Cryptic speciation in Brazilian Epiperipatus (Onychophora: Peripatidae) reveals an underestimated diversity among the peripatid velvet worms.

Abstract

Taxonomical studies of the neotropical Peripatidae (Onychophora, velvet worms) have proven difficult, due to intraspecific variation and uniformity of morphological characters across this onychophoran subgroup. We therefore used molecular approaches, in addition to morphological methods, to explore the diversity of Epiperipatus from the Minas Gerais State of Brazil.
Our analyses revealed three new species. While Epiperipatus diadenoproctussp. nov. can be distinguished from E. adenocryptussp. nov. and E. paurognostussp. nov. based on morphology and specific nucleotide positions in the mitochondrial cytochrome c oxidase subunit I (COI) and small ribosomal subunit RNA gene sequences (12S rRNA), anatomical differences between the two latter species are not evident. However, our phylogenetic analyses of molecular data suggest that they are cryptic species, with high Bayesian posterior probabilities and bootstrap and Bremer support values for each species clade. The sister group relationship of E. adenocryptussp. nov. and E. paurognostussp. nov. in our analyses correlates with the remarkable morphological similarity of these two species. To assess the species status of the new species, we performed a statistical parsimony network analysis based on 582 base pairs of the COI gene in our specimens, with the connection probability set to 95%. Our findings revealed no connections between groups of haplotypes, which have been recognized as allopatric lineages in our phylogenetic analyses, thus supporting our suggestion that they are separate species.
Our findings suggest high cryptic species diversity and endemism among the neotropical Peripatidae and demonstrate that the combination of morphological and molecular approaches is helpful for clarifying the taxonomy and species diversity of this apparently large and diverse onychophoran group.

Full text

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Cryptic Speciation in Brazilian Epiperipatus
(Onychophora: Peripatidae) Reveals an Underestimated
Diversity among the Peripatid Velvet Worms
Ivo S. Oliveira1,3*., Gustavo A. Lacorte2., Cleusa G. Fonseca2, Alfredo H. Wieloch3, Georg Mayer1
1Institute of Biology: Animal Evolution & Development, University of Leipzig, Leipzig, Germany, 2Departmento de Biologia Geral, Instituto de Cie ˆncias Biolo ´gicas,
Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, 3Departamento de Zoologia, Universidade Federal de Minas Gerais, Instituto de Cie ˆncias
Biolo ´gicas, Belo Horizonte, Minas Gerais, Brazil
Abstract
Background: Taxonomical studies of the neotropical Peripatidae (Onychophora, velvet worms) have proven difficult, due to
intraspecific variation and uniformity of morphological characters across this onychophoran subgroup. We therefore used
molecular approaches, in addition to morphological methods, to explore the diversity of Epiperipatus from the Minas Gerais
State of Brazil.
Methodology/Principal Findings: Our analyses revealed three new species. While Epiperipatus diadenoproctus sp. nov. can
be distinguished from E. adenocryptus sp. nov. and E. paurognostus sp. nov. based on morphology and specific nucleotide
positions in the mitochondrial cytochrome c oxidase subunit I (COI) and small ribosomal subunit RNA gene sequences (12S
rRNA), anatomical differences between the two latter species are not evident. However, our phylogenetic analyses of
molecular data suggest that they are cryptic species, with high Bayesian posterior probabilities and bootstrap and Bremer
support values for each species clade. The sister group relationship of E. adenocryptus sp. nov. and E. paurognostus sp. nov.
in our analyses correlates with the remarkable morphological similarity of these two species. To assess the species status of
the new species, we performed a statistical parsimony network analysis based on 582 base pairs of the COI gene in our
specimens, with the connection probability set to 95%. Our findings revealed no connections between groups of
haplotypes, which have been recognized as allopatric lineages in our phylogenetic analyses, thus supporting our
suggestion that they are separate species.
Conclusions/Significance: Our findings suggest high cryptic species diversity and endemism among the neotropical
Peripatidae and demonstrate that the combination of morphological and molecular approaches is helpful for clarifying the
taxonomy and species diversity of this apparently large and diverse onychophoran group.
Citation: Oliveira IS, Lacorte GA, Fonseca CG, Wieloch AH, Mayer G (2011) Cryptic Speciation in Brazilian Epiperipatus (Onychophora: Peripatidae) Reveals an
Underestimated Diversity among the Peripatid Velvet Worms. PLoS ONE 6(6): e19973. doi:10.1371/journal.pone.0019973
Editor: William J. Etges, University of Arkanas, United States of America
Received December 1, 2010; Accepted April 21, 2011; Published June 10, 2011
Copyright: ? 2011 Oliveira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (www.cnpq.br) to AHW (CNPq - Brazil:
481868-2007-0) and ISO (student fellowship CNPq-Brazil, current process 290029/2010-4), the Fundac ¸a ˜o de Amparo a Pesquisa do Estado de Minas Gerais (www.
fapemig.br) to GAL and CGF (FAPEMIG, APQ-01133-08), and the German Research Foundation (www.dfg.de) to GM (DFG: MA 4147/3-1). GM is a Research Group
Leader supported by the DFG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ivo.sena@gmail.com
. These authors contributed equally to this work.
Introduction
The phylogeny and taxonomy of the neotropical Peripatidae is
understudied [1–4] and the estimated number of 70–80 described
species and subspecies apparently does not reflect the actual
diversity of the group [5]. The major difficulties with the peripatid
taxonomy arise from intraspecific variation and uniformity of
morphological characters – an issue that could be addressed by
applying molecular techniques, in addition to classical morpho-
logical methods. While scanning electron microscopy has revealed
a high morphological diversity of the neotropical Peripatidae (e.g.,
[1,2,4]), molecular methods have not been used to clarify the
genetic diversity of the group. However, these methods have
shown that cryptic speciation is a common phenomenon in the
Peripatopsidae, another large onychophoran taxon [6–14].
To provide a basis for future research on the neotropical
Peripatidae, we applied molecular and morphological methods,
including scanning electron microscopy, and analysed specimens
from four different localities of the Minas Gerais State of Brazil.
Our data suggest cryptic speciation and high endemism in the
neotropical Peripatidae and provide evidence of three new species
of Epiperipatus, for which we provide formal descriptions and type
designations to fulfil the requirements of the International Code of
Zoological Nomenclature (ICZN).
