Genetic diversification without obvious genitalic morphological divergence in harvestmen (Opiliones,...

Molecular Phylogenetics and Evolution 61 (2011) 844–853

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Molecular Phylogenetics and Evolution

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Genetic diversification without obvious genitalic morphological divergence inharvestmen (Opiliones, Laniatores, Sclerobunus robustus) from montane skyislands of western North America

Shahan Derkarabetian a,⇑, Joel Ledford b, Marshal Hedin a

a Department of Biology, San Diego State University, San Diego, CA 92182-4614, USAb Department of Entomology, California Academy of Sciences, San Francisco, CA 94118, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 March 2011Revised 30 July 2011Accepted 2 August 2011Available online 16 August 2011

Keywords:PhylogeographyCryptic diversificationMorphological stasisPopulation subdivisionGenealogical sorting index

1055-7903/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ympev.2011.08.004

⇑ Corresponding author. Present address: DepartmUniversity, P.O. Box 751, Portland, OR 97207, USA

E-mail address: [email protected] (S. Der

The southern Rocky Mountains and adjacent Intermontane Plateau Highlands region of western NorthAmerica is a geographically diverse area with an active geologic history. Given the topological complexityand extensive geologic activity, organisms inhabiting this region are expected to show some degree ofmorphological and genetic divergence, especially populations found on the southern montane ‘skyislands’ of this region. Here we examine the phylogeographic history and diversification of a montane for-est 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 robustusrobustus, is recovered as six genetically distinct, geographically cohesive mitochondrial phylogroups.Gene tree data for a single nuclear gene reveals congruent, albeit slightly more conservative, patternsof genetic divergence. Despite high levels of genetic divergence throughout their distribution, phylo-groups show extreme conservation in somatic and reproductive morphology. This uncoupling of morpho-logical and genetic differentiation may be due to morphological conservatism associated with aconserved microhabitat preference. Based on these data, it is obvious that S. robustus has undergone somelevel of cryptic diversification.

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1. Introduction

The southern Rocky Mountains and adjacent Intermontane Pla-teau Highlands (SRMIP) region of the southwestern United States isa geographically and geologically diverse area. Today, this area ischaracterized by isolated forested mountain ranges (‘sky islands’)in southern Arizona and New Mexico, transitioning to more contig-uous mountainous regions in the southern Rocky Mountains.Mountain building in western North America took place duringtwo main periods: the Laramide and Neogene orogenies (Wilsonand Pitts, 2010). More recently, climatic fluctuations have resultedin shifts in vegetation and forest availability, with major fluctua-tions occurring during the Miocene (5 Ma) and the Plio–Pleistoceneglacial cycles (van Devender and Spaulding, 1979; Lomolino et al.,1989).

Given diverse topography and obvious temporal climatic varia-tion, we would expect phylogeographic diversification and/or spe-ciation in different lineages distributed across this region. Indeed,

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ent of Biology, Portland State

karabetian).

there are numerous studies that have shown recent phylogeo-graphic divergence in an array of organisms that inhabit some orall 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 withinthis region, but exceptions include land snails (Sonorella, McCord,1994) and flightless montane grasshoppers (Melanoplus, Knowlesand Otte, 2000). When comparing these studies, it is importantto note that all taxa showing more recent (phylogeographic) diver-gence are considered to be relatively good dispersers, while thosethat have speciated within this region are considered to have lowvagility.

The Opiliones, commonly called harvestmen, comprise an orderof arachnids with over 6000 described species (Machado et al.,2007). Opiliones diversity is divided into four morphologically diver-gent, highly supported clades: Cyphophthalmi, Eupnoi, Dyspnoi, andLaniatores (summarized in Giribet and Kury, 2007). The majority ofnorth temperate Laniatores taxa are short-legged cryptozoic habitatspecialists, usually occupying moist, dark microhabitats. The

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western United States contains much of the diversity of North Amer-ican Laniatores, including the Sclerobunines, which includes thewidespread genus Sclerobunus Banks 1893. Sclerobunus robustus isa small-bodied (2–3 mm) harvestmen species typically found underrocks or partially rotted logs in high elevation forests throughout theSRMIP (Fig. 1). At southern latitudes (e.g., Arizona and New Mexico),Sclerobunus are found in the isolated mountain ranges separated bylow elevation grassland and desert habitats. Further north, in themore contiguous southern Rocky Mountains of Colorado, popula-tions may be found at slightly lower elevations. Given these generalbiological features, and a paradoxically large distribution, we predictthat this taxon has a high potential for phylogeographicdiversification.

