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Molecular phylogeny of the harvestmen genus Sabacon (Arachnida: Opiliones:Dyspnoi) reveals multiple Eocene–Oligocene intercontinental dispersal eventsin the Holarctic

Axel L. Schönhofer a,⇑, Maureen McCormack b, Nobuo Tsurusaki c, Jochen Martens d, Marshal Hedin a

a Department of Biology, San Diego State University, San Diego, CA 92182-4614, USAb Wisconsin State Lab of Hygiene, University of Wisconsin-Madison, Madison, WI 53706, USAc Laboratory of Biology, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japand Institute of Zoology, Johannes Gutenberg University Mainz, D-55099 Mainz, Germany

a r t i c l e i n f o

Article history:Received 9 June 2012Revised 6 September 2012Accepted 3 October 2012Available online 17 October 2012

Keywords:Molecular systematicsHolarcticIntercontinental dispersalDivergence time estimationAncestral range reconstructionParametric biogeographic modelling

a b s t r a c t

We investigated the phylogeny and biogeographic history of the Holarctic harvestmen genus Sabacon,which shows an intercontinental disjunct distribution and is presumed to be a relatively old taxon.Molecular phylogenetic relationships of Sabacon were estimated using multiple gene regions and Bayes-ian inference for a comprehensive Sabacon sample. Molecular clock analyses, using relaxed clock modelsimplemented in BEAST, are applied to date divergence events. Biogeographic scenarios utilizing S-DIVAand Lagrange C++ are reconstructed over sets of Bayesian trees, allowing for the incorporation of phylo-genetic uncertainty and quantification of alternative reconstructions over time. Four primary well-sup-ported subclades are recovered within Sabacon: (1) restricted to western North America; (2) easternNorth American S. mitchelli and sampled Japanese taxa; (3) a second western North American groupand taxa from Nepal and China; and (4) eastern North American S. cavicolens with sampled European Sab-acon species. Three of four regional faunas (wNA, eNA, East Asia) are thereby non-monophyletic, andthree clades include intercontinental disjuncts. Molecular clock analyses and biogeographic reconstruc-tions support nearly simultaneous intercontinental dispersal coincident with the Eocene–Oligocene tran-sition. We hypothesize that biogeographic exchange in the mid-Tertiary is likely correlated with theonset of global cooling, allowing cryophilic Sabacon taxa to disperse within and among continents. Mor-phological variation supports the divergent genetic clades observed in Sabacon, and suggests that a tax-onomic revision (e.g., splitting Sabacon into multiple genera) may be warranted.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

1.1. Biogeographic studies of the Holarctic

Over the past 20 years the biogeographic history of the North-ern Hemisphere has been the subject of several detailed studiesand meta-analyses (e.g. Donoghue and Smith, 2004; Enghoff,1995; Sanmartín et al., 2001). The Holarctic is the largest planetaryecozone, spanning a large proportion of the Northern Hemisphereincluding parts of Europe, Asia, and North America, and many sub-divisions exist according to ecology, geography, floristic and fau-nistic characteristics. For most biogeographic studies spanningthe Holarctic, four major infraregions have been applied (e.g.Donoghue and Smith, 2004; Enghoff, 1995; Sanmartín et al.,

2001) corresponding to zones of temperate forest endemism inthe western Palearctic (Europe), eastern Palearctic (eastern Asia)and the Nearctic (eastern and western North America; see Fig. 1in Donoghue and Smith, 2004).

The temporal connectivity between the four major Holarcticinfraregions follows a complex reticulate pattern (Smith et al.,1994; summarized in Sanmartín et al., 2001). Mid- to late Creta-ceous (100–80 Ma) geographic settings were characterized by theconnection of Europe with eastern North America, and Asia withwestern North America. At the end of the Cretaceous (70–65 Ma)the American mid-Continental Seaway closed and the North Amer-ican continent appeared as it does today. The opening of the Atlan-tic (�90 Ma) isolated North America and Europe, but major landbridges still connected these two continents, most importantly,the Thulean Bridge around 55–50 Ma. Subsequent land bridgeswere apparently less suitable for biotic interchange, especially forwarm-adapted species (McKenna, 1983; Sanmartín et al., 2001;Tiffney, 1985). North America and Asia were repeatedly connectedvia the Bering Bridge, with biotic interchange thought to be

1055-7903/$ - see front matter � 2012 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ympev.2012.10.001

⇑ Corresponding author.E-mail addresses: [email protected] (A.L. Schönhofer), mccmaureen@

gmail.com (M. McCormack), [email protected] (N. Tsurusaki), [email protected](J. Martens), [email protected] (M. Hedin).