Results
General anatomy of the specimens studied
Since there are gaps in our knowledge of morphological
characters in representatives of Epiperipatus (Table S1), we examined
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and compared in detail Epiperipatus specimens from four different
localities in the Minas Gerais State of Brazil (numbered I to IV in
Figure 1A, B). The ground colour of all specimens is in vivo brown,
with numerous light-brown papillae spread over the body surface
(Figure 2A, C). In addition, there is a repeated pattern of bilateral
light-brown arcs on each side of the dark-brown dorsal midline,
which consist of five to six large and numerous small light-brown
dermal papillae (Figure 2C–E; Figure S1A, C). The arcs form
repeated circles enclosing one or two pairs of additional large, light-
brown primary papillae that are, however, missing in some circles
(Figure 2C–E). The dorsal body surface of fixed specimens is
greyish-brown and shows the same pattern as in living specimens.
The ventral body surface is in vivo pinkish-beige. The ventral organs
are brighter and clearly visible (Figure 2B; Figure S1B, D).
The antennal tip consists of 13 rings (including the terminal
button), with the 9th, 11thand 13thrings thinner than the others.
The eyes and the frontal organs are well-developed. The mouth is
surrounded by six to seven pairs of oral lips and one unpaired,
large anterior lip (Figure 3A, B). The dorsomedian furrow is
distinct along the entire body and the hyaline organs are present.
The dorsal integument shows 12 plicae per segment, four of which
(2ndwith 3rdand 11thwith 12th) anostomose with each other
towards each side of the body (Figures 3C, D and 4A). Thus, only
10 plicae per segment are seen laterally and only seven of them
(3rdto 9th) pass to the ventral side between each two subsequent leg
pairs (Figure 3C, D).
There are one to two accessory papillae between each two
primary papillae in the dorsal integument (Figure 4A), but a
variable number of two to six adjacent accessory papillae are
found along the dorsal midline (Figure S2A–F). The primary
papillae show roundish bases and vary in size. A distinct
constriction separates the apical and basal pieces (Figure 4B, C).
The basal pieces possess five to six lateral and seven to eight
anterior scale ranks (Figure 4B). The apical pieces are asymmet-
rical, with three to four anterior and two to three posterior scale
ranks (Figure 4B, C). Sensory bristles are thorn-shaped and
displaced posteriorly (Figure 4C). The primary papillae at the level
of legs and their apical pieces are elongated and possess slender
scales.
Each leg shows eight plical rings and four complete spinous pads
and a fifth fragmented pad (Figure 5A), but two posterior leg pairs
are reduced in size and bear only three complete spinous pads and
a fourth fragmented pad. Most feet have two anterior and one
posterior foot papillae, but some of them show only one anterior
and one posterior or only two anterior papillae. Each proximal
and distal setiform ridges on the ventral surface of the foot possess
one or two bristles. Eversible coxal vesicles are present at the bases
of most legs, except for the fourth and fifth leg pairs, which show
nephridial tubercles in a distal position between the third and
fourth spinous pads. The fourth pad is arched and complete (not
divided by the nephridial tubercle) in these leg pairs (Figure 5B).
Single crural tubercles are present in two pre-genital leg pairs in
males. The genital opening lies mid-ventrally in the segment of the
penultimate leg pair. The male genital pad is divided by a single
longitudinal furrow in two compartments whereas the female
genital pad is divided by two perpendicular furrows in four
compartments.
Morphological differences between the specimens
studied
Despite our detailed morphological analysis, including scanning
electron microscopy, we found only a few morphological
characters that differ between the specimens from different
localities. In particular, the anatomy of the anal gland papillae
differs in males from the Particular Reserve of Natural Patrimony
(=RPPN) Mata do Sossego (E. diadenoproctus sp. nov.: locality I in
Figure 1A, B) compared to those found at the three other localities
(E. adenocryptus sp. nov.: locality II, and E. paurognostus sp. nov.:
localities III and IV in Figure 1A, B). The anal gland papillae in
specimens of E. diadenoproctus sp. nov. are large, roundish and
Figure 1. Distribution of onychophoran species in the Minas Gerais State of Brazil, including the three new species described
herein. A, Overview. Species localities numbered as follows: I, Reserva Particular do Patrimo ˆnio Natural (RPPN) Mata do Sossego (type locality of
Epiperipatus diadenoproctus sp. nov.); II, Co ´rrego dos Ferreiras (type locality of E. adenocryptus sp. nov.); III, Mata do Eremite ´rio (type locality of E.
paurognostus sp. nov.); IV, Rancho Primavera (additional locality of E. paurognostus sp. nov.); V, RPPN Feliciano Miguel Abdala (type locality of E.
machadoi); VI, Estac ¸a ˜o Ecolo ´gica de Tripuı ´ (type locality of E. acacioi); VII, Parque Estadual do Itacolomi (additional locality of E. acacioi). B, Diagram
illustrating air-line distances between the localities of each species occurring in the Minas Gerais State.
doi:10.1371/journal.pone.0019973.g001
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brighter than the surrounding integument (Figure 5C) whereas
they are smaller, bean-shaped and hardly visible in males from
other localities (Figure 5D, E). Furthermore, the specimens of E.
diadenoproctus sp. nov. are characterised by different numbers of
leg pairs, with some males showing 28 leg pairs whereas specimens
of E. adenocryptus sp. nov. and E. paurognostus sp. nov. with the
same number of leg pairs are all females (Table 1). Thus, while E.
diadenoproctus sp. nov. can be distinguished morphologically, we
did not find any unambiguous distinctive characters between E.
adenocryptus sp. nov. and E. paurognostus sp. nov. Although the
numbers of leg pairs in each sex differ between the two species,
with overlapping numbers in E. paurognostus sp. nov. (Table 1),
this result has to be corroborated by using a large number of
specimens. Nevertheless, E. adenocryptus sp. nov. and E.
paurognostus sp. nov. can be distinguished unambiguously by
applying molecular methods.
Analyses of molecular data
The amplified cytochrome c oxidase subunit I (COI) fragments in all
specimens studied were 590 bp long. However, the sequence ends
of some fragments were of suboptimal quality and, therefore, had
to be excluded from our analysis so that the final alignment
contained 582 bp. Among 159 variable sites, 61 were parsimo-
niously informative (Figure S3). The average base frequencies
were A + T biased, in particular in the third codon position. Of
all variable sites, 68% were substituted in the third codon
position, while 20% showed substitutions in the first and 12% in
the second position. The translation of COI nucleotide sequences
into amino acid sequences revealed no stop codons, suggesting
that all sequences belong to functional mitochondrial protein-
coding genes. Furthermore, the alignment of the amino acid
sequences shows that of 194 amino acids, only 42 are variable
(Figure S4). The amplified small ribosomal subunit RNA (12S rRNA)
fragments were 355 bp long. Their alignment revealed 15 gap
sites distributed throughout the fragment lengths, which had to be
excluded from our analysis to avoid the necessity of entering a
new character state. Among 93 variable sites, 61 were
parsimoniously informative (Figure S5). Like in the COI
sequences, an A + T bias was found also in the 12S rRNA
sequences (Table S2).
