranium, 30

2006 ASHER AND HOFREITER—TENREC PHYLOGENY 185
from the jaw and dentition, and 30 from the postcranial
skeleton. Morphological characters were based on Asher
(2000) and coded using specimens noted in Appendix 1.
A nexus file with the morphological data is available at
www.treebase.org (accession S1460).
Olson and Goodman (2003) questioned two coding de-
cisions made by Asher (1999, 2000): occurrence of the fen-
estrate basioccipital in Microgale and morphology of the
nasolacrimal duct (also known as the “lacrimal canal”) in
Limnogale. Olson and Goodman stated that Asher (1999)
coded both as absent, whereas they noted that a fen-
estrate basioccipital occurs in some species of Micro-
gale (Asher [1999] sampled only M. talazaci) and stated
that Limnogale possesses a nasolacrimal duct. As of this
writing, M. talazaci remains the only species of Micro-
gale with nuclear DNA sequences available to us (Malia
et al., 2002). Hence, we still have a limited sample of
this genus, but accept Olson and Goodman’s (2003) ob-
servation and code the genus Microgale as polymorphic
for the fenestrate basioccipital (character no. 35) in this
study.
Concerning the presence of a nasolacrimal duct in
Limnogale, this was in fact not the character cited as a
potential semiaquatic tenrec synapomorphy by Asher
(1999, 2000). Rather, absence of an external lacrimal fora-
men (character no. 53) was coded in both studies, as
depicted here in Figure 2. There is a clear osteologi-
cal difference in the expression of a single, conspicu-
ous lacrimal foramen at the anterior margin of the orbit,
dorsal to the infraorbital canal, in most tenrecs (e.g.,
Setifer, Fig. 2). This region is smooth and without a major
foramen in both potamogalines and Limnogale (Fig. 2).
Hence, we retain the coding of Asher (1999, 2000) for
the present study (see also S´anchez-Villagra and Asher,
2002). Expression of a nasolacrimal duct was coded sepa-
rately fromthe lacrimal foramen in Asher (2000) based on
observations of soft tissue anatomy in histologically pre-
pared anatomical sections (Asher, 2001). To our knowl-
edge, no histological preparation of Limnogale has ever
been made, so we cannot compare the patent, partly
soft tissue nasolacrimal duct in most tenrecs and other
mammals with any such structure in Limnogale. Hence,
we code this character (no. 15) “missing” for Limno-
gale in our morphological data matrix. Coding these
two characters (duct, foramen) independently is justi-
fied by the variable expression of the lacrimal foramen
in taxa with a patent nasolacrimal duct (e.g., Frahnert,
1999).
Phylogenetic Inference
The search strategies described below were applied to
each of the four alignments summarized in Table S3 using
a 43-taxon data set sampling only GHR and a 23-taxon
data set sampling GHR plus morphology.
MP analyses were undertaken with PAUP 4.0b10
(Swofford, 2002); Bayesian algorithms were applied with
MrBayes 3.1 (Huelsenbeck and Ronquist, 2001). For
the full GHR taxon sample, our MP analyses searched
heuristically with at least 100 random addition replicates
with TBR branch swapping, multiple states treated as
polymorphic, and branches with a zero length under any
optimization collapsed. For our Bayesian and likelihood
bootstrap analyses, we used the HKY+I+G model for
the full GHR taxon sample (Fig. 4) and GTR+I+Gasthe
optimal model for the smaller GHR sample (Fig. 5), as in-
dicated by the AIC in MrModeltest 2.1 (Nylander, 2004).
We used the default PAUP commands given in MrMod-
eltest (”DSet distance=JC objective=ME base=equal
rates=equal pinv=0 subst=all negbrlen=setzero; NJ
showtree=no breakties=random”) to obtain an initial
tree, used by PAUP to estimate maximum likelihood
(ML) parameters for the likelihood bootstrap analysis of
the 43-taxon GHR data set (which resulted in the values
“Lset Base=[0.2547 0.3078 0.2293] Nst=2 TRatio=2.1473
Rates=gamma Shape=1.7732 Pinvar=0.1990”). In addi-
tion, the ML bootstrap analysis excluded indels, used 143
pseudoreplicates of an “as-is” addition sequence with
TBR branch swapping, and obtained starting trees with
stepwise addition.
Bayesian analyses of the larger GHR dataset were
based on at least four independent runs, each using a
random starting tree and 1,000,000 generations with one
cold and three heated chains, sampling trees every 100
generations. Bayesian runs of the 43-taxon GHR and
the 23-taxon combined morphology-GHR data sets both
reached stationarity between approximately 10,000 to
14,000 generations, as determined by visually inspecting
asymptotic graphs of likelihood scores across genera-
tions. Our phylogenetic conclusions are based on only
succeeding generations, starting at 15,000, discarding the
first 14,900 (sampling every 100th generation) as “burn-
in.” Each run of 1,000,000 generations converged on a
single, consistent result.
The taxon sample for the smaller, combined GHR-
morphology data set was chosen based on availability
of GHR sequences as well as osteological and soft tis-
sue data from Asher (2000, 2001; see also Appendix 1).
