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Genetic diversification and demographic history of the cactophilic
pseudoscorpion Dinocheirus arizonensis from the Sonoran
Desert
Edward Pfeilera, Ben G. Bitlerb, Sergio Castrezanab,*, Luciano M. Matzkinb,*, and Therese A.
Markowb,*
a Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Guaymas, Apartado Postal 284,
Guaymas, Sonora 85480, México
b Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA
Abstract
Sequence data from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene were
used to examine phylogenetic relationships, estimate gene flow and infer demographic history of the
cactophilic chernetid pseudoscorpion, Dinocheirus arizonensis (Banks, 1901), from the Sonoran
Desert. Phylogenetic trees resolved two clades of D. arizonensis, one from mainland Sonora, Mexico
and southern Arizona (clade I) and the other from the Baja California peninsula and southern Arizona
(clade II). The two clades were separated by a mean genetic distance (d) of ~2.6%. Hierarchical
analysis of molecular variance indicated highly significant population structuring in D. arizonensis
(overall ΦST = 0.860; P < 0.0001), with 80% of the genetic variation distributed among the two
clades. Most pairwise comparisons of ΦST among populations within each clade, however, were not
significant. The results suggest that phoretic dispersal on vagile cactophilic insects such as the neriid
cactus fly Odontoloxozus longicornis (Coquillett, 1904) provides sufficient gene flow to offset the
accumulation of unique haplotypes within each clade of the non-vagile pseudoscorpion. Preliminary
results on dispersal capability of O. longicornis were consistent with this conclusion. Tests designed
to reconstruct demographic history from sequence data indicated that both clades of D.
arizonensis, as well as O. longicornis, have experienced historical population expansions. Potential
barriers to gene flow that may have led to genetic isolation and diversification in clades I and II of
Dinocheirus arizonensis are discussed.
Keywords
cytochrome c oxidase subunit I; historical demography; phoresy; phylogenetic relationships;
population structure; Pseudoscorpiones
To whom correspondence should be addressed: Dr. Edward Pfeiler, CIAD, A.C., Apartado Postal 284, Guaymas, Sonora 85480, México,
E-mail: E-mail: epfeiler@asu.edu, Phone and FAX: +52-622-221-6533.
*Present address: Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0116, USA
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Author Manuscript
Mol Phylogenet Evol. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
Mol Phylogenet Evol. 2009 July ; 52(1): 133–141. doi:10.1016/j.ympev.2008.12.020.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
1. Introduction
Necrotic tissues (rots) of several species of columnar cacti from the Sonoran Desert of
southwestern USA and northwestern Mexico, including saguaro (Carnegiea gigantea), cardón
(Pachycereus pringlei), organ pipe (Stenocereus thurberi), agria (S. gummosus) and senita
(Lophocereus schottii), provide an ideal environment for the feeding, breeding and
development of a host of insects and other arthropods (Ryckman and Olsen, 1963; Castrezana
and Markow, 2001). Although the availability of suitable microhabitat for the cactophilic
arthropods varies with cactus species, in all cases necroses are distributed in a patchy manner
and are ephemeral, with an individual rot lasting anywhere from a few weeks for senita to
several months for saguaro and cardón (Breitmeyer and Markow, 1998). Thus, the ability to
disperse to fresh necroses is of fundamental importance for the survival of organisms dependent
upon this microhabitat. In addition, the different life histories and dispersal abilities of the
diverse community of cactophilic arthropods provide a unique opportunity to examine how
these traits might interact to influence the evolutionary histories of organisms that share the
same habitat.
The chernetid pseudoscorpion, Dinocheirus arizonensis (Banks, 1901), is commonly found in
rotting cactus tissues in the Sonoran Desert, preying upon a variety of cactophilic insects,
especially the cactophilic Drosophila (D. mojavensis, D. arizonae, D. nigrospiracula, D.
mettleri, and D. pachea) (S. Castrezana and T.A. Markow, unpublished). Dispersal of
Dinocheirus arizonensis is facilitated by a behavior termed phoresy in which the
pseudoscorpion attaches to the legs of vagile cactophilic insects and is transported to a new
host cactus when the insect disperses. Phoretic dispersal is also known for several other
members of the order Pseudoscorpiones (Ranius and Douwes, 2002; Moulds et al., 2007;
Murienne et al., 2008). The hitchhiking behavior of D. arizonensis and its transporter has only
been studied in detail on the neriid cactus fly Odontoloxozus longicornis (Coquillett, 1904),
which is also preyed upon by D. arizonensis (Zeh and Zeh, 1992), but in the field we have
observed D. arizonensis attached to cactus beetles (tribe Hololeptini) and to syrphid flies,
suggesting that the symbiosis is not specific (S. Castrezana, unpublished). Phoretic dispersal
provides an obvious benefit to D. arizonensis, which would otherwise be severely limited in
its ability to disperse and colonize new rots. If phoresy is the primary mechanism for dispersal
in D. arizonensis in the wild, and the transporter shows high dispersal capability which is not
compromised by the hitchhiker, then we would predict relatively high gene flow and little
genetic structure among populations of the pseudoscorpion. Conversely, if phoresy represents
only a small fraction of overall dispersal, pseudoscorpion populations should be more highly
structured, given their limited capacity to disperse on their own and the patchy nature of their
microhabitat. In the present study we examine the population structure and demographic
history of the pseudoscorpion D. arizonensis collected from twelve localities in the Sonoran
Desert using DNA sequence data from a single mitochondrial marker, a segment of cytochrome
c oxidase subunit I (COI). We also present preliminary results on population structure of the
neriid O. longicornis, and provide a summary and comparison of the demographic histories of
