Received: 4 October 2018 

|  Revised: 10 December 2018 

|
  Accepted: 10 December 2018

DOI: 10.1111/zsc.12338

ORIGINAL ARTICLE

On the importance of fine‐scale sampling in detecting alpha

taxonomic diversity among saproxylic invertebrates: A velvet
worm (Onychophora: Opisthopatus amaxhosa) template

Aaron Barnes  | Savel R. Daniels

Department of Botany and

Zoology, University of Stellenbosch,
Abstract
Matieland, South Africa The phylogeography and social structure of the narrow endemic velvet worm species
Opisthopatus amaxhosa were investigated by conducting fine‐scale sampling in its
Correspondence
Savel R. Daniels, Department of Botany distribution range in the Eastern Cape province of South Africa. In addition, and as
and Zoology, University of Stellenbosch, part of larger grant on forest biodiversity, Opisthopatus specimens sampled at locali-
Matieland, South Africa.
ties not included during a recent evaluation of the genus were included in a new
Email: srd@sun.ac.za
phylogeny. A total of 89 specimens from 18 sample localities were collected at three
Funding information forest patches for O. amaxhosa samples, while an additional six Opisthopatus sam-
National Research Foundation
ple localities were included. For O. amaxhosa, we sequenced the COI locus for all
specimens, while a subset of specimens was sequenced for two nuclear loci, 18S
rRNA and the fushi tarazu intron (FTz). Phylogenetic analyses using maximum like-
lihood and Bayesian inferences of the latter species revealed the presence of two
highly divergent clades, characterised by marked uncorrected sequence divergence
values. In addition, these two clades did not share any maternal haplotypes, were
characterised by high FST values and fixed nuclear difference for the 18S rRNA locus,
while the FTz intron was genetically invariant. Furthermore, the application of scan-
ning electron microscopy between the two genetically divergent clades also revealed
the presence of fixed ventral and dorsal scale numbers. Collectively, this provides
evidence for a novel species that is present at a fine scale. Divergence time estima-
tions suggest that the two clades diverged during the late and early Pleistocene with
climatic cycling potentially causal to the fragmentation. The social structure was
male‐biased, and samples from the same logs were not always genetically identical.
At the broader scale, the inclusion of new specimens within Opisthopatus revealed
no novel lineages. Fine‐scale sampling appears more important to detect alpha taxo-
nomic diversity compared to broadscale sampling.

KEYWORDS
conservation, fine‐scale, forest fragmentation, novel species, phylogeographic, population‐level

© 2019 Royal Swedish Academy of Sciences     1

Zoologica Scripta. 2019;1–20. wileyonlinelibrary.com/journal/zsc |
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2       BARNES and DANIELS
1  |   IN TRO D U C T ION
Saproxylic environments are defined as decaying habitat
such as logs of wood or leaf litter that are present in for-
ested regions. These habitats harbour a diverse array of in-
vertebrates that are critical in ecosystems function such as
nutrient recycling forming an important link in the terrestrial
food chain (Majka & Pollock, 2006). Generally, the saprox-
ylic invertebrate fauna are habitat specialists, require specific
microclimatic regimes for survival and frequently exhibit
low vagility (Daniels & Ruhberg, 2010; Myburgh & Daniels,
2015). The result is high levels of localised endemism and
marked genetic differentiation, rendering these species prone
to localised extinction (Anderson, 1994; Seibold et al., 2015;
Williams & Pearson, 1997). Due to their life history char-
acteristics, saproxylic invertebrates are ideal non‐model or-
ganisms with which to test hypotheses related to dispersal
and colonisation in fragmented forest habitat. In Australia,
Europe and North America, phylogeographic studies of sap-
roxylic invertebrates have revealed limited dispersal among
conspecific populations, marked population differentiation
and cryptic speciation (Cicconardi, Nardi, Emerson, Frati,
& Fanciulli, 2010; Garrick et al., 2006; Gaublomme et al.,
2013; Jonsson, Kruys, & Ranius, 2005; Ranius, 2006).
Further, phylogeographic studies of saproxylic taxa have in-
vestigated the historical population dynamics and assessed
the impact of demographic drivers such as recolonisation,
extinction and population bottleneck events (Garrick et al.,
2004). Collectively, these studies have argued for the con-
servation of both the taxa and the habitat of saproxylic in-
vertebrates. Nevertheless, in the Afrotropics, Neotropics and
Mesoamerica, saproxylic invertebrate fauna are poorly stud-
ied and their conservation remains limited partially due to a
paucity of taxonomic studies.
The isolation of saproxylic environments within for-
est patches results in similar microclimatic pressures, pro-
moting convergent evolution (Harmon, Kolbe, Cheverud,
& Losos, 2005) resulting in cryptic speciation (Daniels,
Dambire, Klaus, & Sharma, 2016; Daniels, Picker, Cowlin,
& Hamer, 2009; Daniels & Ruhberg, 2010; Trewick, 1997).
Consequently, the alpha taxonomy of most saproxylic taxa
is poor and these groups exhibit high cryptic differentiation.
This highlights the importance of molecular systematic re-
search to document the biodiversity of saproxylic taxa. One
group of saproxylic taxa in South Africa that was recently
subjected to intense systematic scrutiny is the velvet worm
fauna (Daniels et al., 2016, 2009; Daniels, Dreyer, & Sharma,
2017; Daniels, McDonald, & Picker, 2013; McDonald &
Daniels, 2012; Myburgh & Daniels, 2015). Onychophora, or
velvet worms, are soft‐bodied invertebrates, generally con-
fined to saproxylic habitats within forests. The South African
forest biome is the smallest of the seven biomes covering
<0.5% of the countries land surface, is highly fragmented and
restricted to areas of high precipitation (Rutherford, Mucina,
& Powrie, 2006). Velvet worms are confined to saproxylic
environments such as among leaf litter and under decaying
logs and are good indicators of habitat quality (Daniels et
al., 2009; Daniels & Ruhberg, 2010; Hamer, Samways, &
Ruhberg, 1997; McDonald & Daniels, 2012). Two velvet
worm genera, Peripatopsis and Opisthopatus, are present in
South Africa (Hamer et al., 1997). Historically, the latter two
genera contained eight and three species, respectively, with
four of the species being IUCN Red Listed (Hamer et al.,
1997). However, renewed recent interest in the group has re-
sulted in an increased focus on intensive molecular analysis
and scanning electron microscopy (SEM). This resulted in
the discovery and description of nine and five new species
within Peripatopsis and Opithopatus, respectively (Daniels
et al., 2016, 2013; Ruhberg & Daniels, 2013). New IUCN
Red Listing is planned for the recently discovered fauna con-
sidering the narrow endemic distribution of several of the
novel species (S. R. Daniels, personal communication). Since
velvet worm taxa are often cryptic, fine‐scale phylogenetic
study of velvet worms is necessary to understand the popula-
tion genetic structure and the importance of saproxylic hab-
itat conservation, especially, given the high risk of localised
extinction.
Within South Africa, population‐level molecular studies
have provided insight into the dispersal abilities of velvet
worms, generally revealing high levels of population differ-
entiation linked to both ancient and contemporary change
(Daniels, 2011; Daniels et al., 2017; Myburgh & Daniels,
2015). Daniels (2011) demonstrated low haplotypic diver-
sity in the narrow endemic rose pink velvet worm species
Opisthophatus roseus. Similarly, in the velvet worm species
Peripatopsis overbergiensis, distributed in three allopatric
forest fragments in the Overberg district of the Western Cape
province, Myburgh and Daniels (2015) demonstrated the
absence of maternal and paternal dispersal, corroborating a
general trend of high genetic differentiation among conspe-
cific populations (Barclay, Rowell, & Ash, 2000; Daniels,
2011; Daniels et al., 2017). It is thought that velvet worm
dispersal occurs at a natal stage and is male bias, offering a
possible theory for the apparent female bias in group com-
position (Daniels, 2011; Daniels et al., 2017). Although dis-
persal plays a role in shaping group composition, it does not
exclusively govern population divergence or the lack thereof.
Rather migratory dynamics are influenced by physiological
limitations and population size variations which can be di-
rectly related to forest fragment size and habitat availability
(Daniels, 2011; Daniels et al., 2017; Myburgh & Daniels,
2015). It is therefore hypothesised that a lack of vagility and
female bias will be observed among social groups within logs
and that marked genetic structure will be present where for-
ests are fragmented. Further, it is expected that genetic differ-
entiation will increase with increased spatial sampling. The
BARNES and DANIELS
social structure in velvet worms has rarely been considered as
a driver for dispersal. Sex ratio and life histories are two im-
portant components and are determined largely by behaviour
which is more complex than previously conceptualised, as
observed by Reinhard and Rowell (2005). Demographic
variability has been observed between the sexes in the vel-
vet worm species Peripatoides novaezealandiae, where a fe-
male bias sex ratio was observed (Tutt, Daughtery, & Gibbs,
2001). At birth velvet worms exhibit a 1:1 sex ratio, how-
ever, observations of free‐living animals showed a female
bias ratio among sexually mature specimens, a pattern that
has been observed in several studies (Barclay et al., 2000;
Curach & Sunnucks, 1999; Daniels, 2011; Sunnucks et al.,
2000; Tutt et al., 2001; Weldon, Daniels, Clusella‐Trullas,
& Chown, 2013). Therefore, the sex ratio of a population is
possibly dependent on the life stage and the time of sampling
(Tutt et al., 2001).
Opisthopatus amaxhosa, one of five recently de-
scribed velvet worm species, is confined to three allopatric
Afromontane forest fragments around Baziya, Langeni and
Nqadu in the Eastern Cape province of South Africa (Daniels
et al., 2016). Notably, these forests have historically been
very poorly sampled, and the species was originally de-
scribed based on 11 specimens. The habitat of O. amaxhosa
is fragmented and bisected by lower lying dry corridors of
grassland that potentially act as barriers to dispersals. In addi-
tion, recent plantations of Pinus and Eucalyptus have further
restricted the indigenous forest patches to deep valleys and
gorges. Furthermore, anthropomorphic factors such as sub-
sistence sheep and cattle farming by the local communities
have also contributed to the fragmentation and degradation
of these forests, where dead wood is frequently collected
for household usage (S. R. Daniels, personal communica-
tion). Considering the patchy nature of these indigenous
Afrotemperate forests, O. amaxhosa forms the ideal template
taxon with which to test the impact of forest fragmentation on
the phylogeographic structure of the species.
A fine‐scale phylogeographic analysis of O. amaxhosa
has not been conducted to date, limiting our understanding
of the genetic structure and conservation of the saproxylic
environments. In addition, as part of a larger research grant
on the biodiversity of forests in the Eastern Cape province,
we included Opisthopatus sampled from areas that were not
sampled by Daniels et al. (2016) to explore the presence
of additional novel species within the genus. The present
study aims to examine the phylogeography and sex ratio of
O. amaxhosa at three spatial scales: within a decaying log,
between decaying logs within a forest and between the three
major aforementioned forest patches where the species oc-
curs. Considering the limited dispersal capability of the velvet
worms and its preference for specific microclimatic factors,
we hypothesise marked genetic differentiation, with the de-
gree of genetic differentiation increasing with geographic
  