Molecular Phylogenetics and Evolution 66 (2013) 303–315

Contents lists available at SciVerse ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev


moderated mainly by climatic settings. A boreotropic forest con-nected eastern Asia and eastern North America during the warmand humid Eocene, ultimately disrupted by global cooling andorogeny of the Rocky Mountains at the end of this epoch(35 Ma). In the Pleistocene (1.5–1 Ma) the colder climate restrictedBeringian interchange to mostly cold-adapted boreal species.

Development of novel parametric methods in biogeographicstudies allows for a more comprehensive reconstruction of com-plex Holarctic processes than previous methods (Donoghue andSmith, 2004; Sanmartín et al., 2001 and summary herein). MostHolarctic studies implementing parametric biogeographic methodswith molecular phylogenetic data focus on plant taxa, with animalstudies still scarce (e.g., Burbrink and Lawson, 2007; Vieites et al.,2007). Comparisons of codistributed plant and animal taxa oftenreveal contrasting distributional and biogeographic patterns atcomparable phylogenetic levels. For example, the degree anddirection of dispersal events between Holarctic infraregions variesconsiderably, showing extensive interchange between floral ele-ments of eastern North America and eastern Asia, whereas animaldisjuncts across these regions are rare (Donoghue and Smith, 2004;Sanmartín et al., 2001). Holarctic plant genera are generally

widespread with few plant families endemic to the Holarctic.Conversely, many Holarctic arthropod taxa show family-levelendemism at continental or smaller geographic scales. Theincreased structuring seen in arthropods perhaps offers furtherinsight into the complex Holarctic biogeography, particularly whenconducted on groups considered as model taxa for biogeographicanalyses.

1.2. Harvestmen as model taxa for historical biogeography

Recent phylogenetic studies within the arachnid order Opili-ones have shown that the diversification history of many groupsis tightly interconnected with Earth history processes (e.g., Boyeret al., 2007; Giribet et al., 2010; Hedin et al., 2012; Schönhoferand Martens, 2010). The integration of phylogeny and biogeogra-phy has therefore become necessary to delineate centers of Opili-ones endemism and biodiversity, as illustrated by the well-studied mite harvestmen (Boyer and Giribet, 2007; Boyer et al.,2007; Giribet et al., 2012). Despite these many biogeographic con-tributions, few Opiliones groups with primarily Holarctic distribu-tions have been the focus of phylogenetic or biogeographic

Fig. 1. Sabacon habitus images. (A) S. simoni, female, Italy, Passo di Montevaca; (B) S. viscayanus ramblaianus, female, France, Pyrenees, Lac du Bethmale; (C) S. occidentalis,juvenile, USA, CA, Humboldt Co., Berry Glenn; (D) S. paradoxus, female, France, Olargues. All pictures by A.L.S.; and (E) approximate distribution of Sabacon indicated byoutlined areas, with green circles indicating localities of sequenced specimens.

304 A.L. Schönhofer et al. / Molecular Phylogenetics and Evolution 66 (2013) 303–315


attention. Relationships of Holarctic Sironidae have been difficultto resolve (Giribet and Shear, 2010), and this group seems mostlyabsent from continental Asia. Species of Caddo (Eupnoi) provide in-sight into recent biogeographic exchange between eastern NorthAmerica and eastern Asia (Shultz and Regier, 2009). Regional Hol-arctic clades within the long-legged Phalangioidea are still beingdefined (Hedin et al., 2012).

