Molecular Phylogenetics and Evolution 61 (2011) 844–853
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Genetic diversification without obvious genitalic morphological divergence in
harvestmen (Opiliones, Laniatores, Sclerobunus robustus) from montane sky
islands of western North America
Shahan Derkarabetian a,⇑, Joel Ledford b, Marshal Hedin a
a
b
Department of Biology, San Diego State University, San Diego, CA 92182-4614, USA
Department of Entomology, California Academy of Sciences, San Francisco, CA 94118, USA
a r t i c l e
i n f o
Article history:
Received 4 March 2011
Revised 30 July 2011
Accepted 2 August 2011
Available online 16 August 2011
Keywords:
Phylogeography
Cryptic diversification
Morphological stasis
Population subdivision
Genealogical sorting index
a b s t r a c t
The southern Rocky Mountains and adjacent Intermontane Plateau Highlands region of western North
America is a geographically diverse area with an active geologic history. Given the topological complexity
and extensive geologic activity, organisms inhabiting this region are expected to show some degree of
morphological and genetic divergence, especially populations found on the southern montane ‘sky
islands’ of this region. Here we examine the phylogeographic history and diversification of a montane forest inhabiting harvestmen, Sclerobunus robustus, using a combination of genetic and morphological data.
Divergence time estimates indicate that much of the diversification within and between major groups S.
robustus predate the Pleistocene glacial cycles. The most widespread subspecies, Sclerobunus robustus
robustus, is recovered as six genetically distinct, geographically cohesive mitochondrial phylogroups.
Gene tree data for a single nuclear gene reveals congruent, albeit slightly more conservative, patterns
of genetic divergence. Despite high levels of genetic divergence throughout their distribution, phylogroups show extreme conservation in somatic and reproductive morphology. This uncoupling of morphological and genetic differentiation may be due to morphological conservatism associated with a
conserved microhabitat preference. Based on these data, it is obvious that S. robustus has undergone some
level of cryptic diversification.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
The southern Rocky Mountains and adjacent Intermontane Plateau Highlands (SRMIP) region of the southwestern United States is
a geographically and geologically diverse area. Today, this area is
characterized by isolated forested mountain ranges (‘sky islands’)
in southern Arizona and New Mexico, transitioning to more contiguous mountainous regions in the southern Rocky Mountains.
Mountain building in western North America took place during
two main periods: the Laramide and Neogene orogenies (Wilson
and Pitts, 2010). More recently, climatic fluctuations have resulted
in shifts in vegetation and forest availability, with major fluctuations occurring during the Miocene (5 Ma) and the Plio–Pleistocene
glacial cycles (van Devender and Spaulding, 1979; Lomolino et al.,
1989).
Given diverse topography and obvious temporal climatic variation, we would expect phylogeographic diversification and/or speciation in different lineages distributed across this region. Indeed,
⇑ Corresponding author. Present address: Department of Biology, Portland State
University, P.O. Box 751, Portland, OR 97207, USA
E-mail address: [email protected] (S. Derkarabetian).
1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2011.08.004
there are numerous studies that have shown recent phylogeographic divergence in an array of organisms that inhabit some or
all of the SRMIP, including mammals (Neotoma, Sullivan, 1994;
Sciurus, Lamb et al., 1997; Tamiasciurus, Barber, 2007), amphibians
(Hyla, Barber, 1999), invertebrates (Habronattus, Masta, 2000;
Moneilema, Smith and Farrell, 2005; Daktulosphaira, Downie,
2004), and plants (Sedum, DeChaine and Martin, 2005). Conversely,
there are relatively few studies demonstrating speciation within
this region, but exceptions include land snails (Sonorella, McCord,
1994) and flightless montane grasshoppers (Melanoplus, Knowles
and Otte, 2000). When comparing these studies, it is important
to note that all taxa showing more recent (phylogeographic) divergence are considered to be relatively good dispersers, while those
that have speciated within this region are considered to have low
vagility.
The Opiliones, commonly called harvestmen, comprise an order
of arachnids with over 6000 described species (Machado et al.,
2007). Opiliones diversity is divided into four morphologically divergent, highly supported clades: Cyphophthalmi, Eupnoi, Dyspnoi, and
Laniatores (summarized in Giribet and Kury, 2007). The majority of
north temperate Laniatores taxa are short-legged cryptozoic habitat
specialists, usually occupying moist, dark microhabitats. The
S. Derkarabetian et al. / Molecular Phylogenetics and Evolution 61 (2011) 844–853
western United States contains much of the diversity of North American Laniatores, including the Sclerobunines, which includes the
widespread genus Sclerobunus Banks 1893. Sclerobunus robustus is
a small-bodied (2–3 mm) harvestmen species typically found under
rocks or partially rotted logs in high elevation forests throughout the
SRMIP (Fig. 1). At southern latitudes (e.g., Arizona and New Mexico),
Sclerobunus are found in the isolated mountain ranges separated by
low elevation grassland and desert habitats. Further north, in the
more contiguous southern Rocky Mountains of Colorado, populations may be found at slightly lower elevations. Given these general
biological features, and a paradoxically large distribution, we predict
that this taxon has a high potential for phylogeographic
diversification.