|
   3
distance between sampled sites. Hence, the genetic diversity
should increase with increased spatial sampling. The second
aim is to understand the sex ratio, size and composition of
O. amaxhosa specimens within logs in relation to their ge-
netic composition and to make inferences on the social struc-
ture of the species. We hypothesise that the sex ratio should
be female bias and that the sampled logs contain both closely
related maternal lineages as well as genetically highly differ-
entiated specimens.
2  |  M ATERIAL S AND M ETHO D S
2.1  |  Taxon sampling
During April 2017, a total of 78 O. amaxhosa specimens
were collected from three forest fragments (Baziya, Jenca
and Nqadu) in the Eastern Cape province of South Africa
(Figure 1). These samples were combined with 11 O. amax-
hosa specimens used by Daniels et al. (2016) yielding a total
of 89 specimens. Samples were hand collected from decay-
ing logs, and coordinates were recorded using a hand GPS
(Garmin Trek Summit). The number of adults and juveniles
were counted along with the type of social grouping deter-
mined as either “family” or “random” during sample collec-
tion (Table 1). A “family” was defined as a group comprised
of two adults and a large number of juveniles, while a “ran-
dom” group is comprised of two or more adults without any
juveniles occurring in the same log. Specimens were con-
sidered adult when they were >1 cm in length and sexually
mature, while juveniles were <1 cm in length and sexually
immature. Samples were placed in plastic jars and killed by
preservation in absolute ethanol and stored at 4°C in a refrig-
erator. A general collection permit was issued for sampling
(code CRO 60/17 CR). In addition, we included specimens
of Opisthopatus from four new localities (Hogsback, Mboyti,
Mazeppa Bay and Umslanga Nature Reserve) that was un-
sampled by Daniels et al. (2016) to determine their phylo-
genetic placement among the species described by the latter
author.
2.2  |  DNA extraction, PCR and sequencing
Tissue biopsies were taken from the lateral right‐hand side
of specimens, without cutting across the mid‐dorsal line,
preserving morphologically diagnostic characters. DNA was
extracted using a Macherey‐Nagel DNA extraction kit follow-
ing the manufacturer's protocol. Extracted DNA was stored
at 4°C in a refrigerator. DNA extractions were diluted with
deionised water making a 1 µl DNA in 19 µl of water solution.
Microsatellite development remains notoriously difficult
in Onychophorans (Sands, Lancaster, Austin, & Sunnucks,
2009). Further, failures in amplification, high‐frequency null
alleles and failures in microsatellite cloning have led to the
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4       BARNES and DANIELS

F I G U R E 1   Localities where Opisthopatus amaxhosa was collected in the Eastern Cape province of South Africa. The numbers on the map
correspond to the sample localities outlined in Table 1. The colours represent the altitudinal gradients of the region as described in the map legend
[Colour figure can be viewed at wileyonlinelibrary.com]

