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A new hero emerges: another exceptional mammalian spine and its
potential adaptive significance
William T. Stanley, Lynn W. Robbins, Jean M. Malekani, Sylvestre Gambalemoke
Mbalitini, Dudu Akaibe Migurimu, Jean Claude Mukinzi, Jan Hulselmans, Vanya Prévot,
Erik Verheyen, Rainer Hutterer, Jeffrey B. Doty, Benjamin P. Monroe, Yoshinori J.
Nakazawa, Zachary Braden, Darin Carroll, Julian C. Kerbis Peterhans, John M. Bates and
Jacob A. Esselstyn
In our paper we describe a new species of Scutisorex. Here we discuss the methodology
of the analysis of morphological and molecular characters, and provide further details on
the morphology of the holotype of Scutisorex thori. There are four supplementary
figures, and nine references.
S1. MORPHOLOGICAL ANALYSIS
The sole specimen of Scutisorex thori was collected in accordance with American
Society of Mammalogists guidelines [1] and exported from the Democratic Republic of the
Congo to the U.S. with appropriate clearances. The specimen consists of a cleaned skull
and post-cranial skeleton, dried and stuffed skin, and lower gastro-intestinal and
reproductive tracts preserved in alcohol after initial fixation in formalin. The
reproductive tract contains a single embryo. Liver, heart, kidney, brain, lung, spleen and
blood samples were frozen in liquid N2 and are deposited at the Centers for Disease
Control (Atlanta) and Field Museum of Natural History.
We took external measurements with a metric ruler and the weight with a spring
balance (Pesola scale) following Martin, Pine & DeBlase [2] . Cranial measurements were
taken with digital calipers with nomenclature according to Dippenaar [3], van Zyll de
Jong & Kirkland [4], and Carraway [5]. We measured skull height by laying the skull on
a microscope slide and measuring from the bottom of the slide to the top of the skull, and
then subtracting the thickness of the slide.
S2. DNA SEQUENCING AND PHYLOGENETIC ANALYSES
We amplified and sequenced fragments (684 bp each) of the mitochondrial
protein-coding gene Cytochrome b (Cytb) and the nuclear von Willebrand Factor Exon
28 (vWF). Genomic DNA was isolated from frozen liver samples using the Wizard
SV96 kit (Promega Corp., Madison, WI). PCR amplifications used GoTaq Green Master
Mix (Promega Corp., Madison, WI) chemistry in a 20uL reaction volume. We amplified
Cytb with the primers 425F and 1167R [6]. We designed the new primers vWF-F1 (5´ TRGAGGACACCCCGGAGCC - 3´) and vWF-R1 (5´ AGGACAAACRCCACATCCAGGACC - 3´) to amplify a section of exon 28 of the
vWF gene. PCR thermal cycling used an initial denaturing step of 95ºC for 2 minutes,
followed by 35 cycles of 95º for 30 sec., 54º (Cytb) or 57º (vWF) for 30 sec., and 72º for
45 sec., and a final extension step at 72º for 7 min. PCR products were visualized on a
1% agarose gel and cleaned with a 20% dilution of Exosap-It. Amplicons were
sequenced using Big Dye Terminator v3.1 chemistry (Applied Biosystems, California)
[6]. All sequences were deposited in GenBank under accession numbers KF110748–
KF110785.
Sequences were edited and aligned in Geneious v5.6. We examined both
alignments by eye and found them to be unambigous. The Cytb alignment contained no
premature stop codons or indels. The vWF alignment contained no stop codons, but one
indel was present, with a 9 (Myosorex babaulti) or 6 bp (M. jejei) insertion in Myosorex
(or deletion in all other taxa). We fit 88 candidate models of sequence evolution to each
alignment using a maximum likelihood estimate of the phylogeny in jModeltest [7]. We
chose the best-fit model (Cytb: HKY + Γ + I; vWF: TrN + Γ) with the Bayesian
information criterion and then estimated phylogenetic relationships and branch lengths
independently for each gene in BEAST v1.7.4 [8]. Settings included a birth-death tree
prior and relaxed log-normal clock substitution rates. We estimated divergence times and
relationships simultaneously in the Cytb analysis by calibrating the root of the
crocidurine (excludes Myosorex) tree with the oldest known fossil crocidurine (lognormal distribution, offset = 13MYA, mean = 0, s = 2; [9] and a relaxed log-normal clock
(mean = 0.01 substitutions/site/MY, s = 0.01). We chose to analyze the vWF alignment
on an arbitrary time scale (mean = 1.0, s = 0.33) because, due to a lack of knowledge
regarding nuclear substitution rates in shrews, the analysis would necessarily hinge
entirely on a single fossil calibration. We completed four independent MCMC analyses
of 2 × 107 generations with parameters sampled every 2000 generations for each locus.