This sample included all genera of tenrecs, including fos-
sils, plus Orycteropus afer, Procavia capensis, Elephantulus
brachyrhynchus,acomposite golden mole (Chrysospalax
trevelyani GHR, Chrysochloris stuhlmanni and C. asiatica
morphology), Erinaceus europaeus, three soricids, and Ca-
nis latrans, and was rooted with a composite didelphid
(Monodelphis domestica GHR, Didelphis sp. morphology).
Most soft tissue characters remain missing for two of the
extant tenrecs: Limnogale and Oryzorictes.
Combined and GHR-only MP analysis of the smaller
dataset used the same search parameters as described
above, leaving DNA entries missing for the three fos-
sil taxa. MP bootstrap support values were generated
with 1,000 pseudoreplicates, each with 10 random ad-
dition replicates and TBR branch swapping. Bayesian
search parameters for the smaller GHR dataset were
as described above. In the combined Bayesian anal-
ysis of morphology and sequences we used differ-
ent models for each partition: the GTR+I+G for
sequences following the AIC in MrModelTest (Nylander
2004), the binary (restriction site) model for indels
(with LSET CODING=VARIABLE), and Mk for mor-
phology following Lewis (2001) and Nylander et al.

186 SYSTEMATIC BIOLOGY VOL. 55
FIGURE 4. Phylogenetic trees based on GHR sequences. Bayesian tree (left) is a majority rule consensus of 9,850 trees (1,000,000 generations
sampling every 100), excluding the first 150 as burn-in, from alignment 1 (Table S3) using the HKY+I+G substitution model. MP tree (right) is
strict consensus of six trees, each with 2389 steps. Numbers above and below nodes at left indicate, respectively, Bayesian posterior probability
values and ML bootstrap support values. (The latter exclude gap characters.) Numbers below nodes at right indicate MP bootstrap support
values. Malagasy tenrecs are shown in boldface. Branch lengths do not represent divergences.
(2004). We undertook multiple runs using both LSET
CODING=VARIABLE and LSET CODING=ALL com-
mands for the morphological partition. We also ran our
morphological partition with (Mk+G) and without (Mk)
gamma-shaped rate variation (LSET RATES=GAMMA).
R
ESULTS
Affinities of Living Tenrecs
Parsimony, likelihood, and Bayesian methods applied
to our GHR data consistently supported the monophyly
of Malagasy tenrecs to the exclusion of the two living
African genera with high support indices (Fig. 4). In each
case, regardless of the alignment (Table S3) or algorithm
used, and in agreement with Olson and Goodman (2003),
Limnogale was closely related to Microgale, and potamo-
galines were reconstructed as the sister group to other
living tenrecs. The extant Malagasy tenrec clade con-
sisted of two radiations: spiny tenrecs (Tenrecinae) and
soft tenrecs (Oryzorictinae plus Geogale). Less clear were
the positions of Geogale and Oryzorictes within the soft-
tenrec clade, and of Hemicentetes and Tenrec within the
spiny tenrec clade.
Bayesian analysis of sequence data alone favors Ory-
zorictes at the base of a soft-tenrec clade, contradict-
ing oryzorictine monophyly (Fig. 4). However, Bayesian
support for a soft-tenrec clade excluding Oryzorictes
ranged from 54 to 58 across the four alignments; and
trees produced by MP for each of the four alignments
left Oryzorictes and Geogale unresolved at the base of
this clade (Fig. 4). Furthermore, we cannot statistically
reject a monophyletic Oryzorictinae with Geogale as its
sister taxon (Table 4). In contrast, statistical comparisons
based on the GHR-only and combined datasets reject any
sister-group relation between the semiaquatic Malagasy
Limnogale and African potamogalines (Table 4).
Application of MP to the morphological dataset yields
optimal trees similar in some regards to those generated
by sequences alone, such as monophyly of tenrecids and
potamogalines, support for a spiny tenrec clade, and a

2006 ASHER AND HOFREITER—TENREC PHYLOGENY 187
FIGURE
5. Single topology supported by both Bayesian and MP
algorithms using combined GHR and morphological data. Bayesian
tree is a majority rule consensus as described in Figure 4, using the
GTR+I+G substitution model for GHR and Mk (Lewis, 2001) for mor-
phology. This tree was generated without gamma-distributed rate vari-
ation (Mk+G) and uses LSET CODING=ALL for the morphological
partition; optimal trees from additional runs with Mk+G and LSET
CODING=VARIABLE were compatible. MP with all character changes
equally weighted supports a single best tree with 1550 steps. Num-
bers above nodes indicate Bayesian posterior probabilities; numbers
below nodes indicate MP bootstrap support values. Malagasy tenrecs
are shown in boldface, fossils with a dagger.
sister taxon relationship between Echinops and Setifer.
However, in contrast to the GHR signal, morphologi-
cal data support the position of African potamogalines
near Limnogale (Fig. 6). This relationship appears in most
of the optimal trees in the combined MP analysis, but
is unresolved in the strict consensus. Nevertheless, a
Limnogale-potamogaline clade is supported by MP ap-
plied to the living taxa alone with a bootstrap value
of 71 (not figured; see also Asher, 1999), and by Mk
applied to the morphological dataset including fossils
(Fig. 6). Using the morphological dataset alone, the alter-
native topologies summarized in Table 4, including vari-
ants that preserve monophyly of Malagasy tenrecs and a
Limnogale-Microgale clade, are rejected by Templeton and
winning sites tests.