Figure 2. Body colour pattern in onychophoran specimens studied, exemplified by Epiperipatus diadenoproctus sp. nov. Photographs
(A–C) and ink drawings (D, E). A, Dorsal colour pattern in an anaesthetised specimen. B, Ventral colour pattern in the same specimen. Arrows point to
the ventral organs. C, Detail of dorsal colour pattern. D, E, Diagrams showing slight variation of dorsal body colour pattern. D, Pattern with two pairs
of large papillae in each circle. E, Pattern with either missing or a variable number of large papillae in each circle. Abbreviations: at, antenna; go,
genital opening; mo, mouth.
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According to our data, the genetic distances range from 4.4% to
9.6% (including the Brazilian species only) and from 4.4% to
18.6% (including all species studied). Epiperipatus adenocryptus sp.
nov. displays the highest mean intraspecific genetic distance
(2.0%), while E. paurognostus sp. nov. shows the lowest value
(1.0%) (Table S3). Epiperipatus paurognostus sp. nov. and E.
diadenoproctus sp. nov. are the most divergent species, with high
interspecific distance values in all pair-wise comparisons, while E.
adenocryptus sp. nov. and E. paurognostus sp. nov. show the lowest
interspecific genetic distances (Table S3).
We used four different methods, Neighbor-Joining (NJ),
Maximum Parsimony (MP), Maximum Likelihood (ML) and
Bayesian Inference (BI), for phylogenetic analyses, which all
revealed similar topologies and the same monophyletic clades for
the hypothesised species (Figure 6; Figures S6, S7, S8, and S9).
The monophyly of each species is well-supported, except for a low
bootstrap support value (64%) for E. adenocryptus sp. nov. in the
MP topology (Figure S6). In all analyses, E. adenocryptus sp. nov.
sister groups with E. paurognostus sp. nov. The node supporting the
clade uniting E. diadenoproctus sp. nov., E. adenocryptus sp. nov.
and E. paurognostus sp. nov. shows low Bayesian posterior
probabilities (54) (Figure 6; Figure S7) and bootstrap support
values in both MP and NJ topologies (49% and 65%, respectively)
(Figure 6; Figures S6 and S8).
Our statistical parsimony network analyses revealed 22 haplo-
types and five separate networks among the COI sequences of the 26
specimensstudied (Figure7).Eachnetworkincludesspecimensfrom
a single location, except for E. adenocryptus sp. nov., which forms
two separate haplotype networks from the same locality. These
findings correspond well to the results of our phylogenetic analyses
and support the existence of three new allopatric species.
Taken together, the results of our morphological examinations
revealed only two novel morphotypes of Epiperipatus whereas the
molecular analyses provide evidence of three new well-supported
species clades, two of which are therefore cryptic (sensu ref. [15]).
Since the three new lineages are monophyletic in all our
phylogenetic analyses and form separate haplotype networks, we
recognise them as separate species.
Description of three new species of Epiperipatus
Epiperipatus diadenoproctus sp. nov.
org:act:992197FB-7D70-4A9A-A4F3-E3D39135B89D.
Holotype. =, BRAZIL, Minas Gerais, Simone ´sia, RPPN Mata do
Sossego, 1150 m, Atlantic rain forest, 20u049210S & 42u049120W,
15–19 July 2008, I. S. Oliveira & F. N. S. Queiro ´s (UFMG0140).
Paratypes. Same data as for holotype, R, 7–19 March 1999, U.
Caramaschi et al. (MNRJ0012); 1=, 3RR, 28 June 2008, I. S.
Oliveira & S. Genelhu ´ (UFMG0096-99); 29==, 27RR, 15–19 July
2008, I. S. Oliveira & F. N. S. Queiro ´s (UFMG0100-106, 108–
0139 & 0141–0157).
Etymology. The name diadenoproctus is derived from Greek du ´o (=
two), ade ´nez (= glands) and prvkto ´z (= anus) [16], in reference
to paired anal gland papillae present in males (Figure 5C).
Diagnosis. Anal gland papillae well-developed, roundish and
brighter than surrounding integument (Figure 5C); 26–28 leg pairs
in males and 29–30 in females, without overlap between sexes
(Table 1). COI and 12S rRNA sequences as in specimens MS1-MS5
urn:lsid:zoobank.
Figure 3. Diagrammatic arrangement of oral lips and plicae in onychophoran specimens studied. A, Single unpaired and six paired lips
(numbered). Anterior is up. B, Single unpaired and seven paired lips (numbered). Anterior is up. C, Arrangement of plicae (numbered) in lateral view.
D, Arrangement of plicae (numbered) in dorsal view. Note two pairs of anastomosing plicae in each leg-bearing segment. Abbreviations: le, legs; ul,
unpaired anterior lip.
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(Table 2). The species is characterised by 14 specific nucleotide
positions: four in the COI sequence and 10 in the 12S rRNA
sequence (Table 3). Phylogenetic relationship as in Figure 6; intra-
and interspecific distances as in Table S3.
Description. Body length after fixation 10.5–44.5 mm, width 1.6–
5.0 mm, height 1.2–3.5 mm. Juveniles reddish-brown and without
any pattern. Antennae with 38 to 43 rings, 25 to 30 of which in
antennal body and remaining 13 in antennal tip (including
terminal button). Outer jaw blade with one principal tooth and
one or two accessory teeth (Figure S10A); inner jaw blade with one
principal tooth, two accessory teeth and 10 denticles (Figure
S10B); accessory teeth with straight and parallel anterior and
posterior faces and a convex ventral face, forming acute angle with
posterior face and obtuse angle with anterior face. Remaining
characters, shared with E. adenocryptus sp. nov. and E. paurognostus
sp. nov., as described above (see the Results section entitled
‘‘General anatomy of the specimens studied’’).
Remarks on anomalies. One male shows an asymmetrical number
of 27 and 28 legs on each body side (Table 1) and another male
has doubled crural tubercles on a pre-genital leg.