We examined convergence diagnostics among the four runs for each locus using Tracer
v1.5. Both analyses appeared to converge within the first 5 × 105 generations and we
discarded the first 2 × 106 generations as burn-in, leaving 3.6 × 104 samples in the
posterior and effective sample sizes >1900 for all parameters. We summarized the
posterior distribution of trees as a maximum clade credibility tree with branch lengths
presented as medians and 95% highest posterior densities (figure S2).
S3. SPECIMEN DESCRIPTION
External measurements and weight are provided in the main text. The specimen
is an adult based on the fully fused suture between the basioccipital and basisphenoid
bones (figure 1) and the presence of an embryo in the uterus. The vertebral column has
eight corrugated, interlocking lumbar vertebrae with broader but fewer lateral tubercles
than are present on the 10 to 11 lumbar vertebrae in S. somereni (figure 2). The vertebral
tubercles of S. thori are not as extended ventrally as in S. somereni, resulting in a less
dramatic concave trough along the ventral axis of the vertebral column. The dorsal
spinous processes of the lumbar vertebrae of S. thori are wider than those of S. somereni
(figure S3). The ribs are more flattened and broad in S. thori (figure 2).
S4. MOLECULAR PHYLOGENETICS
Although both gene trees support a sister relationship between the two species of
Scutisorex, the relationships of Scutisorex to other genera varied between the analyses
(figure S2). We highlight this difference because neither tree contains high nodal support
across the topology and because our divergence time estimate between Scutisorex and
other genera is dependent on the mitochondrial topology.
S5. SPECIMENS EXAMINED
Scutisorex somereni (FMNH 43860: Democratic Republic of the Congo, Medje;
FMNH 148269-148271, 148941: Uganda, Kibira National Park; FMNH 160177-160178,
FMNH 160180: Uganda, Bwindi Impenetrable National Park; FMNH 189277:
Democratic Republic of the Congo, Kahuzi-Biega National Park); Scutisorex thori n. sp.
(FMNH 219669: Democratic Republic of the Congo, Baleko). See figure S4.
S6. SUPPLEMENTARY REFERENCES
1 Sikes, R. S. et al. 2011 Guidelines of the American Society of Mammalogists for
the use of wild mammals in research. J. Mammal. 92, 235-253.
2
Martin, R. E., Pine, R. H. & DeBlase, A. F. 2001 A manual of Mammalogy : with
keys to families of the world., 3rd edition. New York, NY: McGraw-Hill.
3
Dippenaar, N. J. 1977 Variation in Crocidura mariquensis (A. Smith, 1844) in
southern Africa, Part 1 (Mammalia: Soricidae). Ann. Transvaal Mus. 30, 163-206.
4
van Zyll de Jong, C. G. & Kirkland, Jr., G. L. 1989 A morphometric analysis of
the Sorex cinereus group in central and eastern North America. J. Mammal. 70,
110-122.
5 Carraway, L. N. 1990 A morphologic and morphometric analysis of the “Sorex
vagrans species complex” in the Pacific coast region. Spec. Pub. Mus. Texas Tech
Univ. 32, 1-76.
6 Esselstyn, J. A., Timm, R. M. & Brown, R. M. 2009 Do geological or climatic
processes drive speciation in dynamic archipelagos? The tempo and mode of
diversification in southeast Asian shrews. Evolution. 63, 2595-2610.
7
Posada, D. 2008 jModelTest: phylogenetic model averaging. Mol. Biol.
Evol. 25, 1253-1256.
8
Drummond, A. J., Suchard, M. A., Xie, D. & Rambaut, A. 2012 Bayesian
phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969-1973.
9 Storch, G., Qui, Z. & Zazhigin, V. S. 1998 Fossil history of shrews in Asia. In
Evolution of Shrews (eds J. M. Wójcik J. M.& M. Wolsan), pp. 93–120. Mammal
Research Institute, Polish Academy of Sciences, Białowieża.
S7. SUPPLEMENTARY FIGURE LEGENDS
Figure S1. Study skin of the holotype of Scutisorex thori (FMNH 219669).
Figure S2. Bayesian estimates of phylogeny showing single locus gene trees for (a)
Cytochrome b and (b) von Willebrand Factor. Horizontal bars represent 95% highest
posterior densities on node ages. (a) is calibrated with the oldest known fossil crocidurine
shrew and a mitochondrial clock, while (b) is on an arbitrary time scale. Black diamonds
on nodes indicate posterior probably ≥0.95.
Figure S3. Dorsal view of the lumbar region of Crocidura olivieri (A; FMNH 192912),
Scutisorex somereni (B; FMNH 189277) and Scutisorex thori (C; FMNH 219669).
Figure S4. Map of distribution of Scutisorex, including S. thori n. sp., specimens
examined for this paper and two records of S. somereni outside of the known distribution
of the genus.