D. arizonensis, O. longicornis and the cactophilic Drosophila.
2. Materials and methods
2.1 Sampling
A total of 91 adult pseudoscorpions was collected during May–June, 2002 and January 2008
from necrotic tissue of a variety of columnar cacti (saguaro, cardón, organ pipe, agria and
senita); 43 pseudoscorpions were obtained from eight localities on the Baja California (Baja)
peninsula and 48 were collected from seven localities on the mainland (Sonora and southern
Arizona; Fig. 1; Table 1). The sample included two morphologically distinct species, D.
arizonensis (N = 85) and the cheliferid pseudoscorpion Parachelifer hubbardi (Banks, 1901)
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(N = 6). Although sample size in P. hubbardi was too small for population genetic analyses,
the COI sequence data for this species were included in the phylogenetic analyses. A small
sample (N = 28) of larval and adult neriid flies, O. longicornis, was collected along with D.
arizonensis at three localities on the mainland (Guaymas and San Juanico, Sonora, and Tucson,
Arizona); additional specimens of neriids were taken in the Sierra Ancha, Arizona and at San
Bruno, Baja California Sur (Fig. 1; Table 1).
2.2 DNA extraction and amplification
Total genomic DNA was extracted from tissue samples using the DNeasy™ (QIAGEN Inc.,
Valencia, CA) protocol. The polymerase chain reaction (PCR) was used to amplify a segment
of the COI gene (~700 bp) with primers LCO1490f (5′-
GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198r (5′-
TAAACTTCAGGGTGACCAAAAAATCA-3′) using standard PCR conditions (Folmer et al.,
1994). Verification of successful amplification was assessed by agarose gel electrophoresis.
Sequencing reactions were performed on an Applied Biosystems (Foster City, CA) ABI
3730XL DNA sequencer at the DNA Sequencing Facility, University of Arizona, using the
amplifying primers. Sequences were proofread and aligned in either Sequencher 4.1
(GeneCodes Corp.) or ClustalX 1.81 (Thompson et al., 1997) followed by manual editing.
Sequences were trimmed to remove ambiguous sites, resulting in a final segment of 551 bp in
the pseudoscorpions D. arizonensis and P. hubbardi and 639 bp in the neriid fly O.
longicornis. Aligned sequences were translated in MEGA version 3.1 (Kumar et al., 2004)
using the invertebrate mitochondrial genetic code; no stop codons or indels were found.
Calculations of genetic distances among sequences (uncorrected p-distances and K2P distances
[Kimura, 1980]) were carried out in MEGA. Calculations of genetic diversity indices were
performed in DnaSP version 4.10 (Rozas et al., 2003). Sequences of all unique COI haplotypes
have been deposited in GenBank under the following accession numbers: D. arizonensis
(FJ483786–FJ483812), P. hubbardi (FJ483813–FJ483816) and O. longicornis (FJ532245 –
FJ532254).
2.3 Population genetic analyses
Hierarchical analysis of molecular variance (AMOVA, Excoffier et al., 1992) performed in
ARLEQUIN version 3.1 (Excoffier et al., 2005) was used to test for population structure in D.
arizonensis for populations with N ≥ 3. For the AMOVA, populations were divided into two
groups representing the two clades (I and II) found for D. arizonensis (see Section 3.2). The
hierarchical AMOVA partitioned genetic variation among localities relative to the total sample
(ΦST), among localities within clades (ΦSC), and among clades I and II (ΦCT). The calculation
of significance of the fixation indices ΦST, ΦSC, and ΦCT was based on 10,000 permutations
of the data matrix. The significance of population pairwise comparisons of ΦST was assessed
using a sequential Bonferroni correction for multiple comparisons (Rice, 1989). Pairwise
estimates of the number of migrants per generation (Nm) among populations assumed to be in
mutation-drift equilibrium were also calculated in ARLEQUIN. We also performed AMOVA
on populations of O. longicornis from two widely-separated localities, Tucson and Guaymas
(N = 10 for each), where individuals of D. arizonensis were also collected (Table 1).