Remarks on habitat preference. The juveniles of E. diadenoproctus sp.
nov. inhabit leaf litter whereas the adults occur either under or
within rotten logs. In addition, adult specimens were found in
human rubbish (roof clay tiles) placed in front of a researchers’
accommodation, close to the border of the investigated forest
fragment. According to the locals, the roof tiles and rubbish had
remained untouched for five years and contained a diverse
invertebrate fauna. In contrast to the animals found in human
rubbish, specimens collected in the forest were always solitary and
a gregarious behaviour was not observed.
Distribution.Epiperipatusdiadenoproctussp.nov.occursonlyatthetype
locality, the RPPN Mata do Sossego (locality I in Figure 1A, B). A re-
examination of the material identified as ‘‘Peripatus sp. 3’’ reported
from RPPN Mata do Sossego and from three other localities by
Figure 4. Arrangement of plicae and structure of dermal papillae in onychophoran specimens studied. Scanning electron micrographs.
A, Portion of dorsolateral integument in E. adenocryptus sp. nov. showing plical anastomoses. The plicae are numbered. Anterior is up, median is
right. B, Dorsal primary papilla in E. adenocryptus sp. nov. showing five lateral (black dots) and seven anterior scale ranks (white dots) in the basal
piece and four anterior scale ranks in the apical piece (asterisks). Anterior is up, median is left. Note the posteriorly displaced sensory bristle (arrow).
C, Detail of an asymmetrical apical piece in E. paurognostus sp. nov. showing only two posterior scale ranks (asterisks) and a thorn-shaped and
posteriorly displaced bristle. Abbreviations: ac, accessory papillae; ap, apical piece; bp, basal piece; br, sensory bristle; cs, constriction between apical
and basal pieces; pp, primary papillae.
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Sampaio-Costa et al. [5] revealed that only the specimens from Mata
do Sossego, the type locality, belong to E. diadenoproctus sp. nov.
Epiperipatus adenocryptus sp. nov.
act:D172B531-2CFA-4128-B220-DD45AE2218BB.
Holotype. =, BRAZIL, Minas Gerais, Santa Ba ´rbara do Leste,
Co ´rrego dos Ferreiras, 1050 m, Atlantic rain forest, 42u06946.
97580W & 19u58959.246190S, 17 June 2008, I. S. Oliveira & S.
Genelhu ´ (UFMG0071).
Paratypes. Same data as for holotype, R, 01 November 2003, E.
T. Silva (UFMG0016); 11==, 12RR, 17 June 2008, I. S. Oliveira &
S. Genelhu ´ (UFMG0070; 0072-0091).
Etymology. The name adenocryptus is derived from Greek ade ´nez
(= glands) and kruptoz (= hidden) [16], in reference to hardly
visible anal gland papillae in males (Figure 5D).
Diagnosis. Anal gland papillae poorly developed, bean-shaped,
hardly visible and similar in colour to surrounding integument
(Figure 5D); 26–27 leg pairs in males and 28–30 in females,
without overlap between sexes (Table 1). COI and 12S rRNA
urn:lsid:zoobank.org:
Figure 5. Structure of spinous pads and male anal gland papillae in onychophoran specimens studied. Light micrographs (A, C–E) and
scanning electron micrograph (B). A, Leg from the mid-body of E. diadenoproctus sp. nov. in ventral view. Spinous pads are numbered. Note the
presence of a fifth fragmented pad. B, Distal portion of fifth leg of E. paurognostus sp. nov. showing nephridial tubercle, complete fourth pad and
fragmented fifth pad. C–E, Posterior ends in males of E. diadenoproctus sp. nov. (C), E. adenocryptus sp. nov. (D) and E. paurognostus sp. nov.
(E) showing anal gland papillae (arrows). Note the well-developed, roundish anal gland papillae in E. diadenoproctus sp. nov. Abbreviation: ft, foot;
nt, nephridial tubercle.
doi:10.1371/journal.pone.0019973.g005
Table 1. Numbers of leg pairs in specimens of each sex in the
three new onychophoran species.
SpeciesSexNumber of leg pairs
2627 28 2930
E. diadenoproctus sp. nov.
==
420*
6--
RR
--- 283
E. adenocryptus sp. nov.
==
215---
RR
--3 122
E. paurognostus sp. nov.
==
5 11---
RR
-148-
*Including one male, which shows an asymmetrical number of 27 and 28 legs
on each side of the body.
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sequences as in specimens CS1–CS7 (Table 2). The species is
characterised by six specific nucleotide positions in the COI
sequence and a single position in the 12S rRNA sequence (Table 3).
Phylogenetic relationship as in Figure 6; intra- and interspecific
distances as in Table S3.
Description. Body length after fixation 12.3–43.0 mm, width 1.7–
4.4 mm, height 1.1–3.6 mm. Juveniles reddish-brown and with
the same pattern as in adults. Antennae with 38 to 43 rings, 25 to
30 of which in antennal body and remaining 13 in antennal tip
(including terminal button). Outer jaw blade with one principal
and one accessory teeth (Figure S10C); inner jaw blade with one
principal tooth, one or two accessory teeth and seven denticles
(Figure S10D); first (anterior-most) accessory tooth larger than
second and with straight and parallel anterior and posterior faces;
ventral face straight or convex, forming acute angle with posterior
face and obtuse angle with anterior face; second accessory tooth
thorn-shaped; asymmetric number of accessory teeth (either one or
two) on inner jaws of left and right body sides in some specimens.
Remaining characters, shared with E. diadenoproctus sp. nov. and
E. paurognostus sp. nov., as described above (see the Results section
entitled ‘‘General anatomy of the specimens studied’’).
Remarks on habitat preference. Adults and juveniles of Epiperipatus
adenocryptus sp. nov. are found in leaf litter and inside rotten logs.
Distribution. Epiperipatus adenocryptus sp. nov. has been recorded
only from the type locality, Co ´rrego dos Ferreiras (locality II in
Figure 1A, B).
Epiperipatus paurognostus sp. nov.
act:CC6B56A8-6DA8-4BB6-80FD-E2960EE356F2.
urn:lsid:zoobank.org:
Holotype. =, BRAZIL, Minas Gerais, Piedade de Caratinga,
Mata do Eremite ´rio, 897 m, Atlantic rain forest, 42u05922.
750850W & 19u45933.970230S, 14 June 2008, I. S. Oliveira &
S. Genelhu ´ (UFMG0065).
Paratypes. Same data as for holotype, R, 31 May 2008, H. Coelho
& S. Genelhu ´ (UFMG0057); 2==, 7RR, 14 June 2008, I. S.