Members of the harvestmen suborder Dyspnoi are a promisinggroup for studies of Holarctic biogeography. This clade includesabout 350 species in approximately 40 genera, all with north tem-perate distributions. Most Dyspnoi genera are restricted to singlecontinents (Shear, 2010) while taxa above this rank are distributedacross continents. An exception is the genus Sabacon (Fig. 1), whichspans a larger distributional area than any other Dyspnoi genus,with centers of endemism in eastern Asia (Martens, 1972, 1989;Suzuki, 1974; Tsurusaki and Song, 1993a), eastern and westernNorth America (Shear, 1975), and south-western Europe (Martens,1983; Fig. 1E). In these areas, Sabacon species show strong prefer-ences for permanently cool and moist habitats, occurring in varioussheltered microhabitats in coniferous or mixed forests, in deep al-pine stony gravel, and in caves. Although a phylogenetic analysis ofall Sabacon taxa has never been conducted, morphological similar-ities between European and eastern North American taxa (Martens,1983; Shear, 1975, 1986), as well as between North American andAsian species (Martens, 1983; Cokendolpher, 1984; Shear, 1975),suggest possible intercontinental biogeographic connections.

Existing biogeographic patterns imply that diversification inSabacon must be relatively old. Fossil Sabacon dating to the Eoceneare known from European amber (Dunlop, 2006), indicating bothan old age and morphological stasis for this lineage. Lopez et al.(1980) mentioned the cave-dwelling Sabacon paradoxus as anexample of a Tertiary relict, and Thaler (1976) discussed S. simonias a Holarctic relict taxon. Sabacon includes no transcontinentalspecies, and does not feature typical boreal species, as found forexample in the harvestmen suborder Eupnoi (e.g., Mitopus morio).The pattern of disjunct distributions within species groups, butnarrow endemism of most Sabacon species, suggests generally poordispersal abilities for members of the genus. Dispersal across openoceans is unlikely for Sabacon, and connection of landmasses canbe assumed to be the only means for intercontinental biogeo-graphic exchange.

1.3. Applying novel biogeographic methods to Opiliones

The study of biogeography, in combining distributional patternsof extant taxa and the reconstruction of their phylogenetic history,has undergone considerable changes over the last decade. Utilizingphylogenetic information derived from molecular data, novel mod-elling approaches using parsimony or likelihood reconstructionsover sets of plausible trees (Lagrange: Ree and Smith, 2008; Smith,2009; S-DIVA: Yu et al., 2010) are replacing simple area clado-grams and purely parsimony-based methods. These new method-ologies are able to incorporate age estimates and phylogeneticuncertainty, account for missing phylogenetic data, and add statis-tical confidence to reconstructions (Ree and Smith, 2008; Smith,2009). While molecular phylogenetic analyses are becomingincreasingly common within Opiliones, biogeographic results re-main largely descriptive, and statistical approaches have been re-stricted to estimating divergence times (Boyer and Giribet, 2007;Boyer et al., 2007; Derkarabetian et al., 2010). With few exceptions(e.g., Giribet et al., 2012; Sharma and Giribet, 2011), new biogeo-graphic methodologies have yet to be incorporated into Opilionesresearch.

The current study focuses on the systematics and biogeographichistory of Sabacon based on sampling of major lineages includingmost described species diversity in North America and Europe,

and representative species sampling from Japan, Nepal, and China.We use molecular phylogenetics to investigate relationships with-in Sabacon, and evaluate the systematic position of Sabacon withinthe Dyspnoi. Phylogenetic information is used to infer ancestraldistribution areas of Sabacon clades, as well as the timing anddirectionality of Holarctic intercontinental dispersal. We also sum-marize differences in Sabacon morphology as these relate to recov-ered molecular lineages.