2.4 Phylogenetic analyses
Relationships among COI haplotypes from the entire pseudoscorpion data set were initially
assessed with the neighbor-joining (NJ) algorithm of Saitou and Nei (1987) carried out in
MEGA using a matrix of uncorrected p-distances. This initial analysis revealed that haplotypes
of D. arizonensis partitioned into two monophyletic clades as mentioned previously. All
analyses of demographic history, therefore, were conducted on each clade separately. Relative
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rate tests (Tajima, 1993) of sequence evolution in clades I and II of D. arizonensis were carried
out in MEGA using P. hubbardi as the outgroup.
A subset of COI sequences comprised of each of the haplotypes in D. arizonensis and P.
hubbardi were used to conduct phylogenetic analyses using maximum parsimony (MP) and
Bayesian inference. The MP analyses were carried in MEGA using the CNI heuristic search
option and 100 random additions of sequences. Relative support for tree topology was obtained
by bootstrapping (Felsenstein, 1985) using 1,000 pseudoreplicates. Bayesian methods were
implemented in MrBayes version 3.1 (Huelsenbeck and Ronquist, 2001). The model of
nucleotide substitution that best fit the data set, determined with Modeltest 3.7 (Posada and
Crandall, 1998) using the Akaike Information Criterion, was GTR + Γ. Bayesian analyses were
run under the parameters of this model (nst = “6”; rates = “gamma”) for 1,000,000 generations,
sampled every 250th generation (4,000 trees sampled), using the default random tree option
to begin the analysis. Clade support, expressed as posterior probabilities, was estimated
utilizing a Markov chain Monte Carlo (MCMC) algorithm. Log-likelihood values from four
simultaneous MCMC chains (three hot and one cold) stabilized at about 10,000 generations.
The first 40 trees, therefore, were discarded from the analysis (burnin = 40). The cheliferid
pseudoscorpion, Protochelifer naracoortensis (GenBank accession no. DQ184919), was used
as the outgroup.
2.5 Demographic analyses
Statistical tests designed to assess whether nucleotide polymorphisms deviate from
expectations under neutral theory [Tajima’s (1989) D and Fu’s (1997) FS] were carried out in
ARLEQUIN. These tests also are sensitive to factors other than selection, including population
expansions and bottlenecks, with Fu’s FS being especially sensitive in detecting population
expansions which lead to large negative values in the test statistic (Fu, 1997; Ramos-Onsins
and Rozas, 2002). Significance of D and FS values was determined in ARLEQUIN using 1,000
simulated samples to produce an expected distribution under selective neutrality and population
equilibrium. The cut-off level for statistical significance was 0.05. For Fu’s FS, significance
at the 0.05 level was indicated when P values were < 0.02 (Excoffier et al., 2005).
Significant values for Fu’s FS suggested that both clades of D. arizonensis and O.
longicornis had experienced historical population expansions. Their demographic histories,
therefore, were explored further utilizing three different tests of the sequence data: (1) analysis
of the distribution of pairwise sequence differences (mismatch distribution; Harpending,
1994) performed in ARLEQUIN; (2) Bayesian skyline analysis implemented in BEAST
version 1.2 (Drummond et al., 2005); and (3) estimation of changes in population size carried
out in FLUCTUATE version 1.4 (Kuhner et al., 1998).
For populations which have undergone an historical expansion, plots of the distribution of
pairwise differences among haplotypes are expected to be unimodal, whereas populations in
equilibrium generally show a multimodal distribution (Harpending, 1994). Under the sudden
expansion model the parameters generated are τ, the time to the population expansion (=2ut,
where u is the mutation rate for the entire gene segment and t is the number of generations
since the expansion), and the mutation parameters θ0 and θ1, where θ0 = 2uN0, and θ1 =
2uN1 (N0 and N1 are the population sizes before and after the expansion, respectively) (Rogers
and Harpending, 1992). The significance of the estimated parameters is obtained by calculating
the sum of square deviations (SSD) statistic and the raggedness statistic (rg; Harpending,
1994), and their corresponding P values (Excoffier et al., 2005). The sudden expansion model
is rejected when P < 0.05.
The Bayesian skyline analysis utilizes MCMC sampling of sequence data to estimate a
posterior distribution of effective population size through time (Drummond et al., 2005).
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Bayesian skyline analyses were run under the conditions of the GTR + Γ model (four gamma
categories). The mean mutation rate per site per generation (μ) was set at 1.15 × 10−8. We
arrived at this rate by assuming (i) an average pairwise sequence divergence rate of 2.3% per
million years (Brower, 1994) and (ii) a generation time of one year. The number of grouped
intervals (m) was set to ten. Five million iterations of the MCMC chains were run, sampling
every 1,000 iterations; the first 500,000 chains were discarded as burnin. The Bayesian skyline
plots were generated with TRACER version 1.2.1 (Drummond et al., 2005).