In animals with internal fertilization, it is typically expectedthat population- or species-level divergence is somehow coupledwith differentiation in reproductive morphology. More specifically,divergence in genitalia is expected to occur at the same rate as ge-

Fig. 1. Sampling localities for S. robustus. Locations are colored based on recovered mitocTable 1. The Rio Grande is represented as a dark blue line. (For interpretation of the referarticle.)

netic differentiation and typically faster than other morphologicaldifferentiation (reviewed in Eberhard, 1985). In Opiliones, penismorphology has been used to diagnose clades at various levels(e.g. species, genus, family), and is typically used to differentiateclosely 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 lineagediversity is becoming a common occurrence in microhabitat-specialist harvestmen with low vagility. In fact, morphologicallycryptic diversification in Opiliones is more the rule than the excep-tion – molecular studies have found some level of cryptic diversi-fication with significant genetic sub-structuring in Laniatores(Thomas and Hedin, 2008; Hedin and Thomas, 2010), Dyspnoi(Schönhofer and Martens, 2008, 2010), and Cyphophthalmi (Boyeret al., 2007; Sharma and Giribet, 2009). In order to rigorously doc-ument the presence of cryptic diversification it is important tohave a robust molecular phylogeny reconstructed using relatively

hondrial phylogroups. Locality numbers correspond to those listed in Supplementalences to color in this figure legend, the reader is referred to the web version of this

846 S. Derkarabetian et al. / Molecular Phylogenetics and Evolution 61 (2011) 844–853

fast evolving genes, combined with a detailed examination ofreproductive morphology, for a comprehensive geographic sample.

Here, we present an example of a low vagility, ecologically re-stricted harvestmen species that shows deep within-speciesmolecular divergence and an absence of obvious reproductive mor-phological divergence. Both mitochondrial and nuclear gene treesare used to infer relationships among range-wide samples of S.robustus. In addition, population genetic statistics are calculatedto further clarify the phylogeographic history of this species. Malereproductive morphology (penis structure) is examined using SEMto assess morphological divergence between major phylogeo-graphic lineages. In this taxon we find a situation where, despitedeep and relatively ancient intraspecific genetic divergence, thereis little somatic differentiation, and a lack of obvious divergencein reproductive morphology.

2. Materials and methods

2.1. Sampling

Sclerobunus specimens were collected by hand from beneathrotting logs and woody debris, and sometimes rocks, in moisthigh-elevation (2300–3300 m) mixed conifer or aspen forests.Specimens destined for morphological analysis were preserved in80% EtOH; specimens used for molecular analyses were preservedin 100% EtOH. Sampling was conducted for S. robustus fromthroughout the known range of this species (Fig. 1), with a totalof 46 localities sampled from two regional subspecies (Sclerobunusrobustus glorietus from 5 sites; Sclerobunus robustus robustus from41 sites). These subspecies are differentiated by size and colora-tion: 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 wereincluded: Cave of the Winds (CO), Mallory Cave (CO), and TaosSki Valley (NM). Following previous analyses (Derkarabetianet al., 2010), outgroup sampling included all other Sclerobunus spe-cies (this includes samples of the subspecies Sclerobunus robustusidahoensis, which is clearly not closely related to other S. robustus),Cyptobunus species, and Metanonychus as a distant outgroup. De-tailed collecting information can be found in Supplemental Table 1.

2.2. Molecular data collection

Genomic DNA was extracted following standard procedures(see Derkarabetian et al., 2010). Targets of PCR amplification in-cluded the mitochondrial cytochrome oxidase I (COI) gene, and in-tron sequences from the nuclear EF-1a protein-coding gene (seeHedin et al., 2010). PCR protocols and primers were identical tomethods published previously (Hedin et al., 2010; Derkarabetianet al., 2010). All COI and most EF-1a PCR products were purifiedusing Polyethylene Glycol (PEG) precipitation; some EF-1a ampli-cons were gel purified using the QIAGEN QIAquick Gel ExtractionKit. Sanger sequencing was performed at the San Diego State Uni-versity Microchemical Core Facility (http://www.sci.sdsu.edu/dna-core/sdsu_dnacore.html). Contiguous sequences were assembledusing Sequencher 4.5 (Gene Codes Corporation, MI) and MacCladev 4.06 (Maddison and Maddison, 1992). Heterozygous sites in EF-1a sequences were left unphased and treated as ambiguous usingstandard ambiguity codes.