2. Material and methods

2.1. Taxon sampling

Sabacon species are relatively rare and sometimes difficult tocollect. Many species are only known from high elevation habitatsor caves, and many original descriptions are based upon single orfew specimens. Nevertheless we have obtained a comprehensivesample of North American and European taxa, and a representativesample of Asian taxa. Overall, our sample covers much of the de-scribed morphological and geographical diversity in Sabacon (seeTable 1). Specimens were collected in the field via manual searchand preserved in 100% EtOH, or collected into 80% and later trans-ferred to 100%. Material was then stored at minus 80 �C until DNAextraction. Some older samples from Asia were retrieved from 70%EtOH collections. Adult specimens were used whenever possible,but due to the late-maturing seasonal phenology of Sabacon, inclu-sion of juveniles in this study was also necessary. The identity ofjuveniles was corroborated by morphological comparison withadults from the respective localities, partly loaned from the Califor-nia Academy of Science (CAS), and from our own reference collec-tions (San Diego State University Terrestrial Arthropod collection,University of Mainz Opilionid collections of A.L.S and J.M.).

2.2. Gene data collection, sequence alignment and phylogeneticanalysis

Genomic DNA was extracted from leg tissue using the QiagenDNeasy kit. The polymerase chain reaction (PCR) was used toamplify the following gene fragments: 28S rRNA (28S), using theprimers ZX1, ZR2 (Mallatt and Sullivan, 1998), and the newlydeveloped ZX1elong (50-ACCCGCTGAATTTAAGCATATTAG-30);mitochondrial Cytochrome Oxidase 1 (CO1), using C1-J-1718SPI-DERA and C1-N-2776SPIDER (Vink et al., 2005); nuclear ElongationFactor 1-alpha (EF1a), using OP2BSAB and OPRC4 (Hedin et al.,2010). PCR protocols followed Hedin et al. (2010) for EF1a, Thomasand Hedin (2008) for CO1, and Hedin and Thomas (2010) for 28S,the latter using an annealing temperature of 56 �C. PCR productswere purified on Millipore plates and amplicons were directly se-quenced at Macrogen USA. Sequencher V4.5 was used to assembleand edit sequence contigs, and all ambiguous sites were scored asheterozygous using standard ambiguity codes.

CO1 sequences were aligned manually in MEGA 4.0 (Tamuraet al., 2007) using amino acid translation, while 28S and EF1a se-quences were aligned with MAFFT (vers. 6; http://mafft.cbrc.jp/alignment/software/). The G-INS-i strategy was used for the pro-tein coding EF1a; to incorporate 28S structural information weused Q-INS-i as recommended by Katoh and Toh (2008). To ac-count for alignment uncertainty, 28S and EF1a alignments werefurther trimmed using Gblocks (Castresana, 2000), applying a ‘‘lessstringent’’ criterion with standard settings. Models of DNA se-quence evolution were evaluated using jModelTest 0.1.1 (Posada,2008) under three substitution schemes (JC, HKY, GTR) on a fixedBIONJ tree, allowing for unequal base frequencies and among-siterate variation. Final model selection was based on the Akaike

A.L. Schönhofer et al. / Molecular Phylogenetics and Evolution 66 (2013) 303–315 305

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lens

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kard

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ton

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acka

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n:

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tori

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.,K

oku

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ho,

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edak

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lls,

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iens

isM

arte

ns,

1972

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al:

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.,H

anu

man

teD

anda

,27.

7,86

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–Sa

baco

nm

itch

elli

Cro

sby

&B

ish

op,1

924

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C,M

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ell

Co.

,Roa

nM

tn,3

6.05

,�82

.05

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mit

chel

liC

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y&

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hop

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4O

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chel

lC

o.,R

oan

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,36.

05,�

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anks

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reek

,46.

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anks

,189

4)O

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WA

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itz

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man

yC

reek

,46.

2544

,�12

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con

occi

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alis

(Ban

ks,1

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48.1

94,�

124.

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3670

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noc

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anks

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4)O

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48.1

94,�

124.

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3671

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noc

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anks

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(Ban

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ada:

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um

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i,To

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8,�

131.

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con

para

doxu

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mon

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9A

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Fran

ce:

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oc-R

ouss

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n,D

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larg

ues

,43.

5260

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204

JX57

3584

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3674

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con

paso

nian

usLu

que,

1991

AX

LS10

09Sp

ain

:C

anta

bria

Prov

.,M

iera

,Las

Reg

adas

Cav

e,43

.271

2,�

3.72

357

JX57

3576

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3666

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3628

Saba

con

shea

riC

oken

dolp

her

,198

4O

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6U

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ID,I

dah

oC

o,B

urg

dorf

Rd

13.2

mi,

45.3

831,�

115.