Affinities of Extinct Tenrecs
Application of MP to the combined dataset including
the three fossil tenrecs, regardless of alignment (Table S3)
or analysis parameters, supports a Parageogale-Geogale
FIGURE 6. Phylogenetic trees based on morphological data.
Bayesian tree (left) is a majority rule consensus as described in Figure 4,
using Mk (Lewis, 2001) and LSET CODING=VARIABLE. MP tree
(right) is a strict consensus of six trees, 382 steps, all morphological char-
acter changes treated equally. Numbers above nodes indicate Bayesian
posterior probabilities (left) or MP bootstrap support values (right).
Malagasy tenrecs are shown in boldface, fossils with a dagger.
clade with relatively high confidence, with MP-bootstrap
support values (89) comparable to that for potamogalines
(87; see Fig. 5). Bayesian analyses of the combined dataset
also supported this clade, but with a posterior prob-
ability (80) weaker than that for potamogalines (98).
Erythrozootes and Protenrec are also reconstructed to-
gether, in turn adjacent to Geogale-Parageogale, regard-
less of alignment or tree-building technique. However,
support indices for this clade are much lower (posterior
probability 67, MP bootstrap below 50), as are the sup-
ports for a clade joining the three fossil taxa with Geogale
(posterior probability 76, MP bootstrap below 50; see
Fig. 5).
Several morphological characters support a
Parageogale-Geogale clade, which, following Butler
(1984) may be referred to the Geogalinae. First, the
reduction of its upper molar metacone (character no.
73, state 2), protocone (no. 74, state 1), and of the
lower molar talonid (no. 85, state 1) favor its placement
with other dentally zalambdodont taxa (i.e., in this
sample, tenrecs and golden moles; see Asher and
S´anchez-Villagra [2005] for a definition of anatomical
zalambdodonty). Parageogale and Geogale share a highly
reduced maxillary process of the zygoma (no. 59, state 1;
also present in soricids). Geogalines also possess a broad

188 SYSTEMATIC BIOLOGY VOL. 55
T
ABLE
4. Tests of alternative topologies.
(A) One-tailed Shimodaira-Hasegawa test using 1,000 RELL bootstrap replicates, applied to the 43-taxon GHR dataset (Fig. 4), using HKY+I+G
likelihood model in PAUP. The completely bifurcating tree with the highest likelihood score from alignment 1, run 1 (generation no. 478,200,
see supporting data) was used for comparisons. Alternatives are the same except as indicated. Abbreviations are as follows: Ec, Echinops;
Er, Erythrozootes, FT, fossil tenrecs; Ge, Geogale; He, Hemicentetes; Li, Limnogale; Mi, Microgale;MT, Malagasy tenrecs; On, Oryzorictinae; Or,
Oryzorictes; Pa, Parageogale; Pn, Potamogalinae; Pr, Protenrec; Se, Setifer;Te,Tenrec; Tn, Tenrecinae.
Tree −ln L Diff −ln L SH-test P
best Bayesian tree, align1, run1 12,034.29976 — —
(Ge(Or(Li,Mi))) 12,043.75843 9.45867 0.484
(He,Te) 12,046.09929 11.79953 0.394
((Li,Pn)(Tn,(Or(Ge,Mi)))) 12,163.30139 129.00163 0.000
∗
(Tn(Or(Ge(Mi(Li,Pn))))) 12,141.31100 107.01123 0.000
∗
(B) Templeton and winning sites tests applied to 23-taxon combined data set (Fig. 5) using MP in PAUP. The completely bifurcating tree from
Figure 5 was used for comparisons; alternatives are the same except as indicated.
Tree Length Rank Sums
∗
NzTempleton P Counts Winning Sites P
As in Fig. 5 1550 (best) — — — — —
((Or(Li,Mi))((Er,Pr)(Pa,Ge))) 1551 16.0 7 −0.3780 0.7055 4
−12.0 −3 1.0000
(Pn(Er,Pr)(Pa(MT))) 1557 25.0 7 −1.9332 0.0532 6 0.1250
On paraphyletic −3.0 −1
(Pn(Er,Pr)(Pa(MT))) 1557 77.0 14 −1.6977 0.0896 10 0.1796
On monophyletic −28.0 −4
(Pn((Er,Pr)Pa)(MT)) 1556 21.0 6 −2.4495 0.0143
∗
6 0.0313
∗
On paraphyletic −0.0 0
(Pn(Pa((Er,Pr)(MT)))) 1558 21.0 6 −2.2711 0.0231
∗
6 0.0313
∗
On paraphyletic −0.0 0
((Li,Pn)(Tn,(Or((Ge,FT),Mi)))) 1592 1638.0 62 −5.3340 <0.0001
∗
52 <0.0001
∗
−315.0 −10
(Tn(Or((Ge,FT)(Mi(Li,Pn))))) 1582 1254.0 56 −4.2762 <0.0001
∗
44 <0.0001
∗
−342.0 −12
(Ge((Er,Pr)Pa)) 1552 3.0 2 −1.4142 0.1573 2 0.5000
−0.0 0
gap between the anterior central incisors (character no.