The FLUCTUATE program provides an estimate of long-term female effective population size
(Nef) and evaluates whether Nef has changed or remained stable over time (Kuhner et al.,
1998). The simultaneous maximum-likelihood estimates of the mutation parameter θ (where
θ = 2Nefμ) and the exponential population growth parameter (g) were obtained from a final
extended run of ten short chains of 100,000 steps each and two long chains of 200,000 steps
each, sampling every 20th step. Initial estimates of θ were based on number of segregating sites
(Watterson, 1975), with the random tree default setting selected for the starting genealogy.
3.0 Results
3.1 Genetic diversity
Genetic diversity indices for D. arizonensis, P. hubbardi and O. longicornis are shown in Table
2. In D. arizonensis, values for both haplotype diversity (h) and nucleotide diversity (π) were
greater in clade I than in clade II. For both clades, however, nucleotide diversity was low (π =
0.0044–0.0064) and haplotype diversity was high (h = 0.746–0.897). Overall values for the
combined clades in D. arizonensis are also given in Table 2 for comparison. Fu’s FS was
significant in both individual and combined clades of D. arizonensis, as well as in P.
hubbardi and O. longicornis. Tajima’s D was significant in both clade I and clade II of D.
arizonensis and in O. longicornis. Relative rates tests (Tajima, 1993) were not significant in
D. arizonensis, indicating that a molecular clock could not be rejected.
3.2 Phylogenetic relationships
Initial NJ analyses of pseudoscorpion COI sequences (not shown) showed that P. hubbardi
formed a highly-supported lineage separate from D. arizonensis, as expected for two distantly
related species from different families. Average genetic distance between the two species was
24.6% (uncorrected p-distance) and 29.9% (K2P distance). The two species also showed 41
fixed amino acid differences in the translated COI gene segment of 183 amino acids. The initial
NJ tree also showed D. arizonensis resolving as two well-supported clades (clades I and II).
Maximum parsimony and Bayesian analyses confirmed the partitioning of clades I and II, but
support for the split was weaker, especially in the Bayesian tree (Fig. 2). Clade I was comprised
of individuals of D. arizonensis from mainland Sonora and southeastern Arizona. Clade II was
comprised individuals from the Baja peninsula and southeastern Arizona. Individuals from
both clades were found at Tucson (Fig. 1; Table 1). Mean genetic distance (uncorrected p-
distance and K2P distance) among individuals of clades I and II was 2.6%. There were nine
fixed nucleotide substitutions in the 551 bp gene segment among clades I and II, eight of which
were at the third codon position. A single first codon position substitution at site 413 resulted
in a valine to isoleucine amino acid change in the COI protein segment of clade I individuals,
a substitution also seen in P. hubbardi and the outgroup species Protochelifer
naracoortensis.
3.3 Population structure
The hierarchical AMOVA conducted on combined clade I and II populations of D.
arizonensis (Table 3) revealed significant structure (overall ΦST = 0.860; P < 0.0001), with
80.23% of the genetic variation distributed between clades I and II (ΦCT = 0.802; P = 0.026).
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Most (13 of 16) of the pairwise comparisons of ΦST between populations of clades I and II
were also significant using a sequential Bonferroni correction (Table 4). Only 5.81% of the
genetic variation was distributed among populations within clades, but the corresponding
fixation index (ΦSC) was significant (ΦSC = 0.294; P < 0.0001). However, only three of the
within-clade pairwise comparisons of ΦST were significant (Table 4). For the most part, the
estimated number of migrants per generation (Nm) between populations within each clade was
≥1.0, whereas pairwise values of Nm between populations of clades I and II were all low
(≤0.15). One exception is the clade I population from Organ Pipe Cactus National Monument
(OP) in which the consistently higher within-clade pairwise ΦST values, and lower Nm values,
suggest a pattern of isolation by distance. Overall, the results of the AMOVA, while indicating
some within-clade population structuring, suggest a degree of gene flow among these
populations consistent with phoretic dispersal. Therefore, the four clade I populations and the
four clade II populations were each combined for the tests of demographic history. Results of
AMOVA conducted on the populations of O. longicornis from Guaymas and Tucson revealed
a lack of population structure and high gene flow between these localities (ΦST = 0.004; P =
0.579; Nm = 122.5).
3.4 Historical demography
Plots of the distribution of pairwise differences among COI haplotypes in clades I and II of D.
arizonensis and in O. longicornis (Fig. 3) conformed to expectation for populations that have
undergone expansions. For both species, the mismatch distribution test statistics SSD and rg
were small and not statistically significant at the 0.05 level (Table 5), indicating that the sudden
expansion model could not be rejected. The values of τ (time to the population expansion) in
Table 5 were used to obtain estimates of t, the number of generations since the expansion, using
the equation τ = 2ut and assuming a mean mutation rate per site per generation (μ) of 1.15 ×
10−8 (see Section 2.5). The results are shown in Table 6, along with estimates of t for several
species of cactophilic Drosophila, important prey for Dinocheirus arizonensis and O.
longicornis. The results suggest a range of population expansions beginning about 60,000
generations ago (Drosophila mettleri from the Baja peninsula) to about 700,000 generations
ago (D. mojavensis from the mainland). Estimates for the beginning of the population
expansions in Dinocheirus arizonensis fall within this range (293,000 and 214,000 generations
ago for clades I and II, respectively).