suggestion that the problem lies in the genomes of the group
rather than in the method (Sands et al., 2009). Thus, the use of
microsatellite loci was not considered in this study. Instead,
three partial gene fragments were amplified and sequenced
during the present study. These included the cytochrome c
oxidase one subunit (COI) and two nuclear DNA loci, the
small ribosomal 18S subunit (18S rRNA) and the fushi tarazu
(FTz) intron (Sands et al., 2009). The COI locus has been
extensively used for population‐level analyses among vel-
vet worms (Daniels et al., 2013, 2009; Daniels & Ruhberg,
2010). All samples were sequenced for the COI locus, while
a subset of samples was sequenced for the two nuclear DNA
(18S rRNA and the fushi tarazu ‐ FTz intron) loci. The primer
pairs LCOI‐1490 (5′‐GGT CAA CAAATCA TAAA GAT
ATTG‐3′) and HCOI‐2198 (5′‐TAAA CTT CAGGGT GAC
CAAA AAA TCA‐3′) were used to amplify the COI locus
(Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994). The primer
pairs 5F (5′‐GCG AAA GCA TTT GCC AAG AA‐3′) and 7F
(5′‐GCATCA CAG ACC TGT TAT TGC‐3′) were used for
the 18S rRNA subunit (Giribet, Carranza, Baguna, Riutort,
& Ribera, 1996). For the FTz intron, the forward (5’‐GTG
AGG CTG CTGG AAAAATT‐3’C) and reverse (3’‐CAAA
TCC GTTGTC TGT AAAT ATG‐5’) primer pairs were used
(Sands et al., 2009). The 18S rRNA subunit has been used as
complimentary to the COI subunit when determining velvet
worm species boundaries (Daniels et al., 2016, 2017, 2013,
2009; McDonald & Daniels, 2012; Myburgh & Daniels,
2015). The DNA sequences generated for the COI locus
for the O. amaxhosa specimens generated by Daniels et al.
(2016) were combined with sequences generated during the
BARNES and DANIELS   
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T A B L E 1   List of Opisthopatus
Locality no. Name N Social group Coordinates
amaxhosa specimens collected within the
Baziya forest complex in the Eastern Cape 1 BaziyaOLD* 5 N/A 31.31.250 S 28.24.738 E
province of South Africa 2 Nocu* 6 N/A 31.24.928 S 28.29.990 E
3 BaziyaJan 3 N/A 31.34.034 S 29.24.733 E
4 Baziya A 3 Random 31.34.034 S 29.24.733 E
5 Baziya B 5 Family 31.34.095 S 28.24.689 E
6 Baziya C 7 Family 31.33.892 S 28.24.601 E
7 Baziya D 3 Random 31.33.890 S 28.24.594 E
8 Baziya E 6 Family 31.33.914 S 28.24.580 E
9 Baziya F 4 Random 31.32.901 S 28.26.391 E
10 Baziya G 10 Random 31.34.671 S 28.24.671 E
11 Baziya H 4 Family 31.34.293 S 28.24.585 E
12 Baziya I 5 Family 31.34.312 S 28.24.712 E
13 Baziya J 3 Family 31.33.511 S 28.25.369 E
14 Baziya L 2 Random 31.34.885 S 28.23.696 E
15 Ndlukulu 2 Random 31.31.251 S 28.24.693 E
16 Langeni* 2 Random 31.31.252 S 28.24.692 E
17 Nqadu A 9 Family 31.42.839 S 28.73.539 E
18 Nqadu B 10 Family 31.42.840 S 28.73.539 E
Notes. N, the number of samples. N/A implies the designation for the social group was not determined.
Locality numbers correspond to those in Figure 1. The asterisk (*) denotes samples used by Daniels et al. (2016).

present study. Similarly, for the larger phylogenetic study,
the newly sampled Opisthopatus specimens were included
with those from Daniels et al. (2016).
Polymerase chain reactions (PCRs) were conducted on a
geneAmp PCR System Thermo cycler (Applied Biosystems,
Foster City, CA, USA) under the following conditions for the
COI locus: denatured for 94°C for 4 min, 94°C for 30 s, 42°C
for 40 s, 72°C for 45 s, for 36 cycles, with a final extension at
72°C for 10 min. For the 18S rRNA locus and FTz intron, the
annealing temperatures were 50°C and 52°C, respectively,
the number of cycles increased to 40. All PCRs were con-
ducted for a 25 µl reaction and held at 15°C. All three re-
actions contained 14.9 μl of deionised water, 3 μl of 25 mM
MgCl2, 2.5 μl of 10 × Mg2+ free buffer, 0.5 μl of a 10 mM
dNTP solution and 0.5 μl of the primer sets at 10 mM, 0.1
unit of Taq polymerase and 1–3 μl of extracted DNA. PCR
products were gel purified in a 1% agarose gel over 180 min
at 90 V. Gel bands were explicit under UV light, were re-
sected and purified using a BioFlux gel purification kit.
Sequence‐purified PCR products were then cycled under
standard protocols. An ABI 3,730 XL automated machine
conducted the sequencing.
2.3  |  Phylogenetic analyses
Forward and reverse strands were used to compute a consensus
sequence and to check for base ambiguities using Sequence
Navigator (Applied Biosystems). Sequence alignment was
computed with the use of CLUSTAL X (Thompson, Gibson,
Plewniak, Jeanmougin, & Higgins, 1997). The phylogeny for
Opithsopatus and O. amaxhosa was constructed using maxi-
mum likelihood (ML) and Bayesian inference (BI). The ML
analyses were performed using the CIPRES Science Gateway
version 3.3 (Miller, Pfeiffer, & Schwartz, 2010), using
GARLI 2.01 (Zwickl, 2006) on XSEDE. A single replicate
search was conducted for the best tree. Branch support values
were estimated using a bootstrap analysis (Felsenstein, 1985)
with 1,000 pseudoreplicates and parameters set to default.
Bootstrap values >75% were deemed as sufficient statistical
support for nodes. Analyses were performed using a heuristic
search algorithm starting at a random tree. Four rate catego-
ries were included for gamma, and base frequencies were es-
timated. Bootstrap values were discerned via a 50% majority
rule tree, generated using PAUP* 4.0 (Swofford, 2002).
Bayesian analyses were conducted on the CIPRES Science
Gateway version 3.3 (Miller et al., 2010) with MrBayes ver-
sion 3.2.6 (Ronquist et al., 2012) on XSEDE. Each analysis
comprised of one run of four chains for 10 million genera-
tions, sampling every 1,000 generations using default param-
eters. A random tree was selected for the start of each chain.
Burn‐in was included in the command block, set to 20%. A
consensus tree was generated by MrBayes on CIPRES as part
of the output. The percentage of time spent on node recovery
was used to estimate the posterior probabilities (pP) for each
node. Posterior probabilities (pP) <0.95 were regarded as
poorly supported. For both analyses, the Akaike information
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6       BARNES and DANIELS
criterion (AIC) (Akaike, 1973) ascertained the best‐fit substi-
tution model using jModelTest2 (Posada & Crandall, 1998)
on XSEDE through CIPRES (Miller et al., 2010). PAUP*10b
(Swofford, 2002) computed the uncorrected sequence diver-
gence for the COI locus.
2.4  |  Phylogeographic analyses sourced from
COI data for O. amaxhosa
The haplotype network was constructed using TCS 1.21
using 95% confidence interval (Clement, Posada, & Crandall,
2000). Population genetic structure was computed on arle-
quin version 3.5.1.2 (Excoffier & Lischer, 2010) using the
COI data. A hierarchical analysis of molecular variance
(AMOVA) was conducted over all sample localities, and
then for each of the two COI clades evident from the prelimi-
nary analyses. FST values were computed across all sample
localities. Standard diversity indices were further calculated
at each of the localities within fragments. These indices in-
cluded haplotype diversity (h), the number of polymorphic
sites (Np), the number of haplotypes (Nh) and nucleotide di-
versity (π). Fu's Fs (Fu, 1997) was used to clarify deviations
from neutralities in allele frequencies due to its sensitivity to
population equilibrium fluctuations. Fu's Fs is preferred over
Tajima's D due to its higher sensitivity and significance level
of p < 0.02 (Fu, 1997).
2.5  |  Divergence time estimation for
O. amaxhosa
The complete COI locus dataset was used for estimating di-
vergence times between haplotypes despite of known faults
within the locus (Daniels, 2011; McDonald & Daniels,
2012). The 18S rRNA and FTz intron loci were not con-
sidered for the divergence time estimations due to a lack
of mutation rates for the latter two nuclear loci and be-
cause only a subset of data was generated for these two
loci (Clouse et al., 2015; Daniels et al., 2016; Fernandez
& Giribet, 2014). Divergence time estimations for the COI
locus were conducted using a Bayesian framework, which
makes use of a probabilistic model to define the molecular
sequence divergence of lineages which further makes use
of the MCMC method to estimate clade ages. A relaxed
molecular clock was used (Drummond, Ho, Phillips, &
Rambaut, 2006), and the analyses were run using the pro-
gram beast version 2.4.8 (Drummond & Rambaut, 2007).
The mutation rates of 1.5%–2.3% per million years were
used for the COI locus (Boyer, Baker, & Giribet, 2007;
Brower, 1994; Farrell, 2001; Trewick & Wallis, 2001),
with a mean mutation rate of 1.9% per million years
being selected (McDonald & Daniels, 2012). A multiple
coalescent model was used (Heled & Drummond, 2010).
MODELTEST was used to determine the parameters and
substitution model for the entire COI subset. Ten million
generations were run for 10 independent MCMC chains,
with sampling performed every 1,000 generations. After
appropriate burn‐in, the convergence of the 10 combined
chains was determined by effective sample size for each
parameter in tracer (Rambaut, Suchard, & Drummond,
2013). Trees were amalgamated using logcombiner which
contained 10 chains, after which they were assessed
using treeannotator. The program figtree version 1.4.3
(Rambaut, 2009) was used to formulate a chronogram.
2.6  |  Morphological examination using SEM
for O. amaxhosa
Live O. amaxhosa specimens were photographed using
a Canon EOS 70D camera. The colour was noted, and the
number of oncopods was counted. Five specimens from each
of the two clades (observed during the preliminary phylo-
genetic analyses) were examined using a SEM. Specimens
were dehydrated firstly in absolute ethanol, followed by par-
tial dehydration in a 1:1 solution of hexamethyldisilazane
(HMDS) and absolute ethanol for 30 min. Specimens were
finally dehydrated in 100% HMDS in a glass petri dish for
an additional 30 min, before being left to dry inside a drying
oven at 25°C overnight. Images were captured using a Zeiss
MERLIN Field Emission SEM at the Electron Microbeam
Unit of Stellenbosch University's Central Analytical Facility
in the Department of Geology. Prior to imaging, the sam-
ples were mounted on aluminium stubs with double‐sided
carbon tape. The samples were then gold coated to 10 nm,
using an Edwards S150A Gold Sputter Coater. Beam condi-
tions during surface analysis were 5 kV and approximately
250 nÅ, with a working distance of 6 mm. During the SEM,
we focussed on analysis of the dorsal and ventral integu-
ment structure since previous studies successes fully used
these characters to delineating cryptic velvet worm species
(Daniels et al., 2013; McDonald, Ruhberg, & Daniels, 2012;
Oliveira, Read, & Mayer, 2012; Ruhberg & Daniels, 2013).
Scanning electron micrograph images were examined for dif-
ferences in the number of scale rings for the primary and ac-
cessory papillae on both the dorsal and ventral integument
surface.
2.7  |  Population sex ratio
The sex of O. amaxhosa specimens was determined using
standard light microscopic examination (Hamer et al., 1997).
In addition, the total length (TL), measured from the anterior
to the posterior end, and the diameter of the body (DB) each
specimen in millimetre (mm) were recorded using a digital
vernier calliper for all ethanol fixed samples. The subsequent
results were analysed for significance of ratio for each sex
within each locality using a t test (Quinn & Keough, 2002).
T A B L E 2   Haplotype (H) frequency distribution for the COI locus for Opisthopatus amaxhosa sampled from the Baziya forest complex in the Eastern Cape province of South Africa