9995

JX57

3553

––

Saba

con

sim

oni

Dre

sco,

1952

AX

LS52

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aly:

Prov

.Par

ma,

Pass

odi

Mon

teva

cà,4

4.53

546,

9.60

968

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3585

–JX

5736

31Sa

baco

nsi

mon

iD

resc

o,19

52O

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1Fr

ance

:It

aly,

Prov

.Cu

neo

,Mon

esi

diTr

iora

,44.

0738

1,7.

7502

8JX

5735

86JX

5736

75JX

5736

32Sa

baco

nsi

mon

iD

resc

o,19

52O

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2Fr

ance

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aly,

Prov

.Cu

neo

,Mon

esi

diTr

iora

,44.

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1,7.

7502

8JX

5735

87G

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GQ

8721

70Sa

baco

nsi

skiy

ouSh

ear,

1975

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034

USA

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A,S

iski

you

Co.

,WN

WM

cClo

ud,

41.2

736,�

122.

1750

JX57

3588

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3676

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3633

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con

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iyou

Shea

r,19

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USA

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Co.

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dee,

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40JX

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89JX

5736

77JX

5736

34



Information Criterion (AIC) and individual models were applied torespective partitions in all downstream analyses.

Bayesian inference using MrBayes v3.1.2 (Huelsenbeck andRonquist, 2001; Ronquist and Huelsenbeck, 2003) was applied tosingle gene as well as concatenated datasets. Data were partitionedby gene, and for CO1 and EF1a, by codon position. Bayesian analy-ses were run for 5,000,000 generations, where in all cases the stan-dard deviation of split frequencies had dropped below 0.01(Ronquist et al., 2005). Analyses were repeated to further checkfor convergence. The first 40% of trees were discarded as burn-in,with remaining trees used to reconstruct a 50% majority rule con-sensus tree. Split frequencies were interpreted as posterior proba-bilities (pp) of clades.

We also investigated phylogenetic relationships for a larger setof Dyspnoi taxa to evaluate the relative phylogenetic position anddistinctiveness of Sabacon within the suborder. This analysis wasbased on 28S rRNA, the only marker for which a comprehensivesample of Dyspnoi taxa was available. These data were generated,aligned and analyzed using the above-described methods. 28S se-quences not generated for this study were downloaded from Gen-Bank, based on studies of Giribet et al. (2010) and Schönhofer andMartens (2010; see Fig. 2).

2.3. Divergence time estimation using BEAST

We estimated taxon divergence times from the concatenateddataset using BEAST 1.6.1 (Drummond and Rambaut, 2007). Be-cause initial analyses did not reach convergence, we conductedanalyses on a reduced dataset of 37 specimens, excluding recentlydiverged lineages within the S. occidentalis and S. cavicolens clades.The Bayesian analysis was rerun and the reduced dataset topologycompared to the full dataset topology to check for congruence. Co-don partitioning was applied to CO1 (1, 2, 3) and EF1a ((1 + 2), 3) toaccommodate heterogeneous rates of evolution (Brandley et al.,2011). Models for CO1 and EF1a partitions were set to the secondbest fitting HKY+I+G model; although other priors were high, effec-tive sample size (ESS) values for rare substitutions within parti-tions for a GTR model remained low and analyses did notconverge. Strongly supported nodes (pp = 0.95–1.00) from the fulldataset were constrained to be monophyletic in the reduced data-set, to avoid entrapment in local optima (following Smith, 2009).No treemodel operators were removed, allowing BEAST to re-esti-mate the remaining topology. Priors were set to gamma if requiredto be modified. These settings were applied to all subsequent anal-yses. Analyses were run until ESS values exceeded 200 even forpoorly sampled priors, which was after 100,000,000 generations.Analyses were replicated twice and checked for convergence usingTracer 1.5.