In animals with internal fertilization, it is typically expected
that population- or species-level divergence is somehow coupled
with differentiation in reproductive morphology. More specifically,
divergence in genitalia is expected to occur at the same rate as ge-
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netic differentiation and typically faster than other morphological
differentiation (reviewed in Eberhard, 1985). In Opiliones, penis
morphology has been used to diagnose clades at various levels
(e.g. species, genus, family), and is typically used to differentiate
closely related harvestmen taxa. This is true for Laniatores (e.g.
Martens, 1976, 1986; Ubick and Briggs, 2004, 2008). However,
the discovery and description of morphologically cryptic lineage
diversity is becoming a common occurrence in microhabitatspecialist harvestmen with low vagility. In fact, morphologically
cryptic diversification in Opiliones is more the rule than the exception – molecular studies have found some level of cryptic diversification with significant genetic sub-structuring in Laniatores
(Thomas and Hedin, 2008; Hedin and Thomas, 2010), Dyspnoi
(Schönhofer and Martens, 2008, 2010), and Cyphophthalmi (Boyer
et al., 2007; Sharma and Giribet, 2009). In order to rigorously document the presence of cryptic diversification it is important to
have a robust molecular phylogeny reconstructed using relatively
Fig. 1. Sampling localities for S. robustus. Locations are colored based on recovered mitochondrial phylogroups. Locality numbers correspond to those listed in Supplemental
Table 1. The Rio Grande is represented as a dark blue line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
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fast evolving genes, combined with a detailed examination of
reproductive morphology, for a comprehensive geographic sample.
Here, we present an example of a low vagility, ecologically restricted harvestmen species that shows deep within-species
molecular divergence and an absence of obvious reproductive morphological divergence. Both mitochondrial and nuclear gene trees
are used to infer relationships among range-wide samples of S.
robustus. In addition, population genetic statistics are calculated
to further clarify the phylogeographic history of this species. Male
reproductive morphology (penis structure) is examined using SEM
to assess morphological divergence between major phylogeographic lineages. In this taxon we find a situation where, despite
deep and relatively ancient intraspecific genetic divergence, there
is little somatic differentiation, and a lack of obvious divergence
in reproductive morphology.
2. Materials and methods
2.1. Sampling
Sclerobunus specimens were collected by hand from beneath
rotting logs and woody debris, and sometimes rocks, in moist
high-elevation (2300–3300 m) mixed conifer or aspen forests.
Specimens destined for morphological analysis were preserved in
80% EtOH; specimens used for molecular analyses were preserved
in 100% EtOH. Sampling was conducted for S. robustus from
throughout the known range of this species (Fig. 1), with a total
of 46 localities sampled from two regional subspecies (Sclerobunus
robustus glorietus from 5 sites; Sclerobunus robustus robustus from
41 sites). These subspecies are differentiated by size and coloration: S. r. glorietus is under 2 mm with black pigmentation, and S.
r. robustus is larger than 2 mm with no black pigment (Briggs,
1971). In addition, three highly troglomorphic populations were
included: Cave of the Winds (CO), Mallory Cave (CO), and Taos
Ski Valley (NM). Following previous analyses (Derkarabetian
et al., 2010), outgroup sampling included all other Sclerobunus species (this includes samples of the subspecies Sclerobunus robustus
idahoensis, which is clearly not closely related to other S. robustus),
Cyptobunus species, and Metanonychus as a distant outgroup. Detailed collecting information can be found in Supplemental Table 1.
2003). The manually aligned COI matrix was partitioned using
the optimal partitioning strategy determined by Bayes Factor analyses (Nylander et al., 2004; Brandley et al., 2005). Analyses were
run until the average standard deviation of split frequencies was
below 0.01. The first 40% of trees were discarded as burnin. Posterior probabilities of 0.95 and greater were considered highly supported. Nuclear data were aligned by eye with amino acid
translations and the exon was subsequently removed from analysis. Because of high EF-1a intron divergences among taxa, outgroup sequences were not used. EF-1a intron sequences were
both manually aligned (using MacClade v 4.06, Maddison and
Maddison, 1992) and aligned using ClustalX v 1.83 (Thompson
et al., 1997), with subsequent manual adjustments.
Divergence dates were estimated from the COI matrix using a
relaxed clock with uncorrelated lognormal distribution, implemented in the BEAST v1.6.1 software package (Drummond and
Rambaut, 2007). Two geological calibration points were used in
this analysis. First, the split between Sclerobunus nondimorphicus
from the Cascade Mountains and S. r. idahoensis from the northern
Rocky Mountains, coinciding with the rise of the Cascades (normal
distribution with mean of 3.5 and deviation of 1.0). The Cascades/
Northern Rockies vicariance is a well-documented biogeographic
break found in over 150 different species (Brunsfeld et al., 2001).
Second, the timing of cave passage development for the Cave of
the Winds began 5 MA (Luiszer, 2007); this was used as a minimum age for the divergence of the Cave of the Winds population
from all other S. robustus. Several analyses were run to check for
convergence of tree topology and parameters, with a final analysis
run for a total of 40 million generations (10% burnin) sampled
every 1000 steps. The combined analyses were examined with Tracer v1.5 (Rambaut and Drummond, 2007) to check for sufficient
ESS values for all parameters (BEAST manual), and a tree was
assembled using TreeAnnotator v1.6.1 (Drummond and Rambaut,
2007).