126, state 1), a condition also seen in Erinaceus and in
some specimens of Setifer (here coded as polymorphic).
They also have two premaxillary teeth (no. 67, state 2;
also present in some tenrecines and Erythrozootes). In
contrast to the other nine tenrecid genera, fossil tenrecs
plus Geogale possess a relatively long infraorbital canal
(no. 60, state 0).
Templeton and winning sites tests based on MP re-
ject alternative hypotheses placing all three fossils either
outside of living Tenrecidae or together as the sister-
clade to African potamogalines (Table 4). However, an-
other alternative, placing Parageogale as the sister-taxon
to a (potamogaline (Protenrec Erythrozootes)) clade, again
with all African tenrecsoutside of the Malagasy radiation
(Fig. 1A), cannot be rejected.
Additional Tests of Fossil Tenrec Phylogeny
All three fossil tenrec genera were first described from
the Kenyan Miocene (Butler and Hopwood, 1957) and
remain known only from a few craniodental fragments
(Butler, 1984). Published reviews including these taxa
have generally supported their affinity to modern ten-
recids (Butler, 1969, 1978, 1984, 1985; McKenna and Bell,
1997; Mein and Pickford, 2003; but see Poduschka and
Poduschka, 1985). As is the case for other fossils over
1 million years in age, sequence data cannot be ob-
tained from these specimens (Hofreiter et al., 2001). Of
the 126 characters sampled in our morphological ma-
trix, Erythrozootes and Protenrec are 24% complete and
Parageogale is ca. 18% complete. Nevertheless, the most
poorly known taxon in this study, Parageogale, shows a
relatively well-supported position, consistent with the
hypothesis originally presented by Butler and Hopwood
(1957) that it is the sister-taxon to the living Geogale au-
rita, and contradicting the monophyly of the Malagasy
radiation (Fig. 1A).
To test the hypothesis that the 22 characters sampled
for Parageogale can accurately reconstruct its phylogeny,
we used these same characters to reconstruct the phy-
logeny of other tenrecs in our study. That is, for each of
the 10 living tenrecid genera, we replaced GHR data and
all morphological characters, except for the 22 known
for Parageogale, with missing entries and ran the modified
morphology+GHR dataset using MP, as described above
in Materials and Methods. Stated differently, if a living
tenrecid genus had gone extinct in the early Miocene, and
were known only from cranial fragments similar to those
of Parageogale, would we be able to accurately (as defined
by the full-data sample depicted in Fig. 5) reconstruct
its phylogenetic position? If the respective extant taxon
sampled only for the 22 Parageogale characters appears
in a different part of the tree relative to its position in
the full analysis, we would have less confidence in the
placement of Parageogale.
In fact, the reduced dataset did not greatly change
the position of any extant tenrec (Fig. 7). Out of the 10

2006 ASHER AND HOFREITER—TENREC PHYLOGENY 189
FIGURE7. Results of reduced-character MP analyses of each of the 10 living tenrecid genera (as identified in boldface), with all GHR sequences
and morphological characters, except for the 22 known for Parageogale, coded as missing. Matrices with either Echinops or Setifer coded in this
fashion yield the same topology (top left), also identical to the combined-data topology depicted in Figure 5. Each tree represents either a strict
consensus or a single, most-parsimonious result, as follows: Echinops 1tree 1,539 steps, Geogale 3trees 1,491 steps, Hemicentetes 4trees 1,517 steps,
Limnogale 3trees 1,521 steps, Microgale 4trees 1,521 steps, Micropotamogale 1tree 1,520 steps, Oryzorictes 7trees 1,515 steps, Potamogale 1tree 1,511
steps, Setifer 1tree 1,543 steps, Tenrec 2trees 1,522 steps. Numbers adjacent to nodes represent MP bootstrap support values (100 pseudoreplicates
of a simple addition sequence). Bootstrap values in tree at top left for Echinops are listed above nodes, Setifer below.

190 SYSTEMATIC BIOLOGY VOL. 55
modified datasets (1 for each living tenrecid genus), 2
(Echinops and Setifer) yielded the same tree as the full
sample, and 6 of the remaining 8 yielded varying de-
grees of nonresolution in multiple shortest trees, con-
sensuses of which (Fig. 7) were still compatible with
the topology supported by the full dataset. Only two
cases (Tenrec and Potamogale) yielded optimal trees with
a slightly different topology. The former altered relations
within spiny tenrecs (supporting Tenrec-Setifer rather
than Tenrec-Hemicentetes), and the latter reconstructed
Oryzorictes adjacent to Microgale-Limnogale to the ex-
clusion of Geogale,preserving oryzorictine monophyly.
However, Tenrec bootstrap resampling still supports a
spiny-tenrec clade with a value of 79; and the Tenrec-
Setifer clade has an MP bootstrap support value under 50.
Similarly, for the run using a reduced sample for Potamo-
gale, oryzorictine monophyly is supported with an MP
bootstrap of just 57, and the unmodified, combined-data
sample cannot reject this hypothesis (Table 4). In these
and other cases, bootstrap resampling generally yielded
lower support values compared to the full sample (cf.