Oliveira & S. Genelhu ´ (UFMG0058-0064 & UFMG0066); 1=,
2RR, 22 July 2009, I. S. Oliveira & G. A. Lacorte (UFMG0179–
0181); 1=, Piedade de Caratinga, Rancho Primavera, 830 m,
Atlantic rain forest, 42u3935.540W–19u4594.430S, 23 April 2009,
H. Coelho (UFMG0184).
Etymology. The name paurognostus is derived from Greek pauroz (=
little) and cnvsto ´z (= distinguished) [16], in reference to a
remarkable morphological similarity of E. paurognostus sp. nov. to E.
adenocryptussp.nov.,whichmakesitdifficulttodistinguishthespecies.
Diagnosis. Anal gland papillae poorly developed, bean-shaped,
hardly visible and of the same colour as surrounding integument
(Figure 5E); number of leg pairs overlapping between sexes: 26–27
in males and 27–29 in females (Table 1). COI and 12S rRNA
sequences as in specimens ME1-ME10 (Table 2). The species is
characterised by 10 specific nucleotide positions in the COI
sequence and a single position in the 12S rRNA sequence (Table 3).
Phylogenetic relationship as in Figure 6; intra- and interspecific
distances as in Table S3.
Description. Body length after fixation 11.1–44.1 mm, width 1.7–
3.9 mm, height 1.2–3.3 mm. Colour pattern in juveniles as in E.
adenocryptus sp. nov. Antennae with 37 to 42 rings, 24 to 29 of which
in antennal body and remaining 13 in antennal tip (including
Figure 6. Maximum Likelihood topology amongst onychophoran specimens from different localities. Combined mitochondrial data
sets (COI + 12S rRNA), with E. biolleyi as an outgroup. Bayesian posterior probabilities and bootstrap values are given in the following order: BI/NJ/ML//
MP/Bremer/Relative Bremer index decay. Abbreviations: CS, Co ´rrego dos Ferreiras; FM, RPPN Feliciano Miguel Abdala; ME, Mata do Eremite ´rio; MS,
RPPN Mata do Sossego.
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terminal button). Outer jaw blade with one principal and one
accessory teeth (Figure S10E); inner jaw blade with one principal
tooth, one or two accessory teeth and six to nine denticles (Figure
S10F); accessory teeth and principal tooth of inner jaw blade similar
in shape; accessory tooth either well-developed and with convex
anterior and straight posterior faces or vestigial and thorn-shaped;
asymmetric number of accessory teeth (either one or two) on inner
jawsofleftandrightbodysidesinsomespecimens(asinE.adenocryptus
sp. nov.). Remaining characters, shared with E. diadenoproctus sp.
nov. and E. adenocryptus sp. nov., as described above (see the Results
section entitled ‘‘General anatomy of the specimens studied’’).
Remarks on habitat preference. Specimens of E. paurognostus sp. nov.
are found under rotten logs and in leaf litter close to watercourses.
Juveniles occur within small pieces of rotten wood. Locals
recorded the species in bean straw, which is used as a fertilizer
in coffee plantations bordering the native forest remnant.
Distribution. Epiperipatus paurognostus sp. nov. has been recorded
from the type locality, Mata do Eremite ´rio, and from Rancho
Primavera (localities III and IV in Figure 1A, B).
Discussion
Evidence of three new species of Epiperipatus
So far, only two onychophoran species, Epiperipatus machadoi
(Oliveira & Wieloch, 2005) and E. acacioi (Marcus & Marcus,
1955), have been described from the Minas Gerais State of Brazil
[4], although this region occupies an area about as large as France.
However, a recent report of ‘‘Peripatus sp. 3’’ close to the type
Figure 7. Haplotype networks for the COI sequences of Epiperipatus specimens from different localities. Abbreviations as per Table 2.
The connection probability was set to 95% (see ref. [48]). Each dot indicates one missing or unsampled haplotype. Two or more names in one frame
represent identical genotypes.
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locality of E. machadoi suggests a higher number of species in this
region than have been described thus far [5]. Our data indeed
revealed three additional separate lineages occurring close to the
type locality of E. machadoi. We recognise them as different species
since the lineages are monophyletic in all our analyses and are
delineated by their geographical locations. The molecular
differences and the allopatric distribution indicate that these
species do not hybridise and their recognition as species is
therefore in accordance with both the biological and phylogenetic
species concepts [17,18].
Our statistical parsimony analyses revealed five separate
networks, three of which correspond to the three allopatric species
(E. machadoi, E. diadenoproctus sp. nov. and E. paurognostus sp. nov.),
thus supporting the results of our phylogenetic analyses. In contrast,
specimens of E. adenocryptus sp. nov. fell apart in two unconnected
haplotypenetworks,which is not inaccordance with the assumption
Table 2. Origin of onychophoran specimens sequenced and corresponding GenBank accession numbers.
Taxon Collecting citeSpecimenGenBank accession number (COI)GenBank accession number (12S rRNA)
E. adenocryptus sp. nov. Co ´rrego dos FerreirasCS1 HQ236108HQ236134
CS2HQ236109 HQ236135
CS3 HQ236110HQ236136
CS4 HQ236111 HQ236137
CS5HQ236112 HQ236138
CS6 HQ236113HQ236139
CS7 HQ236114HQ236140
E. diadenoproctus sp. nov. RPPN Mata do Sossego MS1HQ236093HQ236119
MS2HQ236094HQ236120
MS3HQ236095 HQ236121
MS4 HQ236096HQ236122
MS5 HQ236097HQ236123
E. machadoiRPPN Feliciano Miguel Abdala FM1HQ236089 HQ236115
FM2HQ236090 HQ236116
FM3HQ236091HQ236117
FM4 HQ236092HQ236118
E. paurognostus sp. nov.Mata do Eremite ´rio ME1HQ236098 HQ236124
ME2 HQ236099HQ236125
ME3 HQ236100HQ236126
ME4 HQ236101HQ236127
ME5 HQ236102HQ236128
ME6 HQ236103 HQ236129
ME7 HQ236104 HQ236130
ME8 HQ236105HQ236131
ME9 HQ236106HQ236132
ME10HQ236107HQ236133
doi:10.1371/journal.pone.0019973.t002
Table 3. Unambiguous synapomorphies (character states in parentheses) of each Epiperipatus species studied.