Baziya Baziya Baziya Baziya Baziya Baziya Baziya Baziya Baziya Baziya Baziya Nqadu Nqadu
BaziyaOLD Nocu BaziyaJan A B C D E F G H I J L Ndlukulu Langeni A B
H1 1
H2 1
BARNES and DANIELS

H3 1
H4 2
H5 1
H6 1
H7 1
H8 1
H19 7 9
H10 2
H11 1
H12 1 3 1
H13 1 4 4 1 1
H14 1
H15 2
H16 1
H17 1
H18 1
H19 1
H20 1
H21 1
H22 1
H23 1
H24 1
H25 1
H26 1 5 5 3 5
H27 1 1 1 1
H28 1
H29 2 1
H30 2
H31 1
  

H32 2
|   7

Note. The haplotype numbers correspond to Figure 4.

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8       BARNES and DANIELS
BARNES and DANIELS
F I G U R E 2   Bayesian inference tree topology for Opisthopatus amaxhosa derived from the mtDNA COI locus. The nodal values below
branches represent the bootstrap values (>75%) for ML. Posterior probability (pP) values for BI are shown above branches (>0.95pP). Values
below the prescribed threshold are indicated by an asterisk for ML (*) and a hashtag (#) for BI
3  |   R E S U LTS
3.1  |  Phylogenetic analyses of the COI data
for Opisthopatus amaxhosa
A 610‐base pair fragment of the COI locus was amplified
and sequenced for the 78 O. amaxhosa specimens and com-
bined the 11 specimens from Daniels et al. (2016) yielding a
total of 89 specimens. The novel sequences were deposited
in GenBank (Accession numbers MK236395–MK236470).
Both the BI and ML analyses produced near identical tree
topologies, and hence, only the BI topology is shown. The
DNA substitution model selected using the AIC criteria
was TIM2 + I + G (−1nl = 3,106.46; AIC = 6,592.91)
(Akaike, 1973). The base frequencies were A = 33.87%,
C = 17.88%, G = 12.29% and T = 35.97%. Similar results
of an A/T rich base frequency bias have been reported in
other velvet worm studies (Daniels et al., 2009; Daniels
& Ruhberg, 2010, McDonald & Daniels, 2012). The rate
matrix was R(a) [A‐C] = 5.44, R(b) [A‐G] = 22.30, R(c)
[A‐T] = 5.44, R(d) [C‐G] = R(f) [G‐T] = 1.00 and R(e)
[C‐T] = 40.54, the proportion of invariable (I) sites was
0.5320 and the gamma (G) shaped distribution was 0.6990.
The BI topology (Figure 2) retrieved two statistically well‐
supported (>75% and 1.00 = pP) clades. Clade 1 consisted
of all the O. amaxhosa specimens described by Daniels et
al. (2016), plus sample localities from Nqadu A, Nqadu B,
Langeni, Ndlukulu, Baziya C, E and BaziyaJan while clade
2, sister group to Opisthopatus kwazululandi, comprised
specimens from Baziya A, B, C, D, E, F, G, H, I, J and L.
At BaziyaJan, Baziya C and Baziya E, the two clades were
sympatric and genetically diverged indicating maternal
isolation of the two clades. Divergence time estimations
indicate that the two clades diverged 2.82 million years ago
(95% highest posterior density [HPD]: 1.94–3.69 Mya) (to-
pology not shown). These results indicate that the diver-
gence of O. amaxhosa occurred during the late Pliocene
to early Pleistocene. The updated phylogenetic analyses of
the newly collected Opithopatus specimens (Figure 3) did
not reveal any novel lineages, except for those from the
Baziya forest complex (Figure 2). Further the tree topol-
ogy presented (Figure 3) placed the novel lineage within
the species complex of O. kwazululandi, this is most likely
due to the inclusion of the conserved 18S rRNA locus. The
result from the BI analysis including only the COI locus re-
vealed the novel species as sister to the existing combined
clade of O. kwazululandi and O. amaxhosa (Figure 3).
The uncorrected sequence distance between clades 1 and
2 was 19.50%. Within clade 1, the maximum uncorrected
  