2.3. Sequence alignment and phylogenetic analyses

Phylogenetic analyses were conducted on individual genematrices using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003),employing models chosen by the AIC in MrModeltest (Nylander,

2003). The manually aligned COI matrix was partitioned usingthe optimal partitioning strategy determined by Bayes Factor anal-yses (Nylander et al., 2004; Brandley et al., 2005). Analyses wererun until the average standard deviation of split frequencies wasbelow 0.01. The first 40% of trees were discarded as burnin. Poster-ior probabilities of 0.95 and greater were considered highly sup-ported. Nuclear data were aligned by eye with amino acidtranslations and the exon was subsequently removed from analy-sis. Because of high EF-1a intron divergences among taxa, out-group sequences were not used. EF-1a intron sequences wereboth manually aligned (using MacClade v 4.06, Maddison andMaddison, 1992) and aligned using ClustalX v 1.83 (Thompsonet al., 1997), with subsequent manual adjustments.

Divergence dates were estimated from the COI matrix using arelaxed clock with uncorrelated lognormal distribution, imple-mented in the BEAST v1.6.1 software package (Drummond andRambaut, 2007). Two geological calibration points were used inthis analysis. First, the split between Sclerobunus nondimorphicusfrom the Cascade Mountains and S. r. idahoensis from the northernRocky Mountains, coinciding with the rise of the Cascades (normaldistribution with mean of 3.5 and deviation of 1.0). The Cascades/Northern Rockies vicariance is a well-documented biogeographicbreak found in over 150 different species (Brunsfeld et al., 2001).Second, the timing of cave passage development for the Cave ofthe Winds began �5 MA (Luiszer, 2007); this was used as a mini-mum age for the divergence of the Cave of the Winds populationfrom all other S. robustus. Several analyses were run to check forconvergence of tree topology and parameters, with a final analysisrun for a total of 40 million generations (10% burnin) sampledevery 1000 steps. The combined analyses were examined with Tra-cer v1.5 (Rambaut and Drummond, 2007) to check for sufficientESS values for all parameters (BEAST manual), and a tree wasassembled using TreeAnnotator v1.6.1 (Drummond and Rambaut,2007).

2.4. Population structure

To examine the genetic differentiation of recovered mitochon-drial lineages, average intra- and interclade distances werecomputed using PAUP� 4.0b (Swofford, 2002) using the Kimuratwo-parameter model (Kimura, 1980). Several diversity statisticswere calculated for each major mitochondrial lineage, including:number of haplotypes (N), number of polymorphic sites (s), aver-age number of pairwise nucleotide differences (k), and nucleotidediversity (p). All diversity statistics and Tajima’s D (Tajima, 1989)tests were calculated using DnaSP (Librado and Rozas, 2009). Thegenealogical sorting index (gsi) test statistic was used to assessthe level of genealogical exclusivity for particular groups (Cum-mings et al., 2008), and was calculated using the gsi website(http://www.genealogicalsorting.org/) with 10,000 replicates. Thegsi statistic can be used to assess the relative degree of exclusivityfor a group, ranging from no exclusivity (completely random distri-bution of haplotypes – values of 0) to exclusive ancestry (values of1). When comparing two (or more) sampling locations, exclusiveancestry for haplotypes collected from different locations impliesan absence of gene flow among them. In calculating gsi values,COI sequences were grouped by collecting locality (assessing siteexclusivity), and EF-1a sequences were grouped by recoveredmitochondrial COI clades (assessing nuclear/mitochondrialcongruence).

2.5. Penis morphology

Penises were extracted by pushing the penis through the genitaloperculum with an insect pin inserted through the opening of theanal operculum. Extracted penises were soaked in a cold solution