Fig. 5 versus Fig. 7), but in no case did a clade pro-
duced by a reduced-sample analysis contradict a well-
supported clade in the full sample.
D
ISCUSSION
Data Combination and Tenrec Phylogeny
Aprevious morphology-based investigation of tenre-
cid phylogeny published by one of us (Asher, 1999) ar-
gued for a clade of semiaquatic tenrecs, placing Malagasy
Limnogale as the sister-taxon to continental African pota-
mogalines. Character support for this clade was primar-
ily from the skull, including a fenestrate basioccipital (no.
35 in this study), a shortened frontal bone (no. 61), and a
reduced lacrimal foramen (no. 53). Importantly, none of
these character states are consistently found in nontenre-
cid, semiaquatic, faunivorous, small mammals (S´anchez-
Villagra and Asher, 2002), a factor that had previously
led Asher to view the “semiaquatic” tenrec clade with
increased confidence.
As discussed above, morphological data analyzed
alone still yield some support for a semiaquatic clade,
although recoding fenestration in the basioccipital to ac-
count for polymorphism in Microgale (as recommended
by Olson and Goodman, 2003) has eliminated this char-
acter from optimizing unambiguously as a Limnogale-
potamogaline synapomorphy. Furthermore, compared
to the study of Asher (1999), the larger number of char-
acters and sampled tenrecs in this study yields reduced
support for a semiaquatic tenrec clade (Fig. 6).
However, the key reason for the nonrecovery of
a semiaquatic clade in this study is the very strong
sequence-based signal favoring a Limnogale-Microgale
clade. Indeed, with their sample of different loci for mul-
tiple species of Microgale, Olson and Goodman (2003)
found that Limnogale actually nests within that genus,
comprising the sister-taxon to an M. dobsoni–M. talazaci
clade to the exclusion of other Microgale species. The
strength of the signal supporting a Microgale-Limnogale
clade in our study (100 MP bootstrap, 100 ML bootstrap,
and 100 Bayesian posterior probability in the GHR-only
analysis [Fig. 4]; 95 MP bootstrap and 94 Bayesian poste-
rior probabilityin the combined analysis [Fig. 5]) has con-
vinced both of us that the previous interpretation of the
morphological signal as indicative of a semiaquatic ten-
rec clade (Asher, 1999) is incorrect. Due to this unambigu-
ous support from GHR sequences, which is considerably
stronger than that from morphology alone for a semi-
aquatic clade and which prevails in the combined analy-
sis, the cranial characters supporting the “semiaquatic”
clade cited above must be reinterpreted as homoplastic.
If the morphological data used here are misleading
regarding a semiaquatic tenrec clade, why do we then
combine them with our GHR data? The most important
reason for retaining morphology in our dataset is one
of principle: most individual datasets are not in their en-
tirety either “true” or “false”; but are themselves mosaics
of variable character-data that may provide resolution at
different levels in any given tree (Gatesy et al., 2003).
Combined data sets enable recognition of phylogenetic
signals that would remain obscure with the analysis of
subdivisions thereof (Gatesy et al., 1999, 2005). Further-
more, including morphological data in the combined
analysis remains the best means to sample fossil ten-
recs. We cannot be completely sure that the morphology
known for these fossils enables us to accurately under-
stand their phylogenetic history. However, as discussed
above, when used in simulations to replace the complete
morphology-GHR dataset for each of the 10 living ten-
recid genera, the morphological characters known for
the most incomplete of the fossils (Parageogale) yield re-
sults that are largely congruent with the combined-data
topology.
Character Assessment and Hindlimb Function
in Potamogale
One recent study of hindlimb characters (Salton and
Szalay, 2004) has also argued for the inclusion of
Limnogale within the Malagasy radiation. By assess-
ing characters of the tarsal complex in an “ecological
and evolutionary framework,” Salton and Szalay pro-
posed to identify phylogenetically informative charac-
ters: “traits with clear species-specific adaptations are
a potential interference in cladistic analyses and can-
not be meaningfully used without ecology-based charac-
ter assessment” (Salton and Szalay, 2004:73). In regards
to the “semiaquatic” clade, their procedure resulted in
the identification of anatomical differences (e.g., astra-
galar neck-head transition) and similarities (e.g., medi-
ally directed tibial-fibular malleoli) between Limnogale
and Potamogale (they did not include Micropotamogale in
their analysis). In their opinion, the former comprise phy-
logenetic data in support of the “family level distinction”
of Potamogale from other tenrecs, and the latter are inter-
preted as homoplastic.