   9
|
sequence divergence was 9.34%, while within clade 2, the
maximum uncorrected sequence divergence was 4.09%.
Within clade 2, the basal specimen labelled Baziya A4,
identified as O. kwazululandi was 18.36% divergent from
the remainder of the samples in clade 2. The inclusion of
a genus‐wide analysis representing both the COI and 18S
genes for Opisthopatus clarified that the sample Baziya A4
is in fact part of and not sister to the species complex of
O. kwazululandi, hence the highly differential uncorrected
sequence divergence and the classification of this sample
as a third sympatric species.
3.2  |  Population genetics for the COI locus
A total of 32 haplotypes were retrieved for the 89 COI se-
quences using TCS analyses (Table 2; Figure 4). The hap-
lotype network corroborated the phylogenetic analyses and
retrieved two haploclusters. Haplocluster 1 comprised of 20
haplotypes while clade 2 comprised the remaining 13 hap-
lotypes. Due to the classification of the sample Baziya A4
as a separate species, O. kwazululandi (haplotype 3), it was
excluded from the network representation of the popula-
tion‐level analyses for O. amaxhosa (Figure 3). The lack of
connectivity and shared haplotypes between clades 1 and 2
is indicative of the absence of gene flow (Table 3). Within
haplocluster 1, only populations in close geographic proxim-
ity were connected indicating limited maternal dispersal at a
small spatial scale. In contrast, the large number of unsam-
pled or missing mutational steps between haplotypes in hap-
locluster 2 suggests a similar lack of dispersal between more
distantly related sample sites (Table 3).
The AMOVA results over all O. amaxhosa samples re-
vealed that 80.04% variation occurred among populations
(Va = 26.29, df = 17, s.s. = 2,287.00, p < 0.01) while
19.96% variation occurred within populations (Vb = 6.56,
df = 71, s.s. = 465.67, p < 0.01). Within clade 1, 78.14% of
the variation occurred among populations (ΦST = 0.78135,
Va = 14.24, df = 8, s.s. = 464.57, p < 0.01) while 21.86%
variation occurred within populations (Vb = 4.01, df = 28,
s.s. = 112.34, p < 0.01). Within clade 2, 59.88% of the varia-
tion occurred among populations (ΦST = 0.59883, Va = 4.14,
df = 11, s.s. = 223.48, p < 0.01) while 40.12% of the vari-
ation occurred within populations (Vb = 2.77, df = 40, s.s.
110.85, p < 0.01). Pairwise FST values across all sample lo-
calities indicate marked genetic structure, suggesting limiting
maternal dispersal (Table 3). The number of haplotypes (Nh),
number of polymorphic sites (Np), haplotype diversity (h),
nucleotide diversity (π) and Fu's Fs for the 18 sample sites
 |
10       BARNES and DANIELS

F I G U R E 3   Bayesian inference tree topology derived from the combined COI and 18S data for the Opisthopatus genus, containing one
sample from each study site. The nodal values below branches represent the bootstrap values (>75%) for ML. Posterior probability (pP) values for
BI are shown above branches (>0.95pP). Values below the prescribed threshold are indicated by an asterisk for ML (*) and a hashtag (#) for BI.
New sample localities are highlighted in blue and the single sample from the novel clade of the present study is highlighted in red. The abbreviation
“NR” depicts whether the locality is a nature reserve [Colour figure can be viewed at wileyonlinelibrary.com]
BARNES and DANIELS
F I G U R E 4   The haplotype networks derived from the 32 COI haplotypes demonstrating two haploclusters corresponding to the phylogenetic
tree in Figure 2 for Opisthopatus amaxhosa. The numbers within circles represent the haplotype number and correspond to Table 2. Solid black
circles represent missing or unsampled haplotypes [Colour figure can be viewed at wileyonlinelibrary.com]
are summarised in Table 4. The sample locality Baziya A
had the highest nucleotide diversity (Nh), compared to the
remaining samples. This was followed in order of descent by
BaziyaOLD, Baziya C and Baziya E. The highest number of
polymorphic sites (Np) corresponded to the sample localities
where the two clades co‐occurred. Fu's Fs values contained
a mixture of negative and positive values with a bias towards
the latter. The positive values are representative of population
bottlenecks or overdominant selection, shown by a deficiency
of alleles. The negative values are characterised by an excess
in the number of alleles and indicate a recent population ex-
pansion or genetic hitchhiking (Fu, 1997). The latter result
is present at three localities: Baziya G, Nqadu A and Nqadu
B. At the clade level, Fu's Fs values were both positive, in-
dicative of a reduction in gene flow and a potential historical
bottleneck.
3.3  |  nu DNA (FTz intron and 18S rRNA)
For the FTz intron, a 250‐bp fragment was sequenced; how-
ever, the intron was genetically invariant between the two
clades, and hence, this locus was not considered further. The
  
|
   11
GenBank accession numbers for the 10 FTz intron specimens
sequenced were MK236476‐MK236485. For the 18S rRNA
locus, a 427‐bp fragment was amplified and sequenced for
two specimens from each of the clades 1 and 2, respectively
(GenBank Accession numbers MK236486–MK236489).
Within clade 1, the observed uncorrected sequence distance
was 0.23%, while within clade 2, the observed uncorrected
sequence distance was 9.83%. Between clades 1 and 2, the
uncorrected distance was 12.18%.
3.4  |  Morphological examination using SEM
Scanning electron microscopy revealed morphological di-
agnostic differences between the two O. amaxhosa clades
in the number of scale rings for the primary dermal papilla
on the dorsal and ventral integumental areas. Opisthopatus
amaxhosa (clade 1) exhibited six rings on the dorsal primary
papillae and four rings on the ventral primary papillae, while
specimens in clade 2 exhibited eight rings on the dorsal pri-
mary papillae and six rings on the ventral primary papillae
(Figure 5). All specimens had 16 pairs of oncopods, and the
dorsal integument was rose pink with white and burgundy
T A B L E 3   Pairwise FST values for the COI locus across all 18 of the different sample localities for Opisthopatus amaxhosa
|