Results of analyses of COI sequence data using FLUCTUATE generally were consistent with
those of the mismatch distribution. In clade II of D. arizonensis and O. longicornis, the
exponential population growth parameter (g) was positive and significantly different from zero
(Table 7), indicating population growth. In clade I of D. arizonensis, the value for g was positive
but it was not significantly different from zero, or no population growth.
Bayesian skyline plots (Fig. 4), showing the estimated changes in median Nef over time for D.
arizonensis and O. longicornis, were concordant with results from the mismatch distribution
and FLUCTUATE. Figure 4 shows that after a long period of relative population stability, both
clades I and II of D. arizonensis experienced population expansions dating roughly to similar
time periods (~260,000 years before present). The population expansion in O. longicornis dates
to ~100,000 years before present. These values agree well with the time intervals for both
species shown in Table 6, assuming a generation time of one year. The magnitude of the
population increases for D. arizonensis shown in Fig. 4, however, was greater in clade II than
in clade I, in agreement with results from FLUCTUATE (Table 7).
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4.0 Discussion
4.1 Genetic divergence and population structure in Dinocheirus arizonensis
Phylogenetic analyses revealed that populations of D. arizonensis within the Sonoran Desert
partitioned into two clades (clades I and II) separated by a mean genetic distance (d) of 2.6%.
The genetic distance between the two geographically isolated (except in southeastern Arizona)
clades, together with the presence of nine fixed nucleotide differences and one fixed amino
acid substitution in the COI gene segment, suggest that they represent two distinct evolutionary
lineages that have diverged in allopatry. Results from the AMOVA were consistent with this
conclusion, indicating that 80% of the genetic variation in D. arizonensis was partitioned
between the two clades. We attribute the co-occurrence of clades I and II in Arizona to
secondary contact after divergence (see below).
Although only a few molecular studies have been conducted on the order Pseudoscorpiones
(Murienne et al., 2008), these studies are beginning to reveal a high level of intraspecific genetic
differentiation. For example, divergences in the COI gene larger than those seen in D.
arizonensis have been found among different populations of the cheliferid cave pseudoscorpion
Protochelifer cavernarum in Australia (Moulds et al., 2007). Also, in the chernetid
pseudoscorpion Cordylochernes scorpioides from Panama and South America, COI
divergences (K2P distances) ranged from 2.6% between populations from Trinidad and French
Guiana to 13.8% between populations from Panama and South America (Wilcox et al.,
1997). Even within Panama, three highly divergent lineages of C. scorpiodes have been
reported (Zeh et al., 2003). Most population comparisons of C. scorpiodes among regions also
showed 1–3 amino acid substitutions in the COI protein segment (Wilcox et al., 1997),
consistent with the differences seen in clades I and II of D. arizonensis.
Because a molecular clock could not be rejected for sequence evolution in D. arizonensis, the
mean genetic distance between clades I and II can be used to estimate dates of when the two
clades began to diverge. As with the chernetid pseudoscorpion C. scorpioides, however, a
calibrated molecular clock is not available for Dinocheirus. Thus we have applied the
commonly used standard rate of 2.3% pairwise sequence divergence per million years for
mitochondrial DNA in arthropods (Brower, 1994), a rate also used for C. scorpiodes (Zeh et
al., 2003). Because several lines of evidence suggest that the mitochondrial COI gene in some
arthropods evolves at a slower rate of ~0.6–1.5% pairwise sequence divergence per million
years (Farrell, 2001: Pfeiler et al., 2006) we have also estimated dates using an average rate of
1.0%. The mean sequence divergence of 2.6% found among clades I and II suggests that the
two began to split from a common ancestor roughly 1.1 million years ago (Ma) during the mid
Pleistocene using the 2.3% clock, or 2.6 Ma during the mid-to-late Pliocene using the 1.0%
clock. The temporal framework provided by the molecular clocks suggests two scenarios that
may have led to disruption of gene flow in the ancestral population of D. arizonensis. Marine
incursions of the Gulf of California into southeastern California and southwestern Arizona
occurred during the late Miocene and early Pliocene (McDougall et al., 1999; Riddle et al.,
2000; Oskin and Stock, 2003) forming a potential barrier to dispersal of terrestrial organisms
on either side of the seaway. The ability of the neriid fly O. longicornis and other vagile insects
to transport pseudoscorpions over expanses of water by phoresy is unknown, but it seems highly
probable that dispersal of the pseudoscorpions would be diminished in the presence of such a
barrier. Even in the absence of a water barrier, Plio-Pleistocene climate transitions and
associated glacial cycles would probably have affected populations of both the
pseudoscorpions and their aerial transporters, potentially resulting in reduced phoretic dispersal
and increased reproductive isolation between mainland and peninsular populations of the
pseudoscorpions. By the end of the Pleistocene, when climatic conditions became more stable
and the northern Gulf had receded to its present position, the disjunct populations could have
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come into secondary contact, explaining the sympatric occurrence of clades I and II seen today
in southeastern Arizona.