Species
Synapomorphies*
N
COI
12S rRNA
1st**2nd** 3rd**
E. adenocryptus sp. nov.7 457(C); 583(A)479(T)495(G); 666(G) 708(T) 425(G)
E. diadenoproctus sp. nov. 14369(G); 381(A) 534(A); 555(T)127(A); 154(T); 173(A) 237(G); 260(G);
288(A) 294(A); 296(G); 328(G) 337(G)
E. machadoi18238(A) 237(G); 270(T) 273(C); 363(A) 429(G); 417(T)
489(A); 495(T) 510(A); 537(A) 600(T)
204(G); 207(T); 215(A); 269(A); 278(A);
362(T)
E. paurognostus sp. nov.10241(T); 274(T) 242(T); 257(G) 278(T)249(T); 258(C) 261(C); 270(C)425(C)
N = total number of synapomorphies.
*The nucleotide positions are based on complete COI and 12S rRNA sequences of E. biolleyi (GenBank: DQ666064).
**Codon position.
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that the DNA sequences from single species typically stick together
in a single haplotype network [19]. Thisresult might be due to a low
number of specimens sequenced and to numerous missing
haplotypes in our analyses, which have caused the disruption of a
single network in two separate subnetworks. Alternatively, E.
adenocryptus sp. nov. might be a complex of two sympatric species.
Including additional specimens and covering unsampled haplotypes
in future analyses might clarify whether E. adenocryptus sp. nov. is a
single species or a species complex.
All our specimens can be assigned to Epiperipatus, based on the
presence of two anterior and one posterior foot papillae in most leg
pairs, a low number of scale ranks in the basal pieces of dorsal
primary papillae and the presence of crural papillae in two pre-
genital leg pairs in males [1–4]. The three new species differ
morphologically from E. acacioi and E. machadoi in that their males
possess paired anal glands (also called ‘‘male accessory glands’’),
which open to the exterior on specialised papillae close to the anus
(= anal gland papillae). Apart from the three new species, anal
gland papillae have been described among representatives of
Epiperipatus only in E. biolleyi (Bouvier, 1902) from Costa Rica and
in E. edwardsii (Blanchard, 1847) from Sarare, Venezuela (Table
S1). Given that E. edwardsii might be a species complex [5], the
occurrence of anal gland papillae has to be clarified in specimens
of E. edwardsii from other localities.
The paired anal glands and anal gland papillae are present in
outgroup taxa, such as Oroperipatus and Plicatoperipatus from the
neotropics and Typhloperipatus from South-East Asia [20–22] but are
absentinE.acacioiand E.machadoi, whichoccurcloseto the localities
of the three new species [4]. Clarifying whether the anal glands
occur in other species of Epiperipatus, for which the corresponding
data are currently missing (Table S1), will help understand the
evolution of these structures and might provide a useful character
for phylogenetic studies of the neotropical Peripatidae.
Cryptic speciation and high endemism in the neotropical
Peripatidae
The localised distributions of the three new species of Epiperipatus
in a relatively small area of the Minas Gerais State of Brazil confirm
Sampaio-Costa et al.’s [5] suggestion of high species diversity in the
neotropical Peripatidae. In particular, Epiperipatus is according to
our data one of the most speciose onychophoran genera, with
numerous crypticspecies still awaiting formaldescription. However,
we caution that the monophyly of this genus has not been
demonstrated yet and additional morphological (Table S1) and
molecular data are required for a better understanding of species
diversity and phylogeny of this onychophoran group.
The high endemism found in the three new species of Epiperipatus
(Figure 1A, B) is not restricted to this taxon, but is a common
phenomenon of Onychophora as it was also found in representa-
tives of Peripatopsidae [7,9,12,23]. One of the reasons for the high
endemism of the onychophoran species might be their low dispersal
ability since they are confined to microhabitats with high moisture
levels (e.g., [24]). Therefore, the putative wide distribution of some
species, such as E. edwardsii, O. balzani (Camerano, 1897) and O.
eisenii (Wheeler, 1898), over hundreds or even thousands of
kilometres [5,25], is doubtful. The high endemism found amongst
most other onychophoran species argues against the existence of
species showing such a wide distribution. Previous reports of widely
distributed species, thus, might be based on misidentified specimens
and have to be reconsidered.
Conclusion
In this paper, we give formal names and provide combined
morphological and molecular diagnoses for three new species of
Epiperipatus. This approach is uncommon among taxonomists,
which is unfortunate because taxonomical studies based on
morphological data alone apparently underestimate the cryptic
diversity of Onychophora, in particular of the neotropical
Peripatidae. Currently, a combination of morphological and
molecular methods seems to be the best approach for delineating
and identifying the onychophoran species, in particular of those
lineages that show a low number of distinctive morphological
characters. We believe that this approach will help handle the
cryptic diversity and clarify the phylogeny and taxonomy of
Onychophora in future studies. In addition, the potential value of
the so-called ‘‘DNA barcodes’’ has to be considered for species
identification in future studies of Onychophora as this method has
proven useful in other animal groups (see, e.g., refs. [26,27]). This
will accelerate the slow pace, at which new onychophoran species
are currently described, with only five formal species descriptions
published in the last five years [28–32]. This is quite detrimental to
studies of biological diversity because unnamed species are usually
not taken into consideration for conservation programs.
Materials and Methods
Areas studied and collection of specimens
In addition to thetwolocalitiesof E. acacioi and oneof E. machadoi,
four collecting sites were sampled (I–IV in Figure 1A, B): (I) RPPN
Mata do Sossego (municipalities of Simone ´sia and Manhuac ¸u,
20u049200S, 42u049120W, 1150 m); (II) Co ´rrego dos Ferreiras
(municipality of Santa Ba ´rbara do Leste, 42u069460W–19u589590S,
1050 m); (III) Mata do Eremite ´rio (Sa ˜o Jose ´ convent, municipality
of Piedade de Caratinga, 42u059220W–19u459330S, 897 m); and
(IV) Rancho Primavera (municipality of Piedade de Caratinga,
42u3935.540W–19u4594.430S, 830 m). All localities are areas of the
fragmented Atlantic rain forest complex called Caratinga-Sossego.
Specimens were collected from leaf litter and from within or
under rotten logs as described previously [4]. Most specimens from
Mata do Sossego were found in human rubbish (roof clay tiles),
placed in front of a researchers’ accommodation near the border
of the forest remnant. A total of 125 specimens were analysed
(Table 4), including two single specimens from the Museu
Nacional do Rio de Janeiro [MNRJ, National Museum of Rio
de Janeiro] and the collection of the Departamento de Zoologia da
Universidade Federal de Minas Gerais [DZUFMG, Department
of Zoology of Universidade Federal de Minas Gerais]. All
specimens were collected under the Brazilian federal license
(ICMBio) number 10432/3.
Table 4. Number of specimens of new onychophoran species
analysed from each locality.