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
12      

1
0.29097
2
0.38228 0.55472
3
0.80131 0.87931 0.16442
4
0.73653 0.87931 −0.05270 0.18328
5
0.83631 0.91338 0.32533 0.77143 0.22680
6
0.65688 0.74874 0.03454 0.35771 −0.08709 −0.04551
7
0.78792 0.89003 0.14231 0.70714 0.03846 0.00000 −0.15711
8
0.62762 0.73058 −0.00085 0.35666 −0.08485 −0.03448 −0.17321 −0.15385
9
0.80697 0.89447 0.15751 0.68326 0.38381 0.89813 0.15832 0.86047 0.16355
10
0.89504 0.94069 0.50181 0.12586 0.70575 0.96942 0.61871 0.96394 0.62650 0.94237
11
0.75787 0.85217 0.03795 0.31834 −0.18567 0.33594 −0.02331 0.18725 0.00423 0.28148 0.73734
12
0.72986 0.83883 −0.08420 0.13114 −0.33613 0.37824 −0.04797 0.20000 −0.03190 0.24638 0.67729 −0.36565
13
0.70533 0.83391 −0.26599 −0.21495 −0.44134 0.60396 −0.02848 0.41463 −0.03643 0.37173 0.58148 −0.27121 −0.56818
14
−0.00372 0.47071 0.32709 0.91976 0.87940 0.99385 0.71101 0.98978 0.67890 0.97654 0.98759 0.88486 0.86493 0.86500
15
0.46952 0.72579 0.44415 0.92371 0.88582 0.99603 0.71641 0.99339 0.68338 0.98039 0.98881 0.89055 0.87220 0.87440 0.94624
16
0.72152 0.84400 0.79886 0.96857 0.96549 0.99726 0.86141 0.99671 0.85494 0.99187 0.99309 0.95995 0.96089 0.97099 0.98192 0.98866
17
0.74113 0.85643 0.81667 0.97214 0.97004 0.99872 0.87085 0.99849 0.86546 0.99391 0.99433 0.96459 0.96587 0.97550 0.98803 0.99300 0.00454
18
BARNES and DANIELS

Note. Values in bold are statistically significant (p < 0.005). Localities 1–18 correspond to those in Table 1.
BARNES and DANIELS   
   13
|
T A B L E 4   Summary list of the population genetic parameters measured using the COI locus in Opisthopatus amaxhosa

No. of haplotypes No. of polymorphic Haplotype

Locality N (Nh) sites (Np) diversity (h) Nucleotide diversity (π) Fu's Fs
BaziyaOLD* 5 5 56 1.0000 ± 0.1265 0.054098 ± 0.033428 1.08892
Nocu* 6 4 28 0.8667 ± 0.1291 0.026995 ± 0.016234 3.71423
BaziyaJan 3 3 109 1.0000 ± 0.2722 0.121858 ± 0.091558 3.20102
Baziya A 3 3 25 1.0000 ± 0.2722 0.027322 ± 0.021085 1.67558
Baziya B 5 1 0 0.0000 ± 0.0000 0.000000 ± 0.000000 N/A
Baziya C 7 3 106 0.5238 ± 0.2086 0.051444 ± 0.029388 10.36994
Baziya D 3 1 0 0.0000 ± 0.0000 0.000000 ± 0.000000 N/A
Baziya E 6 2 107 0.3333 ± 0.2152 0.058470 ± 0.034414 13.48405
Baziya F 4 2 3 0.6667 ± 0.2041 0.003279 ± 0.002750 2.19722
Baziya G 10 4 3 0.7778 ± 0.0907 0.001785 ± 0.001443 −0.69898
Baziya H 4 3 26 0.8333 ± 0.2224 0.023224 ± 0.015839 3.21925
Baziya I 5 2 24 0.4000 ± 0.2373 0.015738 ± 0.010184 6.77643
Baziya J 3 3 27 1.0000 ± 0.2722 0.030601 ± 0.023530 1.79304
Baziya L 2 2 24 1.0000 ± 0.5000 0.039344 ± 0.040156 3.17805
Langeni* 2 2 3 1.0000 ± 0.5000 0.004918 ± 0.005679 1.09861
Ndlukulu 2 2 2 1.0000 ± 0.5000 0.003279 ± 0.004016 0.69315
Nqadu A 9 3 2 0.4167 ± 0.1907 0.000729 ± 0.000806 −1.08110
Nqadu B 10 2 1 0.2000 ± 0.1541 0.000328 ± 0.000499 −0.33931
Note. The number of samples (N), haplotype number (Nh), number of polymorphic sites (Np), haplotype diversity (h), nucleotide diversity (π) and Fu's Fs of the separate
populations. Localities marked with an asterisk were used by Daniels et al. (2016).

dermal papillae (Figure 6) except for a single specimen from
Baziya A4, which was slate black.
3.5  |  Population sex ratio
A male bias was observed across all sample localities with
females being found only present at five sample localities
(p < 0.05). Juveniles were only present at four sample lo-
calities (Baziya B, C, E and I). Social groupings classified
as families did not comprise of a pair of single‐sex adults
and juveniles, suggesting that social aggregations are ran-
dom. Female specimens were longer and wider compared
to male specimens; however, this trend was non‐statistically
significant (p > 0.05). Morphological data are summarised in
Table 5.
4  |   D IS C U SSION
The present study clearly indicates that at a large spa-
tial scale we did not detect any novel lineages (Figure 3).
However, at a fine spatial scale, our phylogeographic analy-
ses provide evidence that two statistically well‐supported
clades are present within O. amaxhosa (Figure 2). The two
clades within O. amaxhosa are characterised by marked
uncorrected COI distances where sympatric, clearly indi-
cating genetic isolation. In addition, the two clades can fur-
ther be differentiated based on the nuclear 18S rRNA locus
and exhibit fixed morphological differences, based on the
number of scale rings for the primary papillae using SEM.
Collectively, these results suggest the presence of an addi-
tional novel, as yet undescribed species within O. amaxhosa
sensu lato. All the specimens originally used by Daniels et
al. (2016) to describe O. amaxhosa are assigned to clade 1,
suggesting the specimens in clade 2 represent a novel op-
erational taxonomic unit. These results clearly indicate the
importance of fine‐scale sampling in an attempt to uncover
the alpha diversity among saproxylic taxa, a fact that is often
disregarded when taxonomic studies are designed despite its
critical importance.
Our first hypothesis of observing marked levels of genetic
differentiation, increasing with the degree of spatial separa-
tion across three spatial scales, that is, within a decaying log;
between decaying logs within a forest; and between the three
major forest patches, was partially supported within the two
observed clades. Within each of the two clades, the general
trend exhibited was limited maternal dispersal between con-
specific sample localities. We can reject our second hypoth-
esis of a female bias in specimens, since we observed a male
bias in our sampling.
 |
14       BARNES and DANIELS
(a)
(b)
F I G U R E 5   Scanning electron micrographs of primary and accessory dermal papillae for the two Opisthopatus amaxhosa clades. Each white
dot represents a scale ring. (a) Dorsal papilla of O. amaxhosa. (b) Dorsal papilla of clade 2. (c) Ventral papilla of O. amaxhosa. (d) Ventral papilla
of clade 2. The abbreviation “br” is for the sensory bristle
4.1  |  Evidence for a new species
We observed a clear paraphyly with clade 2 being sister to
O. kwazululandi, while clade 1 is monophyletic. Species
paraphyly can be attributed to several factors including
poor taxonomy, introgression (interspecific hybridisation)
and incomplete lineage sorting (Funk & Omland, 2003).
Differentiation between the possible causes of species para-
phyly can be difficult as introgression and incomplete lineage
sorting may result in similar gene tree topologies and for a
brief period in evolutionary time, diverging lineages remain
incompletely sorted making it near impossible to differenti-
ate from poor taxonomy when there are insufficient synapo-
morphies (Funk & Omland, 2003; McKay & Zink, 2010).
The paraphyly and the proximity of genetic relatedness are
further exhibited in Figure 3 whereby the novel lineage from
this study is grouped with O. kwazululandi. This result is
most likely due to the inclusion of the conserved 18S rRNA
locus within the analyses, and a lack of differentiation be-
tween the two species at a nuclear level.
Multiple lines of independent evidence (both genetics
and morphology) are congruent and provide evidence for
(c)
(d)
the presence of two distinct evolutionary lineages present
within what has hitherto been considered a single species,
with clade 2 representing the novel lineage and clade 1 rep-
resenting the nominal O. amaxhosa species. Considering
that these two clades are sympatric at sites Baziya C, E and
BaziyaJan, we can confidently define them as distinct lin-
eages. Within clade 2, a single slate black specimen from
site Baziya A had an uncorrected sequence divergence of
18.36% for the mtDNA COI locus compared to the remain-
der of clade 2. Under further investigation at a broad scale,
this specimen was observed to fall within the O. kwazulu-
landi clade, forming a sister group to the species lineage
from Weza Forest which also contains black specimens
(Daniels et al., 2016) (Figures 2 and 3). This was supported
by both the COI and the 18S markers, grouping the spec-
imen as a sister species to clade 2 of this study (Figures 2
and 3). The marked uncorrected sequence distance values
between the two large clades as well as the distance be-
tween the slate black specimen falls within the region of
what has been used by Daniels et al. (2016) to differentiate
Opisthopatus species. This level of divergence of the COI
locus is high and similar studies investigating Opisthopatus
BARNES and DANIELS
F I G U R E 6   Live photographic images of Opisthopatus
amaxhosa collected at the Baziya forest in the Eastern Cape province
of South Africa. Scale bar = 1 cm [Colour figure can be viewed at
wileyonlinelibrary.com]
sister species with a maximum sequence divergence of
19.27% (Daniels et al., 2016). These two clades could fur-
ther be differentiated based on the nuclear 18S rRNA data,
further validating the presence of two distinct species. We
caution against assigning species recognition solely on se-
quence divergent values and on the basis of a single molec-
ular marker. However, our result is also corroborated by the
18S rRNA locus. Morphologically, the dorsal integument
colour and the leg pair numbers have been shown to be lim-
ited as diagnostic characters between cryptic Opisthopatus
species (Daniels et al., 2016). However, SEM examination
of the primary papillae, on the dorsal and ventral integ-
ument, corroborated the genetic differentiation observed
with the DNA sequence data (Figure 5).
4.2  |  Population genetic structure
The divergence time analysis indicates that the cladogenesis
between the two O. amaxhosa clades occurred during the late
Pliocene to early Pleistocene, 2.82 Mya. This time period is
characterised by intense aridification and sea‐level reduc-
tions induced largely by temperature variations and conse-
quent rainfall regime alterations during glaciation cycles
(Van Andel, 1989; Lawes, 1990). Forest fragmentation was
an end result, with the climate effects being assisted by the
evolution of C4 grasses and subsequent fire regimes (Adie,
  