However, we are concerned that Salton and Szalay
(2004) did not identify a replicable optimality criterion
(e.g., MP, ML) by which they reached their conclusions

2006 ASHER AND HOFREITER—TENREC PHYLOGENY 191
on homology. Furthermore, we believe that Salton and
Szalay have not fully appreciated the function of the
hindlimb in Potamogale. Regarding its locomotion, Salton
and Szalay refer to its “heavy foot thrusts” (p. 90),
and note that “heavy loading in the UAJ [upper an-
kle joint]. . . and UAJ stabilization plays an important
role. . . in the aquatic locomotion of Potamogale” (p. 86). In
regards to calcaneal morphology, Salton and Szalay state
that “Potamogale has an extremely long and narrow calca-
neus with a long tuber, appropriate for strong, dorsolat-
eral aquatic propulsion” (p. 93). In fact, these inferences
of locomotion run counter to published descriptions of
locomotor behavior in Potamogale (e.g., DuChaillu, 1860;
Kingdon, 1974), which indicate that it uses its massive
tail, not its feet, for aquatic propulsion. As in the other
two potamogaline species (Micropotamogale lamottei and
M. ruwenzorii), digits II and III of the hindfoot in Potamo-
gale are syndactyl, and their use in grooming has been
documented (Nicoll, 1985; Kingdon, 1997). As summa-
rized by Nowak (1999), the relatively small, nonwebbed
pes of Potamogale is tucked under its pelvic region dur-
ing swimming and is not used for propulsion. Dobson
(1883:97–98) infers from its anatomy that during loco-
motion, “the sole [of the foot] lies so evenly against the
[pelvic ventrum] as to present the least possible projec-
tion and interfere in the least degree with the rapid pas-
sage of the body through the water, propelled by the
powerful tail.. . . [The tail] is doubtless the sole organ of
propulsion.” Based on field observations, Kingdon ob-
served that in the water, “the animal is propelled entirely
by lateral movements of the back and tail” (Kingdon,
1974:15). In contrast, Limnogale (the “web-footed” ten-
rec), has been observed to use its hindlimbs for semi-
aquatic propulsion (Benstead and Olson, 2003:1272).
Despite this, and without presenting new behavioral
data for either taxon, Salton and Szalay (2004:100) pro-
pose the opposite: “. . . the tarsal complex indicates that
[Limnogale’s] hind limbs are less important for propulsion
than those of Potamogale.”
Hence, we remain skeptical about Salton and Szalay’s
method for distilling phylogenetically informative data
from their morphological observations. Although we
agree with them that Limnogale is not more closely re-
lated to Potamogale than to other Malagasy tenrecs, contra
Asher (1999), we do not believe they presented in their
paper a basis for reaching this conclusion, independent
of the sequence data analyzed by Olson and Goodman
(2003), and confirmed with additional data in this article.
Tenrec Biogeography
Considering the living radiation alone, Malagasy ten-
recs show substantial morphological diversity, yet are
recognized as a single radiation by sequence data, as ob-
served for primates (cf. Yoder, 1992, versus Yoder et al.,
1996) and carnivorans (cf. Veron, 1995, versus Flynn et al.,
2005). Similarly, our results support a cohesive Malagasy
radiation and argue against Malagasy tenrec polyphyly.
However, the living tenrecid radiation is not a complete
picture of this group’s diversity. Although its paleon-
tological record is meager, fossil African tenrecids ap-
pear to have a close relationship with living Geogale. This
relationship makes the Malagasy tenrec radiation para-
phyletic (Figs. 1B, 5).
A similar phylogenetic scenario was presented by
Jansa et al. (1999) for Malagasy nesomyine rodents.Based
on cytochrome b sequences for multiple representatives
of all genera of this group, Jansa et al. (1999) disputed
previous interpretations of polyphyly (Ellerman, 1940,
1941), but argued that two mainland African genera
(Steatomys and Tachyoryctes) nested within the Malagasy
radiation. They suggested that this phylogenetic pattern
would be consistent with colonization of Madagascar by
nesomyines via a single founder event, followed by dis-
persal to Africa from Madagascar. The inclusion of main-
land African muroids within the Malagasy radiation has
subsequently been questioned (Steppan et al., 2004; Jansa
and Weksler, 2004); and nesomyine monophyly remains
possible. A definitive conclusion must await a study that
synthesizes the taxon and character samples discussed
by Jansa et al. (1999), Jansa and Weksler (2004), and
Steppan et al. (2004).
Monophyly of Malagasy tenrecs is also possible. We
have at present no way of knowing how the missing
GHR nucleotides for Parageogale,orcharacters from its
still unknown skeleton, would affect our estimate of its
relationships. Some uncertainty regarding our results
supporting paraphyly (Fig. 1B) is reflected in the nonre-
jection of at least one alternative topology that preserves
Malagasy tenrec monophyly (Table 4); and we eagerly
anticipate how this result is affected by future discov-
eries of better-preserved fossil tenrecid material. Never-
theless, the current hypothesis of a Parageogale-Geogale
clade has support from both MP and Bayesian meth-
ods (Fig. 5). Furthermore, as discussed above, the limited
morphological sample available for Parageogale appears
to perform fairly well when these same characters are
used to reconstruct the phylogeny of each of the ten liv-
ing tenrecid genera, a result that slightly increases our
confidence in the placement of this fossil.
As stated in the introduction, the absence of modern
mammalian orders from Madagascar (and elsewhere)
during the Mesozoic (Krause, 2003), during which time
land connections existed with mainland Africa (until the
late Jurassic) and India (until the early Late Cretaceous),
has led many to favor dispersal as the prime mechanism
by which modern mammals colonized Madagascar (e.g.,
Olson and Goodman, 2003; Yoder et al., 2003). Repeated
monophyly of Madagascar’s endemic radiations is con-
sistent with dispersal, as individual colonization events
are hypothesized to be rare, and a previously unpopu-
lated island may have open adaptive zones into which a
founder can radiate into a diverse clade.