Collecting site
Number of specimens used for
morphological analyses molecular analyses
RPPN Mata do Sossego62*
5
Co ´rrego dos Ferreiras34**
7
Mata do Eremite ´rio 2810
Rancho Primavera1-
*Including one specimen from the MNRJ collection.**Including one specimen
from the DZUFMG collection.
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Morphological analyses
The specimens were photographed in vivo and sacrificed using a
piece of cotton soaked with ether and placed into a Petri dish.
They were then analysed using an Olympus SZ61 stereomicro-
scope. In addition, a piece of dorsal integument and the fifth right
leg from each specimen were fixed, handled and analysed in a
Quanta 200-FEG-FEI-2006 Scanning Electron Microscope (FEI,
Hillsboro, Oregon, USA) as described previously [4]. Additional
data were obtained from the type series of (1) Peripatus heloisae
Carvalho, 1941, (2) Epiperipatus acacioi and (3) E. machadoi, held in
the MNRJ, the Museu de Zoologia da Universidade de Sa ˜o Paulo
[MZUSP, Museum of Zoology of Universidade de Sa ˜o Paulo] and
the DZUFMG, respectively. The terminology of morphological
terms was used according to Oliveira et al. [4].
Molecular and phylogenetic analyses
Tissue samples from 22 specimens from different localities were
used for molecular studies (Table 4). In addition, we included four
specimens of E. machadoi from RPPN Feliciano Miguel Abdala in
our analyses for comparison since this species occurs close to the
type localities of the three new species described herein (Figure 1A,
B; Table 4). Epiperipatus biolleyi was selected as an outgroup since it
was the only species of the genus, for which all required molecular
data were available [33]; GenBank accession number: DQ666064).
The samples were preserved in ethanol. The genomic DNA was
extracted from body pieces (,25 mg) using the DNeasy Tissue Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
protocol. DNA sequences of the mitochondrial COI gene were
amplified using the specific primers COI5584 (59-TGTGA-
CTGGTCATGCATTTGT-39) and COI6174 (59-GAAACTAT-
TCCAAAGCCAGGAA-39), designed for this study using the COI
sequenceofE. biolleyi.Thesequencesofthemitochondrial 12SrRNA
gene were amplified using the primers SR-J-14233 and SR-N-
14588 from Simon et al. [34]. The COI and 12S rRNA loci were
selected because they show numerous variable sites and were used
successfully in studies of genetic variation and cryptic speciation in
Peripatopsidae [8,9,11,12,14].
PCR amplifications were performed in 20 ml reaction volumes
containing 40 ng genomic DNA, Buffer 1B (PhoneutriaH, Belo
Horizonte, Brazil: 1.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl,
0.1% Triton X-100), 0.8 mM dNTPs, 0.3 mM primers, 1% bovine
serum albumin (BSA) and 1 unit Taqpolymerase (PhoneutriaH).After
an initial denaturing step for 5 min at 94uC, the PCR conditions for
the COI and 12S rRNA fragments followed a standard three-step
protocol, with 27 cycles of (1) denaturing for 45 s at 94uC, (2)
annealing for 45 s at 56uC(COI primers)or54uC (12S rRNA primers),
and (3) extensionfor1 min at 72uC,followed bya final extension step
for 5 min at 72uC. The PCR products were purified using a solution
of 20% polyethylene-glycol (PEG 8000) and 2.5M NaCl according to
Sambrook et al. [35]. After purification, the PCR products were
sequenced in both directions using the BigDye Terminator Kit v3
(Applied Biosystems, Foster City, USA) and an ABI3100H automated
sequencer (Applied Biosystems, Foster City, USA). The sequences
were assembled and checked for quality using Phred v.0.20425 [36],
[37] and Phrap v.0.990319 [38] and the assembled chromatograms
were verified and edited using Consed 12.0 [39]. The sequences were
deposited in the GenBank database (Table 2).
The obtained sequences were aligned using the Clustal W
algorithm implemented in MEGA 4.1 [40]. This software was also
used for calculating the intraspecific and interspecific genetic
distances using the Kimura 2-parameter (K2P) model. Modeltest
3.7 [41] was used to select the best-fit model of sequence evolution.
The selected model for the COI data set was the General Time
Reversible Model (GTR + I + C) with gamma distributed (C) rates
a=0.6886 and a proportion of invariant sites I=0.3536. For the
12S rRNA data set, the model GTR + C was selected with
a=0.2944 and I=0. For the combined data set, the selected
model was GTR + I + C, with a=0.8048 and I=0.4049.
For phylogenetic analyses, NJ, MP, ML and BI were used. The
NJ analyses were conducted using PAUP*4.0b10 [42] and the ML
distances were determined using Modeltest 3.7 [41]. The support
for each clade was assessed by 1,000 bootstrap replicates [42]. The
MP analyses were implemented using PAUP*4.0b10 [42],
including 100 replicates of random sequence addition with tree
bisection and reconnection (TBR) branch swapping. The support
for each clade was assessed by 1,000 bootstrap replicates [43] and
by estimating Bremer support values (decay index-DI) of the strict
consensus tree. Unambiguous synapomorphies were identified
after performing the MP analyses and used as diagnostic molecular
characters for each species. The TNT software was used for
estimating Bremer support values and identifying unambiguous
synapomorphies [44]. For the ML inference analyses, the PhyML
software [45] was implemented using the selected model and
support of clades assessed by bootstrap analyses with 1,000
replicates. The BI analysis was performed with four Markov chain
Monte Carlo chains, which run simultaneously for 10,000,000
generations, with trees sampled every 100 generations for a total of
100,000 trees. Posterior probabilities were calculated based on
trees retained after log-likelihood values had stabilised. All BI
analyses were performed with MrBayes v3.0b4 [46].
To calculate the haplotype networks, we performed a statistic
parsimony analysis of the COI sequences from all 22 specimens
sequenced and four additional specimens of E. machadoi using the
TCS v1.21 software [47]. The connection limit excluding the
homoplastic changes was set to 95% according to Hart & Sunday
[19], whose statistic analyses of empirical data have shown that
alignments of DNA sequences typically fall apart into separate
networks corresponding to Linnean species. This suggests that
network parsimony analyses are useful for species detection
using molecular data, in particular the mitochondrial DNA data
sets [19,48].