We predicted that if phoresy plays an important role in dispersal and colonization of patchily
distributed resources in D. arizonensis, populations of the non-vagile pseudoscorpion should
show little structure throughout its range in the Sonoran Desert. When clades I and II are
analyzed separately, our results generally support this conclusion, with populations showing
relatively high genetic connectivity within each of the divergent clades. The prediction
obviously assumes high genetic connectivity of the host transporter over the same geographic
range as the pseudoscorpion, an assumption supported by our preliminary results on O.
longicornis. The high gene flow seen in O. longicornis from the mainland Sonoran Desert is
also in agreement with results obtained for most species of cactophilic Drosophila (Pfeiler and
Markow, 2001; Markow et al., 2002; Hurtado et al., 2004; Ross and Markow, 2006; Reed et
al., 2007).
Although samples of Dinocheirus arizonensis used in the present study were collected from
several species of columnar cacti, we found no evidence for genetic differentiation resulting
from cactus host type. For example, high gene flow was seen in clade II populations from the
Baja peninsula, where samples were obtained from senita, agria, organ pipe and cardón, to
southeastern Arizona where samples were obtained from saguaro.
4.2 Demographic history of Dinocheirus arizonensis
Results of different tests of demographic history were generally congruent and suggested that
both clades I and II of D. arizonensis have experienced similar historical population
expansions. As described earlier, we have assumed a standard 2.3% molecular clock and a
generation time of one year in the tests of demographic history. Generation times of D.
arizonensis in the wild, however, are not known, but it is highly probable that more than one
generation is produced each year. Laboratory experiments have shown that the period of
development from fertilization to adult is about 2–3 months in D. arizonensis (Zeh, 1987). In
addition, females are known to store sperm for extended periods and produce more than one
brood from a single mating (Zeh and Zeh, 1992). Because of the various assumptions associated
with the estimation of μ the mean mutation rate per site per generation for D. arizonensis, the
time axes in the Bayesian skyline plots (Fig. 4) should be considered only rough estimates. But
regardless of the number of generations per year, and assuming it is the same in clades I and
II, it is apparent that each clade began to increase its population size at about the same time
(Table 6; Fig. 4).
Results of different tests of demographic history in O. longicornis also were generally
congruent and suggested that it too has undergone an historical population expansion, similar
to the expansions found for the cactophilic Drosophila which utilize the same necrotic
microhabitat and serve as prey for O. longicornis and Dinocheirus arizonensis (Hurtado et al.,
2004; Machado et al., 2007; Pfeiler et al., 2007). The estimates of number of generations since
the population expansion (Table 6) suggest that in the cactophilic Drosophila (with the
exception of D. mettleri) the population expansions roughly coincide with those seen in clade
I and II of Dinocheirus arizonensis of ~200,000–300,000 generations ago. The population
expansion in O. longicornis began approximately 90,000 generations ago, the same as in
Drosophila mettleri from the mainland. We must emphasize that the values of t shown in Table
6 are only rough estimates given the large confidence intervals surrounding the values of τ and
the fact that we assumed the same COI mutation rate and a one year generation time for all
species. Nonetheless, the historical increases seen in population size of Dinocheirus
arizonensis, O. longicornis and the cactophilic Drosophila suggest a complex interaction and
ecological balance among predator, prey and phoretic dispersal of Dinocheirus arizonensis.
Pfeiler et al. Page 8
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NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Acknowledgments
We thank L.A. Hurtado and T. Watts for technical assistance. L.M.M. was supported by an NIH Postdoctoral Training
Grant to the University of Arizona. This work was supported by NSF grants DEB 00–75312 and OISE–0440648 to
T.A.M.
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Fig. 1.
Map showing collecting localities (black dots) in northwestern Mexico and southwestern USA.
Total number of pseudoscorpions (Dinocheirus arizonensis and Parachelifer hubbardi)
collected at each locality is shown in parentheses; asterisks indicate localities where neriid flies
Odontoloxozus longicornis were taken (see Table 1 for details). Shaded areas represent
approximate geographical distributions of D. arizonensis clade I and clade II inferred from the
molecular data. Abbreviations (Arizona): SA, Sierra Ancha; SU, Superstition Mountains; TC,
Tucson; DM, Arizona-Sonora Desert Museum; OP, Organ Pipe Cactus National Monument;
(Sonora): SJ, San Juanico; SC, San Carlos; GY, Guaymas; (Baja California Sur): LP, La Paz;
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PZ, Pozo 100; PC, Pozo Cota; AR, Armenta; SB, San Bruno; SR, Santa Rosalía; (Baja
California): SE, Sepultura; CA, Cataviña; SF, San Felipe.