Nonmonophyly is also compatible with dispersal, but
requires more (potentially unparsimonious) crossings of
a geographic barrier, in this case the Mozambique chan-
nel. No one will ever know exactly how or why the ten-
reccrossed the channel; but based on our phylogeny
we can estimate how often such an event took place.
Given the combined-data tenrec phylogeny presented in

192 SYSTEMATIC BIOLOGY VOL. 55
Figure 5, we hypothesize that a single founder event of
Madagascar by the common ancestor of Malagasy ten-
recs took place at some point after the Maastrichtian,
during which time a diverse vertebrate fauna shows no
sign of Madagascar’s modern inhabitants (Krause, 2003).
Prior to the Miocene, when fossil tenrecs were present in
east (Butler, 1984) and southwest (Mein and Pickford,
2003) Africa, an additional dispersal of an animal re-
lated to the geogaline common ancestor took place from
Madagascar to continental Africa. The position of Ery-
throzootes and Protenrec (in the Protenrecinae of Butler
[1984]) as sister taxa to Geogale-Parageogale implies that a
protenrecine relative made this back-migration yet again.
However, we note that a Protenrec-Erythrozootes clade
to the exclusion of geogalines has an unimpressive MP
bootstrap value below 50 and a Bayesian posterior prob-
ability of 67 (Fig. 5). An alternative hypothesis, placing
protenrecines as the sister clade to Parageogale within
Geogalinae, would require just a single Madagascar-
Africa dispersal event postdating the initial Madagas-
car colonization. This alternative is just two steps longer
in MP analyses and cannot be statistically rejected (Ta-
ble 4). As stated above, we are cognizant of yet another
alternative that preserves Malagasy tenrec monophyly
(Fig. 1A), also statistically unrejected in Table 4. This sce-
nario would require only a single colonization event of
Madagascar by tenrecs, with no back-migration, again at
some point after the Late Cretaceous.
Nevertheless, the optimal explanation of the data pre-
sented in this article supports paraphyly of Malagasy
tenrecs relative to their mainland relatives (Figs. 1B, 5),
not monophyly (Fig. 1A) or polyphyly (Fig. 1C). DNA
sequence data for extinct, pre-Pleistocene tenrecs will
probably never be available; and even for certain liv-
ing taxa, in particular Geogale and Limnogale, such data
are very difficult to obtain. Our technique for sequencing
nuclear DNA from museum specimens without damag-
ing them eases this constraint, can be applied to other
groups, and greatly reduces fieldwork expense and dis-
turbance of living populations otherwise necessary for
obtaining research material. This highlights yet further
the value of museum collections for basic science (Su´arez
and Tsutsui, 2004).
A
CKNOWLEDGMENTS
We thank Peter Vogel for access to material of Micropotamogale, Judith
Chupasko and the Museum of Comparative Zoology, Harvard Univer-
sity, for access to Geogale, and Roland Pfeiffer and Nadin Rohland for
technical assistance. We thank Rod Page, Ron DeBry, Scott Steppan, and
three anonymous reviewers for helpful comments that improved the
manuscript. We are also grateful to Nils Hoff for help with Figure 1 and
Knut Finstermeier for preparing Figure 3. RJA acknowledges support
from the Deutsche Forschungsgemeinschaft (grant AS 245/2-1), the
European Commission’s Research Infrastructure Action via the SYN-
THESYS Project (GB-TAF 218), and the National Science Foundation
USA (DEB 9800908). MH was supported by the Max Planck Society.
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First submitted 14 March 2005; reviews returned 7 June 2005;
final acceptance 6 September 2005
Associate Editor: Ron DeBry
A
PPENDIX 1
List of osteological specimens examined. The geographic provenance
of specimens is listed in parentheses following the taxon name.