http://www.sci.sdsu.edu/dnacore/sdsu_dnacore.html

http://www.sci.sdsu.edu/dnacore/sdsu_dnacore.html

http://www.genealogicalsorting.org/

Cyptobunus cavicolens OP2143

robustus (19) Pinalero Mtns OP946

robustus (42) Apex Valley Rd OP2687

S. nondimorphicus OP222

Cyptobunus u. ungulatus OP1229

robustus (13) Beaver Creek OP1173

n. sp. (1) Taos Ski Valley OP1171

robustus (45) N of Chama OP2115


n. sp. (7) Mallory Cave OP2568

robustus (30) North Pass OP1188

glorietus (2) Glorieta Canyon OP890

robustus (29) Longs Peak OP1184

robustus (36) Great Sand Dunes OP2105

robustus (27) Wet Mtns OP1165

Cyptobunus cavicolens OP2144

robustus (20) Pinos Altos Mtns OP952

robustus (14) La Sal Mtns OP1203

robustus (45) N of Chama OP2114

robustus (48) Bradford Canyon OP959

robustus (49) Chiricahua Mtns OP1040

robustus (33) Church Creek OP1209

robustus (16) San Fransisco Mtns OP914

robustus (26) Sangre de Cristo OP1145

Cyptobunus u. ungulatus OP1232

robustus (37) S Creede OP2109

robustus (24) Twin Lakes OP1136

robustus (34) nr Garfield OP1214

robustus (35) Weston Pass OP1219

robustus (18) Hannagan Meadow OP935

S. r. idahoensis OP1635

robustus (22) San Fransisco Mtns NM OP2100

robustus (10) Haviland Lake OP1224



glorietus (1) Taos Ski Valley OP2119

robustus (12) Dolores River OP1149robustus (12) Dolores River OP1148

robustus (47) Mimbres Mtns OP929

n. sp. (6) Cave of the Winds OP1127

robustus (39) Fault Cave OP2570

robustus (46) Ski Apache OP893

Cyptobunus u. madhousensis OP240


glorietus (4) Sandia Mtns OP2101

robustus (21) Santa Catalina Mtns OP965

robustus (23) S of Gothic OP1123



robustus (31) NE Deckers OP1193


robustus (32) N Gore Range OP1208


glorietus (4) Sandia Mtns OP2102

robustus (36) Great Sand Dunes OP2104

glorietus (3) Mt. Taylor OP903

robustus (43) Deer Creek Trail OP877



robustus (10) Haviland Lake OP1223

robustus (17) Mogollon Mtns OP923

robustus (24) Twin Lakes OP1135

robustus (13) Beaver Creek OP1174

robustus (15) Alpine Divide OP905

robustus (17) Mogollon Mtns OP921

robustus (31) NE Deckers OP1194

robustus (18) Hannagan Meadow OP938

robustus (16) San Fransisco Mtns OP913



robustus (40) Hanging Lake OP2679

robustus (23) S of Gothic OP1122


robustus (35) Weston Pass OP1218

robustus (25) NE Leadville OP1140

robustus (8) Pagosa Springs OP1179


robustus (11) Abajo Mtns OP1130

robustus (28) Monarch Pass OP1177

glorietus (5) Manzano Mtns OP907

n. sp. (6) Cave of the Winds OP1128

robustus (26) Sange de Cristo OP1146

Cyptobunus u. madhousensis OP239


robustus (9) Ouray OP1199

robustus (34) nr Garfield OP1215



robustus (19) Pinalero Mtns OP945


glorietus (3) Mt. Taylor OP904

robustus (44) Jemez Mtns OP885

robustus (38) Skeleton Cave OP2567


Metanonychus OP985





robustus (30) North Pass OP1189

robustus (47) Mimbres Mtns OP927


robustus (9) Ouray OP1197

robustus (46) Ski Apache OP894


robustus (29) Longs Peak OP1183

robustus (39) Fault Cave OP2569

Metanonychus OP974

robustus (8) Pagosa Springs OP1178


robustus (43) Deer Creek Trail OP878

PLIOCENE PLEIST.MIOCENE

12.09 - 28.57

8.03 - 23.69

S. r. glorietus

S. r. glorietus

COTW/MC

Northwestern

Southwestern

Haviland

Central Eastern

Southeastern

Northeastern

Sclerobunus r. glorietus (1)

Sclerobunus r. robustus (42)

COTW/MC (6)

S. robustus

2.65.3

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 accordingto 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 interpretationof the references to color in this figure legend, the reader is referred to the web version of this article.)



of 10% KOH for several minutes to promote expansion, and to re-move muscle layers for easier viewing. For a small sample of spec-imens we used an alternative technique to promote penisexpansion – here we placed specimens with everted penises intoa hot lactic acid (85%) solution for 2–3 min, then moved these spec-imens into distilled water. This procedure was repeated multipletimes for each specimen.