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Fig. 2.
Most parsimonious tree (length = 233; CI = 0.906; RI = 0.961) obtained using each of the COI
haplotypes found in the pseudoscorpions Dinocheirus arizonensis (Chernetidae) and
Parachelifer hubbardi (Cheliferidae) from northwestern Mexico and southeastern Arizona
(156 parsimony informative sites). The cheliferid Protochelifer naracoortensis was used as
the outgroup. Clade support values are shown on branches; nodes with <50% support were
collapsed. Bootstrap support values for the maximum parsimony tree are shown above the
branches; posterior probability values for the 50% majority rule Bayesian tree are shown below
the branches. Branch terminals are labeled with sample identification number and locality
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abbreviation (see Fig. 1). The number of individuals with the same haplotype at each locality
is given in parentheses.
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Fig. 3.
Distribution of pairwise differences among COI haplotypes (mismatch distribution) in clades
I and II of the pseudoscorpion Dinocheirus arizonensis and in the neriid fly Odontoloxozus
longicornis (vertical bars). Solid lines represent the expected distributions under the sudden
expansion model. The unimodal distribution of observed pairwise differences expected for
populations which have undergone an expansion is seen in each of the plots.
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Fig. 4.
Bayesian skyline plots showing changes in effective female population size (Nef) over time for
clades I and II of the pseudoscorpion Dinocheirus arizonensis and for the neriid fly
Odontoloxozus longicornis. Population size is given on a logarithmic scale. A value of 1.15 ×
10−8 for μ the mean mutation rate per site per generation, was assumed. The thick solid lines
represent the median estimates of population size; the thin solid lines show the 95% HPD
(highest posterior density) intervals. Note the different time scales used in the three plots.
Arrows show estimated dates for the beginning of the population expansions in clades I and II
of D. arizonensis (~260,000 years before present) and in O. longicornis (~100,000 years before
present).
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Table 1
Summary of the geographic distribution and number of individuals collected of the pseudoscorpions Dinocheirus
arizonensis and Parachelifer hubbardi and the neriid fly Odontoloxozus longicornis
Locality Abbrev. D. arizonensis P. hubbardi O. longicornis
Clade I Clade II
Arizona
Sierra Ancha (SA) 4
Superstition Mts. (SU) 2
Tucson (TC) 2 6 10
Desert Museum (DM) 2 1
Organ Pipe NM (OP) 3 1
Sonora
San Juanico (SJ) 9 3
San Carlos (SC) 16
Guaymas (GY) 6 10
Baja California Sur
La Paz (LP) 1
Pozo 100 (PZ) 1
Pozo Cota (PC) 1
Armenta (AR) 2
San Bruno (SB) 1
Santa Rosalía (SR) 1
Baja California
Sepultura (SE) 11 1
Cataviña (CA) 4
San Felipe (SF) 21
Total 36 49 6 28
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Table 2
Summary of genetic diversity indices and results of neutrality tests (Tajima’s D and Fu’s FS) in the COI gene segment in the
pseudoscorpions Dinocheirus arizonensis and Parachelifer hubbardi and the neriid fly Odontoloxozus longicornis
Species N L k K h (± SD) π (± SD) Tajima’s DFu’s FS
D. arizonensis 85 551 52 27 0.898 ± 0.022 0.0161 ± 0.0008 −0.54 −24.65*
Clade I 36 551 26 14 0.897 ± 0.030 0.0064 ± 0.0010 −1.58*−25.98*
Clade II 49 551 24 13 0.746 ± 0.057 0.0044 ± 0.0008 −1.82*−26.71*
P. hubbardi 6 551 9 4 0.800 ± 0.172 0.0067 ± 0.0024 −0.42 −2.81*
O. longicornis 28 639 13 10 0.778 ± 0.066 0.0024 ± 0.0005 −1.76*−27.57*
N, number of sequences; L = sequence length (bp); k = number of variable sites; K, number of haplotypes; h, haplotype diversity; π, nucleotide diversity;
*, significant at the 0.05 level.