2.4. Population structure
Genomic DNA was extracted following standard procedures
(see Derkarabetian et al., 2010). Targets of PCR amplification included the mitochondrial cytochrome oxidase I (COI) gene, and intron sequences from the nuclear EF-1a protein-coding gene (see
Hedin et al., 2010). PCR protocols and primers were identical to
methods published previously (Hedin et al., 2010; Derkarabetian
et al., 2010). All COI and most EF-1a PCR products were purified
using Polyethylene Glycol (PEG) precipitation; some EF-1a amplicons were gel purified using the QIAGEN QIAquick Gel Extraction
Kit. Sanger sequencing was performed at the San Diego State University Microchemical Core Facility (http://www.sci.sdsu.edu/dnacore/sdsu_dnacore.html). Contiguous sequences were assembled
using Sequencher 4.5 (Gene Codes Corporation, MI) and MacClade
v 4.06 (Maddison and Maddison, 1992). Heterozygous sites in EF1a sequences were left unphased and treated as ambiguous using
standard ambiguity codes.
To examine the genetic differentiation of recovered mitochondrial lineages, average intra- and interclade distances were
computed using PAUP 4.0b (Swofford, 2002) using the Kimura
two-parameter model (Kimura, 1980). Several diversity statistics
were calculated for each major mitochondrial lineage, including:
number of haplotypes (N), number of polymorphic sites (s), average number of pairwise nucleotide differences (k), and nucleotide
diversity (p). All diversity statistics and Tajima’s D (Tajima, 1989)
tests were calculated using DnaSP (Librado and Rozas, 2009). The
genealogical sorting index (gsi) test statistic was used to assess
the level of genealogical exclusivity for particular groups (Cummings et al., 2008), and was calculated using the gsi website
(http://www.genealogicalsorting.org/) with 10,000 replicates. The
gsi statistic can be used to assess the relative degree of exclusivity
for a group, ranging from no exclusivity (completely random distribution of haplotypes – values of 0) to exclusive ancestry (values of
1). When comparing two (or more) sampling locations, exclusive
ancestry for haplotypes collected from different locations implies
an absence of gene flow among them. In calculating gsi values,
COI sequences were grouped by collecting locality (assessing site
exclusivity), and EF-1a sequences were grouped by recovered
mitochondrial COI clades (assessing nuclear/mitochondrial
congruence).
2.3. Sequence alignment and phylogenetic analyses
2.5. Penis morphology
Phylogenetic analyses were conducted on individual gene
matrices using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003),
employing models chosen by the AIC in MrModeltest (Nylander,
Penises were extracted by pushing the penis through the genital
operculum with an insect pin inserted through the opening of the
anal operculum. Extracted penises were soaked in a cold solution
2.2. Molecular data collection
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Metanonychus OP985
Metanonychus OP974
S. nondimorphicus OP222
S. nondimorphicus OP1056
S. nondimorphicus OP120
S. r. idahoensis OP1635
S. r. idahoensis OP1649
S. r. idahoensis OP1633
Cyptobunus cavicolens OP2144
Cyptobunus cavicolens OP2143
Cyptobunus u. madhousensis OP240
Cyptobunus u. madhousensis OP239
Cyptobunus u. ungulatus OP1229
Cyptobunus u. ungulatus OP1232
glorietus (2) Glorieta Canyon OP890
glorietus (2) Glorieta Canyon OP891
n. sp. (7) Mallory Cave OP2568
n. sp. (6) Cave of the Winds OP1127
n. sp. (6) Cave of the Winds OP1128
glorietus (3) Mt. Taylor OP903
glorietus (3) Mt. Taylor OP904
glorietus (5) Manzano Mtns OP907
glorietus (5) Manzano Mtns OP908
glorietus (4) Sandia Mtns OP2101
glorietus (4) Sandia Mtns OP2102
n. sp. (1) Taos Ski Valley OP972
n. sp. (1) Taos Ski Valley OP1171
glorietus (1) Taos Ski Valley OP1169
glorietus (1) Taos Ski Valley OP882
glorietus (1) Taos Ski Valley OP2119
glorietus (1) Taos Ski Valley OP1170
glorietus (1) Taos Ski Valley OP881
glorietus (1) Taos Ski Valley OP883
robustus (11) Abajo Mtns OP1130
robustus (11) Abajo Mtns OP1131
robustus (14) La Sal Mtns OP1202
robustus (14) La Sal Mtns OP1203
robustus (12) Dolores River OP1149
robustus (12) Dolores River OP1148
robustus (13) Beaver Creek OP1174
robustus (13) Beaver Creek OP1173
robustus (18) Hannagan Meadow OP935
robustus (18) Hannagan Meadow OP938
robustus (22) San Fransisco Mtns NM OP2100
robustus (19) Pinalero Mtns OP945
robustus (19) Pinalero Mtns OP946
robustus (21) Santa Catalina Mtns OP964
robustus (21) Santa Catalina Mtns OP965
robustus (15) Alpine Divide OP906
robustus (15) Alpine Divide OP905
robustus (16) San Fransisco Mtns OP913