Specimens treated with 10% KOH were cleaned prior to SEMexamination (Leo 1450VP SEM) using an ultrasonicator, criticalpoint dried, then mounted on Ted Pella� aluminum SEM stubsusing copper-backed electrical tape. All major mitochondrial lin-eages of S. robustus were examined for penis morphology. SEMsamples could not be obtained for Mallory Cave, as only one (male)specimen is currently known, and for the troglomorphic form fromTaos Ski Valley because males for this population (likely an unde-scribed species) are unknown.

3. Results

GenBank accession numbers for newly generated sequences areprovided in Supplemental Table 1. Alignments and summary treesfrom all analyses (e.g., Bayesian majority rule consensus trees)have been deposited at TreeBASE II. A Google Earth kmz file of allsampled locations is available upon request from the correspond-ing author.

Table 1Results of BEAST divergence analyses. Values in millions of years.

Clade Tmrca

Mean95%minimum

95%maximum

Root 19.84 12.09 28.57Sclerobunus 13.54 8.86 18.91S. robustus (all) 9.31 6.2 12.9S. r. glorietus (without Glorieta

Canyon)5.11 2.86 7.47

COTW/MC 5.8 5 (fixed) 7.4S. r. robustus 5.48 3.54 7.55Western 3.79 2.34 5.37Northwestern 1.34 0.73 2.02Southwestern 2.87 1.75 4.1Haviland 3.09 1.6 4.76Eastern 4.59 2.93 6.42Southeastern 2.75 1.65 3.97Central Eastern 1.51 0.79 2.31Northeastern 2.85 1.74 4.09

3.1. COI gene trees

A total of 103 COI sequences were analyzed, 97 of which weregenerated from S. robustus specimens (14 S. r. glorietus, 78 S. r.robustus, and 5 S r. spp.). COI sequences were obtained from twoindividuals 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 begenerated for Spring Creek samples (site 41, Fig. 1). Not includingoutgroups, 767 characters were constant, and 360 were parsi-mony-informative. The results of the COI BEAST analysis are shownin Fig. 2, with estimated divergence times summarized in Table 1.The BEAST topology is largely congruent with the COI MrBayestopology (Supplemental Fig. 1), except for the placement of theHaviland clade, which is weakly supported in both analyses.S. robustus is recovered as monophyletic with high support. S. r. glo-rietus is paraphyletic, with the type locality (Glorieta Canyon, NM)strongly supported as sister to all other S. robustus, while all otherpopulations morphologically identified as S. r. glorietus form aclade. A Bayes Factor analysis was run (Kass and Raftery, 1995;Nylander et al., 2004) in which all S. r. glorietus were constrainedto form a monophyletic group, but a BF of 10.48 is very strong evi-dence against this alternative topology. The highly troglomorphicCOTW/MC clade was recovered as sister to all S. robustus withthe exception of the Glorieta Canyon population. The most recentcommon 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 is5.48 Ma (95% HPD 3.54–7.55).

Within S. r. robustus, six highly supported major mitochondrialsubclades were found (support values shown on SupplementalFig. 1), including Haviland, Northwestern (NW), Southwestern(SW), Northeastern (NE), Central Eastern (CE), and Southeastern(SE). The geographic distribution of these subclades is shown inFig. 1. Diversification date estimates for these major subcladesare Pleistocene in age (ranging from 1.34 to 1.51 Ma) for the CEand 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 eachhighly supported, the relationships among them are not. All col-

lecting localities where multiple haplotypes were sampled werefound to be reciprocally monophyletic (all highly supported) ex-cept 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 presentedin Supplemental Table 2. Most interclade divergences are above10%, while intraclade divergences range from 3.1% to 9.2%. WithinS. r. robustus, southern ‘sky islands’ subclades (SW and SE) showrelatively high intraclade divergences, while the northern subc-lades (NW, NE, and CE) distributed in more contiguous mountain-ous regions have lower intraclade divergences. Diversity statisticsand demographic history tests for all major clades are shown in Ta-ble 2. Results show high numbers of unique haplotypes within S.robustus (77 unique haplotypes from 97 specimens) and high levelsof intraclade nucleotide diversity. Tajimas’s D values were found tobe non-significant.

3.3. EF-1a gene trees

The EF-1a intron was successfully sequenced for 27 S. robustusspecimens, representing all major mitochondrial lineages. Manysequences contained poly-AT regions near the 50 and 30 ends;ambiguous regions of alignment in these sections were removedfrom subsequent analysis. The manual and ClustalX with manualadjustments 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 adjust-ments alignment, with an alignment length of 605 base pairs.