|
   15
Kotze, & Lawes, 2017; Hughes, Möller, Bellstedt, Edwards,
& Villiers, 2005). Aridification of the area suggest that once
continuous forest were forced into refugia among the shel-
tered valleys of the adjacent escarpment, thus creating islands
of suitable habitat for onychophoran taxa. These refugia are
characterised and limited by their conformity to favourable
Afromontane forest conditions such as increased precipita-
tion and cooler temperatures when compared to the surround-
ing grasslands, which enhance refugia limitation through fire.
The grasslands create a fundamental‐realised niche effect
whereby the forest is driven further into refugia than what
climate permits, augmenting isolation. Thus, refugia would
have been local centres from which the habitat would expand
once climate during the Holocene produced wetter conditions
(Eeley, Lawes, & Piper, 1999; Lawes, 1990).
The extent of forest contractions and expansions for the
greater area must also be considered due to the inclusion of
the sample from Baziya A4 and its identification as part of
the O. kwazululandi species complex. Due to the observed
limited dispersal of the animals, it is highly unlikely for
movement to take place at such a large scale without suit-
able habitat connectivity. Therefore, an assumption can be
made that the forests between the sample sites including the
specimens from those such as Weza and Ixopo containing
O. kwazululandi were once more continuous and connected
to those in Baziya. The Eastern Cape is hypothesised to be
a place of Afrotemperate forest refuge during glacial maxi-
mums due to the combination of climate and the surrounding
geography (Hughes et al., 2005; Lawes, 1990; Lawes, Eeley,
Findlay, & Forbes, 2007). Hughes et al. (2005, Lawes (1990,
and Lawes et al. (2007 noted a trend of the recolonisation of
Afrotemperate patches and associated animal and plant gen-
era radiating north‐east into KwaZulu‐Natal, sourcing from
the southern refugia found in the Eastern Cape. Species with
low vagility such as O. kwazululandi must have had their
movement facilitated by the extended distributions of forest
patches, enhancing connectivity. Assessing the topology of
the area, it is most likely forests followed a route along the
escarpment of the Natal Drakensburg mountains, remaining
in areas of suitable climate refuge when conditions were ap-
propriate (Lawes, 1990), facilitating migration events and
the co‐distribution of O. kwazululandi.
A trend of a reduction in species richness was also ob-
served among various taxa as the distance increased from
the refugial sites in the Eastern Cape (Lawes et al., 2007).
Thus, refugial expansion and a reduction in species richness
with a northward trend may provide further evidence for an
expansion of O. kwazululandi into KwaZulu‐Natal from the
Eastern Cape refugia, especially under conditions of higher
connectivity. The impact of the refuge as a sink for biodi-
versity during climate ameliorations may alter how we con-
ceive the origin of species and may explain the observed high
level of diversity at Baziya. Thus, it is possible that the sites
 |
16      

T A B L E 5   Summary of the morphological characters of specimens examined from the 14 Opisthopatus amaxhosa sample localities of the current study

Mean Mean Mean

female juvenile juvenile
Locality Male (N) Mean male length Mean male width Female (N) length Mean female width Juvenile (N) length width Social grouping
Baziya A 3 19.16 2.92 Random
Baziya B 4 17.25 2.87 1 13.42 1.90 Family
Baziya C 2 21.42 3.33 1 26.02 3.27 3 13.70 1.65 Family
Baziya D 3 21.99 2.66 Random
Baziya E 5 19.43 2.87 2 13.29 2.52 Family
Baziya F 5 22.47 3.13 Random
Baziya G 8 31.36 2.69 3 28.95 2.16 Random
Baziya H 3 16.99 2.23 1 13.12 2.12 Family
Baziya I 3 18.98 3.05 1 14.27 2.67 1 10.63 0.58 Family
Baziya J 3 20.53 3.63 Family
Baziya L 2 17.85 2.36 1 21.38 2.89 Random
Ndlukulu 2 14.20 2.26 Random
Langeni 2 11.72 1.97 Random
Total 45 21.13 2.80 7 23.6 2.56 7 13.1 1.78
Note. All measurements are in millimetres (mm). Number of samples = N.
BARNES and DANIELS
BARNES and DANIELS
in Baziya experienced the enforced sympatry of O. kwazu-
lulandi and O. amaxhosa due to its historical role as a ref-
uge. The subsequent fine‐scale cryptic speciation between
O. amaxhosa and the novel lineage likely occurred during the
early Pleistocene/late Pliocene climate oscillations as a re-
sult of increased aridification. Thus, a phenomenon of forest
patch isolation at two levels is hypothesised with broadscale
forest contractions taking place between the two provinces,
isolating organisms from O. kwazululandi within Baziya, fol-
lowed by the fine‐scale contractions within the Baziya forest
complex itself, which then facilitated the origin of O. amax-
hosa and the novel lineage showcased as clade 2 in this study.
The two species from clades 1 and 2 follow a north to south
trend with O. amaxhosa (clade 1) being found frequently in the
northern sample localities and clade 2 being more frequently
found in the southern localities (Figures 1 and 2). This trend
may hint at how the region followed the pattern of forest con-
traction–expansion and to which fragment each of the species
was assigned. The lack of current dispersal, as indicated by the
significant FST values between populations within clades of the
main forest patches, is suggestive of short distance isolation,
inducing high genetic structure (Avise, 2000). The AMOVA
results showed that more variation occurred among popula-
tions rather than within them further indicating the high levels
of isolation between them. This isolation has been induced by
recent anthropogenic reductions in forest size and the frag-
mentation of larger patches (Cawe & McKenzie, 1989; Eeley
et al., 1999; Hughes et al., 2005), limiting the suitability of
areas between microhabitats, inhibiting migration (Barclay et
al., 2000; Woodman, Cooper, & Haritos, 2007). This historical
climatic reconnection followed by anthropogenic detachment
is further supported by the high, positive Fu's Fs values, in-
dicative of a population bottlenecks after becoming disjointed,
suggesting that even at localities with close proximity, there is
no contemporary maternal gene flow between them. Recent
change due to fragmentation is supported by the samples from
Nqadu which represent the highest sequence divergence in
clade 1, suggesting that the fragment has recently separated.
Nqadu (including both localities) being the most isolated for-
est patch displayed the lowest haplotype and nucleotide diver-
sity (Table 4) indicative of population instability. These were
the only sites apart from Baziya G that had a negative Fu's
Fs value, indicative of a population expansion (Fu, 1997).
Nqadu therefore contains two break‐off populations which
harbour four separate haplotypes with high amounts of disper-
sal within the patch but are however unstable. The pairwise
FST results for the sample localities at Nqadu were significant
when compared to every other locality from the present study.
These two sites are thus the only two of those sampled from
O. amaxhosa that showcase little limitation on their dispersal
capabilities within the forest patch and seem to have migrated
recently but with enough elapsed time to be captured as a pri-
vate haplocluster.
  