robustus (16) San Fransisco Mtns OP914
robustus (17) Mogollon Mtns OP921
robustus (17) Mogollon Mtns OP923
robustus (20) Pinos Altos Mtns OP951
robustus (20) Pinos Altos Mtns OP952
robustus (8) Pagosa Springs OP1178
robustus (8) Pagosa Springs OP1179
robustus (10) Haviland Lake OP1223
robustus (10) Haviland Lake OP1224
robustus (9) Ouray OP1197
robustus (9) Ouray OP1199
robustus (44) Jemez Mtns OP885
robustus (44) Jemez Mtns OP887
robustus (43) Deer Creek Trail OP878
robustus (43) Deer Creek Trail OP877
robustus (45) N of Chama OP2114
robustus (45) N of Chama OP2115
robustus (49) Chiricahua Mtns OP1040
robustus (49) Chiricahua Mtns OP1039
robustus (47) Mimbres Mtns OP927
robustus (47) Mimbres Mtns OP929
robustus (46) Ski Apache OP893
robustus (46) Ski Apache OP894
robustus (48) Bradford Canyon OP956
robustus (48) Bradford Canyon OP959
robustus (26) Sangre de Cristo OP1145
robustus (26) Sange de Cristo OP1146
robustus (36) Great Sand Dunes OP2104
robustus (36) Great Sand Dunes OP2105
robustus (37) S Creede OP2108
robustus (37) S Creede OP2109
robustus (30) North Pass OP1189
robustus (30) North Pass OP1188
robustus (33) Church Creek OP1209
robustus (33) Church Creek OP1210
robustus (28) Monarch Pass OP1177
robustus (34) nr Garfield OP1214
robustus (34) nr Garfield OP1215
robustus (35) Weston Pass OP1219
robustus (25) NE Leadville OP1140
robustus (25) NE Leadville OP1141
robustus (24) Twin Lakes OP1136
robustus (24) Twin Lakes OP1135
robustus (35) Weston Pass OP1218
robustus (31) NE Deckers OP1193
robustus (31) NE Deckers OP1194
robustus (32) N Gore Range OP1208
robustus (42) Apex Valley Rd OP2686
robustus (42) Apex Valley Rd OP2687
robustus (39) Fault Cave OP2569
robustus (39) Fault Cave OP2570
robustus (29) Longs Peak OP1183
robustus (29) Longs Peak OP1184
robustus (27) Wet Mtns OP1164
robustus (27) Wet Mtns OP1165
robustus (23) S of Gothic OP1122
robustus (23) S of Gothic OP1123
robustus (40) Hanging Lake OP2680
robustus (38) Skeleton Cave OP2567
robustus (40) Hanging Lake OP2679
8.03 - 23.69
12.09 - 28.57
S. robustus
Sclerobunus r. glorietus (1)
COTW/MC (6)
Sclerobunus r. robustus (42)
MIOCENE
S. r. glorietus
COTW/MC
S. r. glorietus
Northwestern
Southwestern
Haviland
Central Eastern
Southeastern
Northeastern
PLIOCENE PLEIST.
2.6
5.3
Fig. 2. Results of BEAST divergence analyses. Locality numbers are in parentheses for each taxon. Node bars represent 95% HPD of divergence date and are colored according
to posterior probability support: blue (posterior probability of >0.95) and red (0.50–0.95). Nodes without bars correspond to a posterior probability <0.50. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
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of 10% KOH for several minutes to promote expansion, and to remove muscle layers for easier viewing. For a small sample of specimens we used an alternative technique to promote penis
expansion – here we placed specimens with everted penises into
a hot lactic acid (85%) solution for 2–3 min, then moved these specimens into distilled water. This procedure was repeated multiple
times for each specimen.
Specimens treated with 10% KOH were cleaned prior to SEM
examination (Leo 1450VP SEM) using an ultrasonicator, critical
point dried, then mounted on Ted PellaÓ aluminum SEM stubs
using copper-backed electrical tape. All major mitochondrial lineages of S. robustus were examined for penis morphology. SEM
samples could not be obtained for Mallory Cave, as only one (male)
specimen is currently known, and for the troglomorphic form from
Taos Ski Valley because males for this population (likely an undescribed species) are unknown.
3. Results
GenBank accession numbers for newly generated sequences are
provided in Supplemental Table 1. Alignments and summary trees
from all analyses (e.g., Bayesian majority rule consensus trees)
have been deposited at TreeBASE II. A Google Earth kmz file of all
sampled locations is available upon request from the corresponding author.
3.1. COI gene trees
A total of 103 COI sequences were analyzed, 97 of which were
generated from S. robustus specimens (14 S. r. glorietus, 78 S. r.
robustus, and 5 S r. spp.). COI sequences were obtained from two
individuals from each collecting locality, with few exceptions
(San Francisco Mountains, NM; Monarch Pass, CO; N Gore Range,
CO; Skeleton and Mallory Caves, CO). COI sequences could not be
generated for Spring Creek samples (site 41, Fig. 1). Not including
outgroups, 767 characters were constant, and 360 were parsimony-informative. The results of the COI BEAST analysis are shown
in Fig. 2, with estimated divergence times summarized in Table 1.