The EF-1a gene tree is unrooted, but if we make the assumptionthat the root falls on a branch that separates primary groups, thenthis gene tree recovers many clades also found in COI analyses(Fig. 3). All populations identified as S. r. glorietus were recoveredin a single clade with high support. Similarly, the COTW/MalloryCave clade and Eastern and Western clades of S. r. robustus wereeach recovered with high support (>0.95 posterior probability).The SW clade was recovered with high support, embedded withina paraphyletic NW clade. The NE and SE clades were recoveredwith moderate support (0.90–0.95 pp) while the CE clade wasparaphyletic with respect to the SE clade. The single Haviland sam-ple 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 treeresults suggest two sets of southern ‘sky island’ groups derivedfrom more northern locations: the SW clade derived from the

0.02

robustus (41) Spring Creek OP2683


n. sp. (7) Mallory Cave OP2568








robustus (16) San Francisco Mtns OP913


robustus (45) N Chama OP2115

n. sp. (6) COTW OP1127


robustus (23) S Gothic OP1122




robustus (12) Dolores River OP1149


robustus (34) Garfield OP1215





robustus (10) Haviland OP1223

1.0

.99

.91

1.0

1.0

1.0 1.0

1.0

1.0

1.0

.90

.93

.65.74

.74

.92

.69

.91

.67

.93

S. r. glorietus 1 (<0.001)

COTW/MC: 1 (0.019)

NW: 0.639 (0.007)

SW: 1 (<0.001)

CE: 0.48 (0.035)

SE: 1 (0.022)

NE: 0.84 (<0.001)

HAV

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 mitochondrialphylogroups as grouping variables), with p values in parentheses.


NW, and the SE clade derived from the CE (Fig. 3). Using mitochon-drial clades as a priori groups, EF-1a gsi values were one (=exclu-sive ancestry) with the exception of the NE and CE clades (allvalues are statistically significant; Fig. 3).

Table 2Mitochondrial genetic diversity statistics for S. robustus.

3.4. Penis morphology

Specimens treated in 10% KOH versus lactic acid resulted insimilar degrees of penis expansion. SEM images of penises ex-panded in KOH were generated for 12 specimens representing allmajor mitochondrial lineages of S. robustus. The penis size of S. r.robustus is nearly 50% larger than that of S. r. glorietus, but this isperhaps expected given the body size difference between thesesubspecies. Penis morphology of these two subspecies is structur-ally very similar (Fig. 4). Within S. r. robustus, there is remarkablylittle differentiation in penis morphology between specimens rep-resenting divergent molecular clades (Fig. 4). All penis images havebeen deposited at Morphbank (http://www.morphbank.net/).

Subclade n N s k p Tajima’sD

S. robustus (total) 97 77 191 46.405 0.07117 -S. r. glorietus (no Glorieta

Canyon)14 9 129 50.582 0.06621 0.59577

COTW/MC 3 3 69 46.000 0.04868 n/aHaviland 6 4 73 38.600 0.04843 1.23092Northwestern 8 7 50 21.893 0.02743 0.49345Southwestern 15 15 131 44.638 0.05905 -0.19893Southeastern 8 8 136 59.571 0.06104 0.27722Central Eastern 6 5 44 22.733 0.02922 0.98167Northeastern 35 27 127 29.435 0.04117 -0.68114

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 ofnucleotide differences; p, nucleotide diversity.

4. Discussion

4.1. Phylogeography

Laniatorean harvestmen are attractive candidates for phylogeo-graphical studies due to their low vagility and microhabitat spe-cialization. S. robustus is particularly useful because, in additionto those characteristics, this species has a paradoxically wide dis-tribution. There have been numerous phylogeographic studiesfocusing on organisms inhabiting the SRMIP that show populationsub-structuring reflecting the geological and climatic history ofthis area. A smaller fraction of these studies focus on animals that

occupy the montane forest habitats throughout the SRMIP (e.g. Sul-livan, 1994; Lamb et al., 1997; Barber, 1999; Masta, 2000; Smithand Farrell, 2005; DeChaine and Martin, 2005; Barber, 2007). Thegreat majority of those phylogeographical or biogeographical stud-ies have used vertebrate taxa as the focal species, with few excep-tions including spiders (Masta, 2000) and beetles (Smith andFarrell, 2005), both of which occupy only the extreme southwest(i.e. ‘sky islands’ of southern Arizona and New Mexico). We con-tend that in order to better understand the biogeographical historyof the SRMIP it is important to include all types of organisms (i.e.,vertebrate, invertebrate, and plants), and include low-dispersaltaxa that are distributed throughout the SRMIP.