|
   17
Where there is gene flow among populations, it is only
among adjacent populations thus representing limited but ex-
istent migration in clade 1 and a near similar lack of disper-
sal in clade 2 (Figure 4). This may be due to the proximity
of sample sites, for example, the clustering of only adjacent
sampling localities within the haplotypes 28 and 30. Due to
limited dispersal, the p distances within the clades are mod-
erate. When compared, clade 1 showcased uncorrected diver-
gence of 9.34% within the clade and clade 2 showcased an
uncorrected divergence of 4.09%. This value is vastly higher
than values found for another Opisthopatus species (O. ro-
seus) analysed at relatively fine scale in the study by Daniels
(2011) however is skewed by the presence of the second spe-
cies within the clade, O. kwazululandi. Clade 1 exhibits a
lack of expansive haploclusters coupled with a high number
of private alleles which is supportive of a lack of maternal
dispersal at relatively small ranges. This is presumably due
to unsuitable environmental conditions induced by fragmen-
tation with migration routes being compromised via anthro-
pogenic influence.
4.3  |  Population sex structure
The sex ratio did not exhibit the typical female bias ob-
served in the literature (Barclay et al., 2000; Curach
& Sunnucks, 1999; Sunnucks et al., 2000; Tutt et al.,
2001). Sunnucks et al. (2000) observed that logs inhab-
ited by few velvet worms typically had a male bias due
to recent migrations, in contrast logs with more individu-
als showed a female bias. This pattern did not hold with
our results; however, it may explain the lack of females
found since the sample sizes averaged at four individuals.
Further, male bias is often attributed to newly colonised
logs (Curach & Sunnucks, 1999); however, the positive
Fu's Fs values for the majority of the sample sites suggest
population bottlenecks. Therefore, the lack of females is
probably a result of stochasticity or the time of sampling.
The timing of sampling plays a role in the observed sex
ratios and the presence of juvenile numbers since velvet
worms typically exhibit complex life histories and behav-
iour which are largely dependent on environmental condi-
tions and cues. The reproductive cycle in velvet worms
is largely associated with their microhabitat, which can
be different within each of the sample localities; thus, the
number of observed juveniles is dependent on these condi-
tions (Monge‐Nájera 2015). Therefore, there is very little
to be extrapolated from the number of juveniles observed
other than a recent lack of suitable birthing conditions,
since females have the ability to store sperm (Sunnucks et
al., 2000). Although females have been observed to reject
migratory males (Reinhard & Rowell, 2005), the aggrega-
tion composition within a log is evident to be largely influ-
enced by the abiotic environment while migratory patterns
 |
18       BARNES and DANIELS
appear to be determined by climate rather than social in-
centive and acceptance.
4.4  |  Conservation implications
The onychophora as a habitat specialist group is negatively
impacted by the removal of suitable forest habitat, and the
decaying logs that collect on the forest floor. To date, stud-
ies involving the conservation of velvet worms in South
Africa have analysed the genetic structure and impacts on
genetic diversity of velvet worms at a broad scale, assess-
ing separate forest patches and species (Daniels & Ruhberg,
2010; McDonald & Daniels, 2012; Myburgh & Daniels,
2015). Only a single study has focused on O. roseus in the
Ngele forest and detected shallow genetic structure for the
species (Daniels, 2011). During the present study, the use of
fine‐scale sampling detected the presence of a second line-
age where historically only a single species was known to
occur. Within each lineage a large number of distinct hap-
lotypes were present suggesting limited maternal gene flow
among conspecific localities of both species. This result sug-
gests that at a smaller spatial scale, decaying wood logs in
forests are important units for conservation. The main result
evident from the present study is the need for intensive sam-
pling in forested areas since the biodiversity of saproxylic
fauna remains poorly studied. The presence of three sympa-
tric species within Baziya reinforces the need for intensive
sampling within forest complexes for appropriate diversity
detection. In terms of velvet worms and saproxylic taxa with
low vagility, it would be logical to follow a fine‐scale ap-
proach when making efforts towards identification for con-
servation but also for studies assessing the social and genetic
interactions between these taxa. Hence, we suggest fine‐scale
sampling in areas where velvet worms have previously been
poorly collected to determine the biodiversity of the group.
Furthermore, we argue for the limited removal of decaying
wood logs in forested areas and the assistance of migration
via suitable habitat condition. The results observed in the pre-
sent study is a pattern that is also likely present in other low
sedentary inverterbates with specific microclimatic regimes
such as free‐living flatworms (Turbellaria), as well as in the
Annelida. Indeed, several fine‐scale studies of the latter ani-
mal groups have revealed similar levels of high endemism,
corroborating the importance of scale in sample design when
planning for conservation and taxonomic surveys.
ACKNOWLEDGEMENTS
The Foundational Biodiversity Initiative Programme, through
the South African National Biodiversity Initiative under
the NRF, is thanked for funding the Forest Research grant.
Madelaine Frazenburg is thanked for assistance during and co-
ordination of SEM procedures. Theo Busschau is thanked for
assistance with tree development and design as well as for the
live photography of the animals. Ilse Boonzaaier is thanked for
help with GIS and map design. The environmental affairs de-
partment of the Cacadu district is thanked for issuing collection
permits.
ORCID
Savel R. Daniels  https://orcid.org/0000-0003-2956-3256
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How to cite this article: Barnes A, Daniels SR. On
the importance of fine‐scale sampling in detecting
alpha taxonomic diversity among saproxylic
invertebrates: A velvet worm (Onychophora:
Opisthopatus amaxhosa) template. Zool Scr.
2019;00:1–20. https://doi.org/10.1111/zsc.12338