The BEAST topology is largely congruent with the COI MrBayes
topology (Supplemental Fig. 1), except for the placement of the
Haviland clade, which is weakly supported in both analyses.
S. robustus is recovered as monophyletic with high support. S. r. glorietus is paraphyletic, with the type locality (Glorieta Canyon, NM)
strongly supported as sister to all other S. robustus, while all other
populations morphologically identified as S. r. glorietus form a
clade. A Bayes Factor analysis was run (Kass and Raftery, 1995;
Nylander et al., 2004) in which all S. r. glorietus were constrained
to form a monophyletic group, but a BF of 10.48 is very strong evidence against this alternative topology. The highly troglomorphic
COTW/MC clade was recovered as sister to all S. robustus with
the exception of the Glorieta Canyon population. The most recent
common ancestor (MRCA) for S. robustus is estimated at 9.31 Ma
(95% HPD 6.2–12.9 Ma), while the MRCA for S. r. robustus is
5.48 Ma (95% HPD 3.54–7.55).
Within S. r. robustus, six highly supported major mitochondrial
subclades were found (support values shown on Supplemental
Fig. 1), including Haviland, Northwestern (NW), Southwestern
(SW), Northeastern (NE), Central Eastern (CE), and Southeastern
(SE). The geographic distribution of these subclades is shown in
Fig. 1. Diversification date estimates for these major subclades
are Pleistocene in age (ranging from 1.34 to 1.51 Ma) for the CE
and NW clades, and Pliocene in age (2.75–3.09 Ma) for the SW,
NE, and Haviland clades. Although the Western (NW and SW),
Eastern (NE, CE, SE), and Haviland lineages of S. r. robustus are each
highly supported, the relationships among them are not. All col-
lecting localities where multiple haplotypes were sampled were
found to be reciprocally monophyletic (all highly supported) except for three sites in the NE clade (Weston Pass, NE Leadville,
and Twin Lakes).
3.2. Mitochondrial population structure
Average intra- and interclade COI K2P distances are presented
in Supplemental Table 2. Most interclade divergences are above
10%, while intraclade divergences range from 3.1% to 9.2%. Within
S. r. robustus, southern ‘sky islands’ subclades (SW and SE) show
relatively high intraclade divergences, while the northern subclades (NW, NE, and CE) distributed in more contiguous mountainous regions have lower intraclade divergences. Diversity statistics
and demographic history tests for all major clades are shown in Table 2. Results show high numbers of unique haplotypes within S.
robustus (77 unique haplotypes from 97 specimens) and high levels
of intraclade nucleotide diversity. Tajimas’s D values were found to
be non-significant.
3.3. EF-1a gene trees
The EF-1a intron was successfully sequenced for 27 S. robustus
specimens, representing all major mitochondrial lineages. Many
sequences contained poly-AT regions near the 50 and 30 ends;
ambiguous regions of alignment in these sections were removed
from subsequent analysis. The manual and ClustalX with manual
adjustments produced identical alignments and tree topologies,
and resulted in taxon bipartitions with very similar support values.
The final analysis was run on the ClustalX with manual adjustments alignment, with an alignment length of 605 base pairs.
The EF-1a gene tree is unrooted, but if we make the assumption
that the root falls on a branch that separates primary groups, then
this gene tree recovers many clades also found in COI analyses
(Fig. 3). All populations identified as S. r. glorietus were recovered
in a single clade with high support. Similarly, the COTW/Mallory
Cave clade and Eastern and Western clades of S. r. robustus were
each recovered with high support (>0.95 posterior probability).
The SW clade was recovered with high support, embedded within
a paraphyletic NW clade. The NE and SE clades were recovered
with moderate support (0.90–0.95 pp) while the CE clade was
paraphyletic with respect to the SE clade. The single Haviland sample was found nested within the geographically adjacent NE clade.
The Spring Creek sample, for which no COI data could be collected,
was recovered within the NE clade. Overall, the nuclear gene tree
results suggest two sets of southern ‘sky island’ groups derived
from more northern locations: the SW clade derived from the
Table 1
Results of BEAST divergence analyses. Values in millions of years.