Crosses denote extinct taxa; asterisks denote taxa sampled for soft tis-
sue characters using an uncataloged collection of histological speci-
mens (see table 2 of Asher, 2001). Institutional abbreviations are as
follows:
AMNH, American Museum of Natural History, New York
BMNH, The Natural History Museum, London
FMNH, Field Museum of Natural History, Chicago
IZEA, Institut de Zoologie et d’Ecologie Animale, Lausanne
MCZ, Museum of Comparative Zoology, Harvard University
MNHN, Museum Nationale d’Histoire Naturelle, Paris
USBA, University at Stony Brook, Department of Anatomical Sciences
USNM, United States National Museum, Washington
ZIUT, Zoologisches Institut, Universit¨at T ¨ubingen
ZMB, Zoologisches Museum Berlin
1. *Didelphis sp. (Mexico, Nicaragua, USA): AMNH 28408, 28962,
29255, 70082, 145630, 146551, 148959, 201327; USBA MMr1, MMr4,
MMr5
2. *Blarina brevicauda (USA): AMNH 95256, 95297, 98912, 144485,
144486, 144487, 144488, 207017, 207018, 207019, 207020, 207754,
207755, 212504; FMNH 108390, 121194
3. *Canis latrans (USA): AMNH 5392, 99653, 131833, 131865, 208363,
208367, 208371; USBA MCn28
4. *Chrysochloris stuhlmani (Zaire, Uganda, South Africa, Burundi):
AMNH 82399, 167615, 167963, 180909, 180911, 180912, 180913,
236000; USNM 49896; FMNH 26352, 26353, 26355, 127361, 148200,
148201, 148917
5. *Crocidura olivieri (Zaire, Malawi, Burundi, Cameroon, Ghana):
AMNH 48490, 48491,48497, 161848, 236229, 239320, 239321, 239326;
FMNH 137591, 137592
6. *Echinops telfairi (Madagascar): AMNH 31270, 100751, 100753,
100760, 100767, 100808, 170602, 170605, 170606, 170607, 170608,
170599, 170609, 170610, 170611, 207717, 207718, 212918, 212919;
USNM 464980
7. *Elephantulus branchyrhynchus (Kenya): AMNH 86554, 86555,
86556, 86557, 86577, 86578, 86580, 86581, 86582, 86583, 86584; ZIUT
3860, 3861
8. *Erinaceus europaeus (France, Italy, England, Germany): AMNH
3770, 42561, 42563, 57219, 70613, 140469, 140470, 160470, 201230,
215299; USNM 251763, 251764, ZIUT M140
9. †Erythrozootes chamerpes (Kenya): BMNH M14314, M21831; Butler,
1969, 1984
10. *Geogale aurita (Madagascar): MCZ 37807, 45496, 46274; FMNH
151947, 156551, 15652, 156553, 159732, 159733; MNHN 1912-110A,
1912-110B, 1962-2518, 1962-2519, 1981-1374, 1987-110, 1991-1450
11. *Hemicentetes semispinosus (Madagascar): AMNH 90421, 100777,
100780, 100781, 100783, 206755, 207711, 207712, 207713, 207714,
212921, 212922, 212935, 212938, 212940; USNM 83658; ZMB 71599
12. Limnogale mergulus (Madagascar): AMNH 100688, 100689, MCZ
45050, 45054, 45055; BMNH 35.1.8.255, 35.1.8.256, 48.89, 48.90,
97.9.1.161; MNHN 1962-2511, 1962-2513, 1984-521; ZMB 35258
13. *Microgale talazaci (Madagascar): AMNH 100708, 100709, 100714,
100799, 119216, 207003, 207077; USNM 520881; FMNH 154582,
154583, 154584, 154585, 154586, 154587, 154588, 154589; MNHN
1961-198, 1977-42, 1983-898, 1984-856, 1984-857
14. *Micropotamogale sp. (Congo, Ivory Coast, Liberia): IZEA 4942,
4975; BMNH 67.213, 73.170; MNHN 1970-514, 1976-397, 1980-52,
1980-53, 1980-57
15. Oryzorictes sp. (Madagascar): AMNH 31243, 31257; USNM 578789;
FMNH 5637, 5639, 5640, 5641, 156226; MNHN 1897-520, 1912-111A,
1912-112A, 1941-34, 1962-2501, 1984-523, 1984-525, 1987-108
16. *Orycteropus afer (Zaire): AMNH 34866, 51370, 51372, 51374, 51905,
51906, 51907, 51908, 51909, 51010, 70036, 70189
17. †Parageogale aletris (Kenya): BMNH M33046, Butler, 1984
18. *Potamogale velox (Zaire, Cameroon, Congo, Gabon): AMNH 51161,
51162, 51164, 51165, 51319, 51322, 51324, 51334, 51344, 51348, 51368,
55203, 55204, 120250, 240968; USNM 266897, MCZ 35321, 35322;
FMNH 72831, 25973; BMNH 26.11.1.62; MNHN 1892-2064, 1892-
2065, 1898-1576, 1947-864, 1947-865, 1947-866, 1962-2520; ZMB
46588
19. †Protenrec sp. (Kenya, Uganda) BMNH M34149, M34150, M33036,
M34153, M43551, M43552
20. *Procavia capensis (Kenya, Zaire, Central African Republic, South
Africa): AMNH 53777, 53781, 53784, 53785, 83411, 83412, 80997,
80998, 80999, 88418; USBA Mhy1, Mhy4, Mhy5
21. *Setifer setosus (Madagascar): AMNH 100749, 100750, 100762,
170532, 170533, 170534, 170535, 170537, 170540, 170547, 170548,
170579, 170581, 170582, 170612, 207005, 207076; USNM 578790;
BMNH 93.12.6.8; ZMB 44586
22. *Sorex sp. (Canada, Finland, England, Sweden): AMNH
126007, 126990, 126991, 141626, 148521, 115593, 115594, 115595,
115597
23. *Tenrec ecaudatus (Madagascar): AMNH 100729, 100732, 100733,
100735, 100738, 100809, 170502, 170511, 212913; USNM 19361,
577051; BMNH 70.3.10.5
NOTE: We wish to acknowledge the recent study of Poux et al.
(2005), published after the completion of this paper, on the colo-
nization of Madagascar by terrestrial mammals. Poux et al. sampled
most Recent genera of tenrecids (except Potamogale and Geogale),
plus a large sample of other endemic Malagasy genera, and report
a tenrecid phylogeny congruent with that discussed here and by
Olson and Goodman (2003), for example in supporting a Limnogale-
Microgale clade. However, they did not address the phylogeny of fossil
taxa.