Within S. r. robustus the majority of localities sampled are genea-logically exclusive for mitochondrial COI sequences (Supplemental

http://www.morphbank.net/

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 imagescorrespond to mitochondrial phylogroups of S. r. robustus. Scale bars = 100 lm.


Table 3), indicating an absence of female-based gene flow at thegeographic scale of our sampling regime. Northern clades (NW, NE,and CE) show lower levels of nucleotide and genetic diversity ascompared to southern clades (SW and SE), most likely due to thegreater connectedness of the mountain ranges they inhabit (Table 2).The ‘sky islands’ in the south are more isolated and separated bygreater distances as compared to the more contiguous northernranges. 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 expansionwithin the NE clade, centered in the southern San Juan and Sangre deCristo Mountains, with subsequent northward phylogeographicnesting. 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 inour study. This suggests either higher levels of gene flow in the morecontinuous northern locales, or relatively recent range expansion.Taken together, the mitochondrial and nuclear data suggest thatmid-latitude highlands (e.g., in southern UT/CO, northern NM)may have served as regions for multiple historical refugia forSclerobunus.

There have been numerous other phylogeographic studies oforganisms inhabiting the SRMIP, but the vast majority of these stud-ies are limited in their geographic breadth. An exception is theanalysis of the tassel–eared squirrel, Sciurus aberti (Lamb et al.,1997). These authors report eastern and western phylogeographic


clades within this species, with similar distributions to the east-ern and western clades recovered in Sclerobunus, except for theinclusion of the Abajo Mountains (southeastern UT) populationin the eastern Sciurus clade. Similar geographic breaks are seen inTamiasciurus squirrels (Barber, 2007), with three clades found inthe 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 estimatingdivergence dates from molecular data (see Rutschmann, 2006 andreferences therein). Similarly, there is a great deal of uncertainty,and a lack of consensus, regarding the geologic history and datesof mountain building in western North America. Based on anexamination of previous geologic and biogeographic literatureregarding mountain uplift and desert formation throughout wes-tern North America, Wilson and Pitts (2010) provide conservativeestimates 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 andFig. 2) suggest that diversification within Sclerobunus occurredwithin this general range of Neogene uplift. Due to the broad rangeof dates attributed to the Neogene (some of which occur as recentas 2 Ma), more recent divergences ranging from 3 Ma to the pres-ent can either be attributed to mountain building or Plio–Pleisto-cene glacial cycles. Most divergence date estimates within S.robustus are pre-Pleistocene – this contrasts with other reporteddivergence estimates for ‘sky island’ taxa, which also show muchlower 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 restrictedto the Wet Tropic montane ‘sky islands’ of northeastern Australia.For example, high levels of population structure consistent withpredicted refugia have been reported in Temnoplectron beetles (Bellet al., 2007), Gnarosophia land snails (Hugall et al., 2002), and ver-tebrates (Schneider et al., 1998). Additionally, divergence esti-mates for many of these taxa, including vertebrates, areconsistent with Pliocene divergences.

4.2. Genetic divergence without morphological divergence

High levels of genetic divergence are found in S. robustus at alllevels. Most sampled S. robustus populations are exclusive for mito-chondrial haplotypes, indicating a lack of female-based gene flow.The S. r. robustus subspecies shows much internal genetic differen-tiation, with six highly supported mitochondrial phylogroups, all ofwhich are distributed in allopatry and show high interclade diver-gence levels. Estimated divergence times for these allopatric cladesare mostly pre-Pleistocene. The observation of nuclear gene treecongruence, which is expected to occur relatively late in the diver-gence process (Moore, 1995; Avise, 2000), further supports rela-tively ancient diversification.

Given this obvious lack of genetic cohesiveness, we expected toobserve coincident morphological differentiation. The two subspe-cies 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 distin-guished using morphometrics analyses (Derkarabetian et al.,2010). However, there is surprisingly little difference in reproduc-tive morphology between these subspecies (Fig. 4). Within thegenetically divergent, allopatric clades of S. r. robustus there is al-most a complete absence of both somatic and reproductive mor-phological differences. This conservatism was also noted byBriggs (1971), who in reference to sky island populations statedthat ‘‘the isolated populations are remarkably undifferentiated’’.