Clade
Tmrca
Mean
95%
minimum
95%
maximum
Root
Sclerobunus
S. robustus (all)
S. r. glorietus (without Glorieta
Canyon)
COTW/MC
S. r. robustus
Western
Northwestern
Southwestern
Haviland
Eastern
Southeastern
Central Eastern
Northeastern
19.84
13.54
9.31
5.11
12.09
8.86
6.2
2.86
28.57
18.91
12.9
7.47
5.8
5.48
3.79
1.34
2.87
3.09
4.59
2.75
1.51
2.85
5 (fixed)
3.54
2.34
0.73
1.75
1.6
2.93
1.65
0.79
1.74
7.4
7.55
5.37
2.02
4.1
4.76
6.42
3.97
2.31
4.09
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glorietus (2) Glorieta Canyon OP890
.99
glorietus (5) Manzano Mtns OP907
1.0
S. r. glorietus 1 (<0.001)
n. sp. (1) Taos Ski Valley OP972
.91
glorietus (1) Taos Ski Valley OP882
n. sp. (6) COTW OP1127
1.0
COTW/MC: 1 (0.019)
n. sp. (7) Mallory Cave OP2568
robustus (12) Dolores River OP1149
1.0
1.0
robustus (11) Abajo Mtns OP1130
1.0
NW: 0.639 (0.007)
robustus (14) La Sal Mtns OP1203
.93
robustus (16) San Francisco Mtns OP913
1.0
robustus (20) Pinos Altos Mtns OP951
.65
robustus (15) Alpine Divide OP905
.74
.90
SW: 1 (<0.001)
robustus (21) Santa Catalina Mtns OP964
robustus (44) Jemez Mtns OP885
.74
CE: 0.48 (0.035)
robustus (45) N Chama OP2115
robustus (49) Chiricahua Mtns OP1039
.92
1.0
robustus (48) Bradford Canyon OP956
SE: 1 (0.022)
robustus (25) NE Leadville OP1140
robustus (33) Church Creek OP1210
.69
robustus (27) Wet Mtns OP1164
1.0
robustus (34) Garfield OP1215
.91
robustus (42) Apex Valley Rd OP2686
NE: 0.84 (<0.001)
robustus (41) Spring Creek OP2683
HAV
robustus (10) Haviland OP1223
0.02
.67
robustus (37) S Creede OP2109
.93
robustus (23) S Gothic OP1122
robustus (40) Hanging Lake OP2679
Fig. 3. EF-1a intron 50% majority rule Bayesian consensus phylogram. Posterior probabilities appear to the left of each node. EF-1a gsi values shown (using mitochondrial
phylogroups as grouping variables), with p values in parentheses.
NW, and the SE clade derived from the CE (Fig. 3). Using mitochondrial clades as a priori groups, EF-1a gsi values were one (=exclusive ancestry) with the exception of the NE and CE clades (all
values are statistically significant; Fig. 3).
3.4. Penis morphology
Specimens treated in 10% KOH versus lactic acid resulted in
similar degrees of penis expansion. SEM images of penises expanded in KOH were generated for 12 specimens representing all
major mitochondrial lineages of S. robustus. The penis size of S. r.
robustus is nearly 50% larger than that of S. r. glorietus, but this is
perhaps expected given the body size difference between these
subspecies. Penis morphology of these two subspecies is structurally very similar (Fig. 4). Within S. r. robustus, there is remarkably
little differentiation in penis morphology between specimens representing divergent molecular clades (Fig. 4). All penis images have
been deposited at Morphbank (http://www.morphbank.net/).
4. Discussion
4.1. Phylogeography
Laniatorean harvestmen are attractive candidates for phylogeographical studies due to their low vagility and microhabitat specialization. S. robustus is particularly useful because, in addition
to those characteristics, this species has a paradoxically wide distribution. There have been numerous phylogeographic studies
focusing on organisms inhabiting the SRMIP that show population
sub-structuring reflecting the geological and climatic history of
this area. A smaller fraction of these studies focus on animals that
occupy the montane forest habitats throughout the SRMIP (e.g. Sullivan, 1994; Lamb et al., 1997; Barber, 1999; Masta, 2000; Smith
and Farrell, 2005; DeChaine and Martin, 2005; Barber, 2007). The
great majority of those phylogeographical or biogeographical studies have used vertebrate taxa as the focal species, with few exceptions including spiders (Masta, 2000) and beetles (Smith and
Farrell, 2005), both of which occupy only the extreme southwest
(i.e. ‘sky islands’ of southern Arizona and New Mexico). We contend that in order to better understand the biogeographical history
of the SRMIP it is important to include all types of organisms (i.e.,
vertebrate, invertebrate, and plants), and include low-dispersal
taxa that are distributed throughout the SRMIP.
Within S. r. robustus the majority of localities sampled are genealogically exclusive for mitochondrial COI sequences (Supplemental
Table 2
Mitochondrial genetic diversity statistics for S. robustus.
Subclade
n
N
s
k
p
S. robustus (total)
S. r. glorietus (no Glorieta
Canyon)
COTW/MC
Haviland
Northwestern
Southwestern
Southeastern
Central Eastern
Northeastern
97
14
77
9
191
129
46.405
50.582
0.07117
0.06621
0.59577
3
6
8
15
8
6
35
3
4
7
15
8
5
27
69
73
50
131
136
44
127
46.000
38.600
21.893
44.638
59.571
22.733
29.435
0.04868
0.04843
0.02743
0.05905
0.06104
0.02922
0.04117
n/a
1.23092
0.49345
-0.19893
0.27722
0.98167
-0.68114
Tajima’s
D
Notes: COTW/MC, Cave of the Winds and Mallory Cave. n, number of sequences; N,
number of haplotypes; s, number of segregating sites; k, average number of
nucleotide differences; p, nucleotide diversity.