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Table 3
Hierarchical analysis of molecular variance (AMOVA) for populations of Dinocheirus arizonensis grouped by clade
I and clade II
Source of variation df Sum of squares Variance components % of variation
Among groups 1 229.697 5.93892 Va 80.23
Among populations within
groups 6 27.772 0.43024 Vb 5.81
Within populations 68 70.242 1.03296 Vc 13.95
Total 75 327.711 7.40212
Fixation indices
ΦST = 0.860* (P < 0.0001)
ΦSC = 0.294* (P < 0.0001)
ΦCT = 0.802* (P = 0.026)
*, significant at the 0.05 level
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Table 4
Pairwise comparisons of ΦST (below the diagonal) and number of migrants per generation (Nm; above the diagonal) for populations of
Dinocheirus arizonensis
Clade I Clade II
GY (6) SC (16) SJ (9) OP (3) SE (11) CA (4) SF (21) TC (6)
GY ---- 1.79 0.99 0.27 0.04 0.07 0.05 0.09
SC 0.22 ---- 2.81 0.55 0.08 0.10 0.08 0.11
SJ 0.33* 0.15 ---- 0.61 0.09 0.13 0.08 0.15
OP 0.65 0.48* 0.45 ---- 0.04 0.06 0.06 0.11
SE 0.92* 0.86* 0.85* 0.93* ---- 1.04 1.27 1.89
CA 0.88 0.83* 0.79* 0.89 0.32 ---- 0.94 2.67
SF 0.90* 0.87* 0.86* 0.90* 0.28* 0.35 ---- 1.20
TC 0.84* 0.82* 0.77* 0.82 0.21 0.16 0.29 ----
Significant pairwise ΦST values after a sequential Bonferroni correction (P < 0.0025) are indicated with asterisks. Number of individuals from each locality is shown in parentheses. Populations with a
sample size < 3 [i.e., the clade I population from Tucson and four clade II populations from the Baja peninsula and southern Arizona (Table 1)] were omitted from the analysis. Locality abbreviations
are given in Table 1.
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Table 5
Results of the mismatch distribution of COI sequences in clades I and II of the pseudoscorpion Dinocheirus arizonensis and the neriid
fly Odontoloxozus longicornis
Species τ (95% CI) θ0θ1SSD rg
D. arizonensis
Clade I 3.71 (1.67, 5.14) 0.000 32.93 0.025 (P = 0.052) 0.058 (P = 0.089)
Clade II 2.71 (0.00, 5.52) 0.004 3.64 0.022 (P = 0.32) 0.074 (P = 0.44)
O. longicornis 1.34 (0.38, 2.22) 0.004 >1000 0.017 (P = 0.15) 0.114 (P = 0.14)
See Materials and methods for explanation of abbreviations.
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Table 6
Estimates of number of generations since the population expansion (t) in Dinocheirus arizonensis, the neriid fly Odontoloxozus
longicornis and the cactophilic Drosophila based on the mismatch distribution
Species NCOI (bp) uτ (95% CI) t (generations)
D. arizonensis
Clade I 36 551 6.34 × 10−63.71 (1.67, 5.14) 2.93 × 105
Clade II 49 551 6.34 × 10−62.71 (0.00, 5.52) 2.14 × 105
O. longicornis (Baja and mainland) 28 639 7.35 × 10−61.34 (0.38, 2.22) 0.91 × 105
Drosophila
nigrospiracula (Baja and mainland) 94 655 7.53 × 10−63.00 (0.36, 3.50) 1.99 × 105
pachea (Baja) 142 661 7.60 × 10−65.39 (1.62, 9.62) 3.55 × 105
mettleri (mainland) 45 662 7.61 × 10−61.39 (0.00, 3.68) 0.91 × 105
mettleri (Baja) 50 662 7.61 × 10−60.91 (0.46, 1.60) 0.60 × 105
mojavensis (mainland) 47 658 7.57 × 10−610.47 (0.47, 64.00) 6.92 × 105
mojavensis (Baja) 63 658 7.57 × 10−62.70 (0.00, 5.91) 1.78 × 105
Mismatch distribution parameters for the cactophilic Drosophila were calculated from the COI data of Hurtado et al. (2004) and Reed et al. (2007). Populations of Drosophila that showed significant
structure within a region were omitted from the analysis. A mean mutation rate per site per generation (μ) of 1.15 × 10−8 was assumed. The parameter u is the mutation rate for the entire gene segment
[i.e. μ times the number of base pairs (bp)]. The parameter t (generations) was calculated from the equation τ = 2ut (Rogers and Harpending, 1992).
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Table 7
Effective female population sizes (Nef) and exponential growth rates (g) in clades
I and II of the pseudoscorpion D. arizonensis and the neriid fly Odontoloxozus
longicornis calculated with FLUCTUATE
Species No. of COI
sequences θNef g (1/μ generations)
D. arizonensis
Clade I 36 0.0167 (± 0.0057) 7.26 × 10572 (± 99)
Clade II 49 0.0180 (± 0.0064) 7.83 × 105271 (± 200)
O. longicornis 28 0.0266 (± 0.0175) 2.31 × 1061,703 (± 814)
Values for maximum-likelihood estimates of θ and g (± 1.96 standard deviations) are shown. A neutral mutation rate per site per generation (μ) of 1.15 ×
10−8 was assumed.
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