Taxonomic Registration and Digital Archiving
The electronic version of this document does not represent a
published work according to the International Code of Zoological
Nomenclature (ICZN), and hence the nomenclatural acts
contained in the electronic version are not available under that
Code from the electronic edition. Therefore, a separate edition of
this document was produced by a method that assures numerous
identical and durable copies, and those copies were simultaneously
obtainable (from the publication date noted on the first page of this
article) for the purpose of providing a public and permanent
scientific record, in accordance with Article 8.1 of the Code. The
separate print-only edition is available on request from PLoS by
sending a request to PLoS ONE, Public Library of Science, 1160
Battery Street, Suite 100, San Francisco, CA 94111, USA along
with a check for $10 (to cover printing and postage) payable to
"Public Library of Science".
The online version of the article is archived and available from the
following digital repositories: PubMedCentral (www.pubmedcentral.
nih.gov/), and LOCKSS (http://www.lockss.org/lockss/). In addi-
tion, this published work and the nomenclatural acts it contains have
been registered in ZooBank, the proposed online registration system
for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be
resolved and the associated information viewed through anystandard
web browser by appending the LSID to the prefix "http://zoobank.
org/". The ZooBank LSID for this publication is: urn:lsid:zoobank.
org:pub:E0156AE2-CD2D-415B-94C8-6278B4A12FD2.
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Supporting Information
Figure S1
adenocryptus sp. nov. (A, B) and E. paurognostus sp.
nov. (C, D). A, C, Specimens in dorsal view. B, D, Specimens in
ventral view. Arrows indicate the ventral organs. Abbreviation:
mo, mouth.
(TIF)
Body colour pattern in living specimens of E.
Figure S2
primary papillae (black circles) along dorsal midline in
the new onychophoran species. Female paratypes of E.
diadenoproctus sp. nov. (A, B), E. adenocryptus sp. nov. (C, D) and E.
paurognostus sp. nov. (E, F).
(TIF)
Arrangement of accessory (white dots) and
Figure S3
mitochondrial COI gene in the sampled taxa. Dots indicate
similar bases between the specimens studied and Epipepipatus
machadoi (FM1). The first nucleotide corresponds to the first codon
position. Abbreviations as per Table 2.
(TIF)
Alignment of nucleotide sequences of the
Figure S4
from COI nucleotide sequences. Dots indicate similar amino
acids between the specimens studied and Epipepipatus machadoi
(FM1). Abbreviations as per Table 2.
(TIF)
Alignment of amino acid sequences inferred
Figure S5
mitochondrial 12S rRNA gene in the sampled taxa. Dots
indicate similar bases between the specimens studied and
Epipepipatus machadoi (FM1). Abbreviations as per Table 2.
(TIF)
Alignment of nucleotide sequences of the
Figure S6
combined mitochondrial data sets (COI + 12S rRNA)
amongst Epiperipatus specimens studied. Epiperipatus
biolleyi was used as an outgroup. Upper numbers at each node
represent bootstrap support values, lower numbers are absolute
and relative Bremer support values. Abbreviations as per Table 2.
(TIF)
Maximum Parsimony (MP) topology for
Figure S7
bined mitochondrial data sets (COI + 12S rRNA)
amongst Epiperipatus specimens studied. Epiperipatus
biolleyi was used as an outgroup. Numbers at each node are
Bayesian posterior probabilities. Scale bar represents genetic
distance (substitutions per site). Abbreviations as per Table 2.
(TIF)
Bayesian Inference (BI) topology for com-
Figure S8
mitochondrial data sets (COI + 12S rRNA) amongst
Epiperipatus specimens studied. Epiperipatus biolleyi was used
as an outgroup. Numbers at each node are bootstrap support
values. Scale bar represents genetic distance (substitutions per site).
Abbreviations as per Table 2.
(TIF)
Neighbor-Joining (NJ) topology for combined
Figure S9
combined mitochondrial data sets (COI + + 12S rRNA)
amongst Epiperipatus specimens studied. Epiperipatus
biolleyi was used as an outgroup. Numbers at each node are
bootstrap support values. Scale bar represents genetic distance
(substitutions per site). Abbreviations as per Table 2.
(TIF)
Maximum Likelihood (ML) topology for
Figure S10
the new onychophoran species. Light micrographs. A, B,
Epiperipatus diadenoproctus sp. nov. C, D, E. adenocryptus sp. nov. E,
F, E. paurognostus sp. nov. Anterior is up, ventral is left in all
images. A, Outer jaw blade with a principal tooth and two
accessory teeth. B, Inner jaw blade with a principal tooth, two
accessory teeth and ten denticles. C, Outer jaw blade with a
principal tooth and one accessory tooth. D, Inner jaw blade with a
principal tooth, two accessory teeth and six denticles. E, Outer jaw
blade with a principal tooth and one accessory tooth. F, Inner jaw
blade with a principal tooth, two accessory teeth, and seven
denticles. Abbreviations: at, accessory tooth/teeth; dt, denticles;
pt, principal tooth.
(TIF)
Structure of outer and inner jaw blades in
Table S1
ipatus species described thus far.
(DOC)
Comparison of anatomical features in Epiper-
Table S2
(DOC)
Summary of statistics for sequence data.
Table S3
onychophoran taxa (COI + + 12S rRNA) according to the
Kimura 2-parameter model.
(DOC)
Average genetic distances within and between
Acknowledgments
We are thankful to Adriano Kury, Amazonas Chagas Ju ´nior and Cristiano
Sampaio-Costa (National Museum of Rio de Janeiro—MNRJ), for
providing access to additional specimens used for comparison. We thank
Adalberto Jose ´ dos Santos for providing equipment and his help with multi-
focus light micrographs. The staff of the Centre of Microscopy at the
UFMG is thanked for providing equipment and technical support for
experiments involving scanning electron microscopy. The staff of the
Instituto Chico Mendes de Conservac ¸a ˜o da Biodiversidade (ICMBio)
provided the collecting permits. Special thanks to Mario Cozzuol and
Rodrigo Redondo for their help with phylogenetic analyses, to Felipe
Natali, Sebastia ˜o Genelhu ´, Harley Coelho, Emanuel Teixeira da Silva and
his father Jose ´ Ferreira da Silva for their help with the fieldwork, to
Kendroa and Kenderson for their hospitality, and to Christoph Bleidorn
and Lars Podsiadlowski for fruitful discussions and useful suggestions on an
earlier version of the manuscript. Two anonymous reviewers provided
useful comments, which helped improve the manuscript.
Author Contributions
Conceived and designed the experiments: ISO GAL GM. Performed the
experiments: ISO GAL. Analyzed the data: ISO GAL GM. Contributed
reagents/materials/analysis tools: ISO GAL AHW CGF. Wrote the paper:
ISO GAL GM.
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PLoS ONE | www.plosone.org13 June 2011 | Volume 6 | Issue 6 | e19973