850
S. Derkarabetian et al. / Molecular Phylogenetics and Evolution 61 (2011) 844–853
Fig. 4. SEM images of lateral and ventral views of penes for representatives from mitochondrial phylogroups of S. robustus. (A–B) S. r. glorietus, Taos Ski Valley; (C–D) S. r.
glorietus, Manzano Mountains; (E–F) S. r. robustus, NW: Dolores River (12); (G–H) S. r. robustus, SW: Santa Catalina Mountains (21); (I–J) S. r. robustus, NE: Church Creek (33);
(K–L) S. r. robustus, SE: Ski Apache (46); (M–N) S. r. robustus, CE: N Chama (45), (O–P) S. r. robustus, HAV: Haviland Campground (10). Colored dots at bottom left of images
correspond to mitochondrial phylogroups of S. r. robustus. Scale bars = 100 lm.
Table 3), indicating an absence of female-based gene flow at the
geographic scale of our sampling regime. Northern clades (NW, NE,
and CE) show lower levels of nucleotide and genetic diversity as
compared to southern clades (SW and SE), most likely due to the
greater connectedness of the mountain ranges they inhabit (Table 2).
The ‘sky islands’ in the south are more isolated and separated by
greater distances as compared to the more contiguous northern
ranges. Nuclear data suggest dual origins of the two southern clades,
SW and SE, from NW and CE northern clades, respectively (Fig. 3).
The mitochondrial data show a contrasting south to north expansion
within the NE clade, centered in the southern San Juan and Sangre de
Cristo Mountains, with subsequent northward phylogeographic
nesting. All localities that lack exclusivity for mitochondrial
haplotypes (gsi values less than 1) were found within the NE clade
(Supplemental Table 3), including the northern-most samples in
our study. This suggests either higher levels of gene flow in the more
continuous northern locales, or relatively recent range expansion.
Taken together, the mitochondrial and nuclear data suggest that
mid-latitude highlands (e.g., in southern UT/CO, northern NM)
may have served as regions for multiple historical refugia for
Sclerobunus.
There have been numerous other phylogeographic studies of
organisms inhabiting the SRMIP, but the vast majority of these studies are limited in their geographic breadth. An exception is the
analysis of the tassel–eared squirrel, Sciurus aberti (Lamb et al.,
1997). These authors report eastern and western phylogeographic
S. Derkarabetian et al. / Molecular Phylogenetics and Evolution 61 (2011) 844–853
clades within this species, with similar distributions to the eastern and western clades recovered in Sclerobunus, except for the
inclusion of the Abajo Mountains (southeastern UT) population
in the eastern Sciurus clade. Similar geographic breaks are seen in
Tamiasciurus squirrels (Barber, 2007), with three clades found in
the SRMIP which roughly correspond to the SW, SE, and CE clades,
and with an eastern and western split as seen in S. robustus.
There are many concerns associated with accurately estimating
divergence dates from molecular data (see Rutschmann, 2006 and
references therein). Similarly, there is a great deal of uncertainty,
and a lack of consensus, regarding the geologic history and dates
of mountain building in western North America. Based on an
examination of previous geologic and biogeographic literature
regarding mountain uplift and desert formation throughout western North America, Wilson and Pitts (2010) provide conservative
estimates for the Neogene mountain building episode at 15–
2 Ma, while estimates for the Laramide Orogeny are much older
(at least 50 Ma). Our divergence time estimates (Table 1 and
Fig. 2) suggest that diversification within Sclerobunus occurred
within this general range of Neogene uplift. Due to the broad range
of dates attributed to the Neogene (some of which occur as recent
as 2 Ma), more recent divergences ranging from 3 Ma to the present can either be attributed to mountain building or Plio–Pleistocene glacial cycles. Most divergence date estimates within S.
robustus are pre-Pleistocene – this contrasts with other reported
divergence estimates for ‘sky island’ taxa, which also show much
lower levels of genetic divergence as compared to S. robustus
(e.g., Lamb et al., 1997; Masta, 2000; DeChaine and Martin,
2005). Similar findings have been reported in organisms restricted
to the Wet Tropic montane ‘sky islands’ of northeastern Australia.
For example, high levels of population structure consistent with
predicted refugia have been reported in Temnoplectron beetles (Bell
et al., 2007), Gnarosophia land snails (Hugall et al., 2002), and vertebrates (Schneider et al., 1998). Additionally, divergence estimates for many of these taxa, including vertebrates, are
consistent with Pliocene divergences.
4.2. Genetic divergence without morphological divergence
High levels of genetic divergence are found in S. robustus at all
levels. Most sampled S. robustus populations are exclusive for mitochondrial haplotypes, indicating a lack of female-based gene flow.
The S. r. robustus subspecies shows much internal genetic differentiation, with six highly supported mitochondrial phylogroups, all of
which are distributed in allopatry and show high interclade divergence levels. Estimated divergence times for these allopatric clades
are mostly pre-Pleistocene. The observation of nuclear gene tree
congruence, which is expected to occur relatively late in the divergence process (Moore, 1995; Avise, 2000), further supports relatively ancient diversification.