Under a cryptic female choice model (Eberhard, 1985; reviewedin Hosken and Stockley, 2004; Song and Bucheli, 2010 and refer-ences therein), some reproductive morphological divergence is ex-

pected between separate arthropod lineages because of ‘‘rapid anddivergent’’ genitalic evolution. In the case of S. robustus, we do notobserve noticeable genital divergence in a system with obviousgeographic isolation that appears to be relatively old. One canask, what is maintaining morphological cohesiveness across spacein this taxon? Gene flow is an unlikely cohesive force, and morpho-logical similarity due to recent divergence is not supported. Infor-mally, S. robustus populations are found in very similarmicrohabitats throughout their range, suggesting conservation inthe occupied niche. It is not unusual to find closely related taxathat are morphologically conserved due to ecological niche conser-vation (i.e. niche conservatism promotes morphological conserva-tism, see Bond et al., 2001; Bond and Stockman, 2008). What isunusual is the absence of differentiation in reproductive morphol-ogy, as these traits are not ‘‘adaptive’’ per se, and hence not ex-pected to be affected by ecological niche constraints.

Studies of other arthropod taxa have reported similar findingsregarding a decoupling of genetic and genitalic divergence. Bondet al. (2003) report a gradual evolution of genitalia in Anadenobolusmillipeds, where male genitalia was less structured than expectedgiven the levels of genetic divergence. The authors suggest that, be-cause some overlap existed in genitalic morphological space, notenough time has passed for the genitalia to sort to reciprocalmonophyly. Other studies have also reported a decoupling of gen-italic 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 lineageshas been observed in other harvestmen taxa. Examples include theEuropean genus Trogulus, where more recently evolved speciesgroups show conservation in penis morphology, while relativelyolder lineages show more divergence (Schönhofer and Martens,2010). Similarly, the New Zealand cyphophthalmid species Aorakidenticulata denticulata shows highly conserved morphology acrossits distribution, despite low vagility (an absence of genetic ex-change) and high genetic divergences (Boyer et al., 2007). Wenoted 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, andsomatic morphological differences (Derkarabetian et al., 2010).

4.3. How to proceed from here?

A decoupling of genetic divergence and reproductive morpho-logical divergence poses obvious difficulties for species delimita-tion in harvestmen, as variation in penis morphology representsan important character source for species delimitation. Given theamount of genetic differentiation seen within S. robustus and thecommon occurrence of cryptic divergence in harvestmen, it is pos-sible that this decoupling of genetic divergence and morphologicaldivergence is happening at the species level. By requiring that dif-ferent harvestmen species have different penis morphologies, weare 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 ofapparent genitalic stasis. First, we need to better understand howthe genitalia work from a functional perspective. Are there ‘‘hid-den’’ genitalic characters that we are simply not observing usingthe methods employed here? Although this possibility always re-mains a potential criticism, we note that we have used commonlyemployed techniques for the study of harvestmen penises, and in-deed tried multiple methods to best reveal penis microstructures.A second avenue for future research involves additional develop-ment and study of nuclear gene divergence. The EF-1a intron dataindicates partial (but conservative) nuclear gene divergence coinci-dent with mitochondrial gene divergence in this system. Expand-ing this nuclear gene perspective to many more genes would be


informative, allowing greater insight into historical demography,the true extent of morphological conservatism, and a better under-standing of species limits in these arachnids.

Acknowledgments

This research was supported by the Cave Conservancy Founda-tion (Fellowship in Karst Studies), Society for Integrative and Com-parative Biology (Grants in Aid of Research), and the AmericanArachnological Society (Vincent Roth Fund for Systematic Re-search). For loans of material we thank the California Academy ofSciences, American Museum of Natural History, and the DenverMuseum of Nature and Science. For assistance in fieldwork wewould like to thank Joe Deas, Damian Elias, Ryan Fawcett, Lars He-din, Maureen McCormack, Casey Richart, and Steven Thomas. SEMaccess was provided by the California Academy of Sciences withsupport from the Harriet-Exline Frizzel Fund. Comments by CaseyRichart, Axel Schönhofer, and two anonymous reviewers helped toimprove the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2011.08.004.

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