Given this obvious lack of genetic cohesiveness, we expected to
observe coincident morphological differentiation. The two subspecies differ in size, coloration, and in other subtle somatic characters
(e.g., spination of the palpal femur and leg I femur, Briggs, 1971;
structure of the coxal apophyses, pers. obs.), and are easily distinguished using morphometrics analyses (Derkarabetian et al.,
2010). However, there is surprisingly little difference in reproductive morphology between these subspecies (Fig. 4). Within the
genetically divergent, allopatric clades of S. r. robustus there is almost a complete absence of both somatic and reproductive morphological differences. This conservatism was also noted by
Briggs (1971), who in reference to sky island populations stated
that ‘‘the isolated populations are remarkably undifferentiated’’.
Under a cryptic female choice model (Eberhard, 1985; reviewed
in Hosken and Stockley, 2004; Song and Bucheli, 2010 and references therein), some reproductive morphological divergence is ex-
851
pected between separate arthropod lineages because of ‘‘rapid and
divergent’’ genitalic evolution. In the case of S. robustus, we do not
observe noticeable genital divergence in a system with obvious
geographic isolation that appears to be relatively old. One can
ask, what is maintaining morphological cohesiveness across space
in this taxon? Gene flow is an unlikely cohesive force, and morphological similarity due to recent divergence is not supported. Informally, S. robustus populations are found in very similar
microhabitats throughout their range, suggesting conservation in
the occupied niche. It is not unusual to find closely related taxa
that are morphologically conserved due to ecological niche conservation (i.e. niche conservatism promotes morphological conservatism, see Bond et al., 2001; Bond and Stockman, 2008). What is
unusual is the absence of differentiation in reproductive morphology, as these traits are not ‘‘adaptive’’ per se, and hence not expected to be affected by ecological niche constraints.
Studies of other arthropod taxa have reported similar findings
regarding a decoupling of genetic and genitalic divergence. Bond
et al. (2003) report a gradual evolution of genitalia in Anadenobolus
millipeds, where male genitalia was less structured than expected
given the levels of genetic divergence. The authors suggest that, because some overlap existed in genitalic morphological space, not
enough time has passed for the genitalia to sort to reciprocal
monophyly. Other studies have also reported a decoupling of genitalic and genetic divergence in spiders (Hedin, 1997; Bond et al.,
2001; Huber et al., 2005) and scorpions (Jacob et al., 2004). Finally,
conservation of penis morphology in genetically divergent lineages
has been observed in other harvestmen taxa. Examples include the
European genus Trogulus, where more recently evolved species
groups show conservation in penis morphology, while relatively
older lineages show more divergence (Schönhofer and Martens,
2010). Similarly, the New Zealand cyphophthalmid species Aoraki
denticulata denticulata shows highly conserved morphology across
its distribution, despite low vagility (an absence of genetic exchange) and high genetic divergences (Boyer et al., 2007). We
noted previously that the penis morphologies of the sister taxa S.
nondimorphicus and S. r. idahoensis are nearly identical in structure,
despite geographic isolation, significant genetic divergence, and
somatic morphological differences (Derkarabetian et al., 2010).
4.3. How to proceed from here?
A decoupling of genetic divergence and reproductive morphological divergence poses obvious difficulties for species delimitation in harvestmen, as variation in penis morphology represents
an important character source for species delimitation. Given the
amount of genetic differentiation seen within S. robustus and the
common occurrence of cryptic divergence in harvestmen, it is possible that this decoupling of genetic divergence and morphological
divergence is happening at the species level. By requiring that different harvestmen species have different penis morphologies, we
are likely underestimating species diversity, perhaps greatly so.
We see several opportunities for further research in Sclerobunus,
and in other harvestmen groups that show similar patterns of
apparent genitalic stasis. First, we need to better understand how
the genitalia work from a functional perspective. Are there ‘‘hidden’’ genitalic characters that we are simply not observing using
the methods employed here? Although this possibility always remains a potential criticism, we note that we have used commonly
employed techniques for the study of harvestmen penises, and indeed tried multiple methods to best reveal penis microstructures.
A second avenue for future research involves additional development and study of nuclear gene divergence. The EF-1a intron data
indicates partial (but conservative) nuclear gene divergence coincident with mitochondrial gene divergence in this system. Expanding this nuclear gene perspective to many more genes would be
852
S. Derkarabetian et al. / Molecular Phylogenetics and Evolution 61 (2011) 844–853
informative, allowing greater insight into historical demography,
the true extent of morphological conservatism, and a better understanding of species limits in these arachnids.
Acknowledgments
This research was supported by the Cave Conservancy Foundation (Fellowship in Karst Studies), Society for Integrative and Comparative Biology (Grants in Aid of Research), and the American
Arachnological Society (Vincent Roth Fund for Systematic Research). For loans of material we thank the California Academy of
Sciences, American Museum of Natural History, and the Denver
Museum of Nature and Science. For assistance in fieldwork we
would like to thank Joe Deas, Damian Elias, Ryan Fawcett, Lars Hedin, Maureen McCormack, Casey Richart, and Steven Thomas. SEM
access was provided by the California Academy of Sciences with
support from the Harriet-Exline Frizzel Fund. Comments by Casey
Richart, Axel Schönhofer, and two anonymous reviewers helped to
improve the manuscript.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2011.08.004.
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