We checked for rate heterogeneity in all datasets using the ran-dom local clock model implemented in BEAST. This clock modelestimates rate changes for each branch individually and identifieslineage specific rate changes (Drummond and Suchard, 2010). Anuncalibrated log-normal analysis estimated different clock modelsfor the three gene partitions, and the standard deviation of the par-tition frequency (stdev) was investigated for deviation from clock-like behavior.

For final analyses in BEAST a Death-Birth model of speciationand a relaxed log-normal clock were applied. Clock rates where un-linked and estimated for individual partitions. We simultaneouslyapplied both rate and fossil calibrations for a final BEAST recon-struction. For the clocklike CO1 (see Section 3.3.) we specified aclock rate based on the standard arthropod clock from Brower(1994; 2.3% per Ma) and a newly reported arthropod clock rate(Papadopoulou et al., 2010; 2.69%/Ma). To accommodate differ-ences of these rates a uniform prior was chosen for CO1. Meanrateto include the range of 0.0115–0.0145/Ma. The fossil taxon Sabacon

Saba

con

sp.

MC

ZDN

A10

0711

Fran

ce:

Dép

.Gar

d,Pa

rcN

atio

nal

des

Cév

enn

es,4

4.08

,3.5

9G

Q91

2770

bG

Q91

2866

b–

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con

sp.

OP7

90U

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CA

,Men

doci

no

Co.

,Hw

y12

8,38

.919

56,�

123.

2964

4JX

5735

90JX

5736

78JX

5736

35Sa

baco

nsp

.C

JM66

08N

epal

:M

anas

luH

imal

,upp

erM

yagd

iK

hol

aV

all.,

28.5

8,84

.66

JX57

3597

––

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con

sp.

CJM

6697

Ch

ina:

Sich

uan

,Pao

ma,

29.9

262,

101.

9795

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3598

––

Saba

con

sp.

CJM

6710

Ch

ina:

Gan

su,L

ian

hu

aSh

an,S

hah

etan

,33.

43,1

06.9

9JX

5735

99–

–Sa

baco

nsp

.C

JM66

95C

hin

a:Si

chu

an,M

uge

Cu

o,30

.146

2,10

1.86

05JX

5736

00–

–Sa

baco

nvi

scay

anus

ram

blay

anus

Mar

ten

s,19

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XLS

663

Fran

ce:

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i-Py

rén

ées,

Dép

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tes-

Pyré

née

s,V

allé

ede

l’Ou

zom

,43.

0270

8,�

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581

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3679

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3636

Tara

cus

sp.

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asta

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e,40

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.(20

10).

bG

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al.(

2010

).



claviger from European amber was used as a calibration point forthe minimum age of the cavicolens-Europe clade (see Section 3.2.).This taxon dates to the Eocene (�55–34 Ma; Dunlop, 2006) forwhich a log-normal distributed prior was set with a mean of2.53, a Stdev of 0.9, and an offset of 32 so the 2.5% percentile wouldbe 34 Ma and the median 44.18 Ma, constraining the minimum ageof the node but allowing it to be significantly older.

2.4. Ancestral range reconstructions using S-DIVA and Lagrange

Ancestral distributions were reconstructed using S-DIVA (Yuet al., 2010), which evaluates the most parsimonious reconstruc-tions of dispersal and vicariance events and integrates uncertaintyover a set of analyzed trees. Here we used 5000 post burn-in treesresulting from Bayesian analysis of the full concatenated dataset.Default settings were modified for ‘‘Allowed Reconstruction’’(checked) and Hold set to 32.767 (to include all alternative recon-structions per node, see Ronquist (1997), DIVA 0.1.1 manual). Wespecified five distributional areas for terminal taxa, including A:continental Asia (including Nepal and China only), B: westernNorth America, C: eastern North America, E: Europe, and J: Japan.We conducted analyses with maximum alternative scenarios ateach node set to 5, 4 and 3 to examine the effect of constraintson reconstructed ancestral distributions (Bendiksby et al., 2010;Kodandaramaiah, 2010).

Likelihood reconstruction of ancestral areas using Lagrange(C++ version, Bendiksby et al., 2010; http://code.google.com/p/la-grange/downloads/detail?name=lagrange_cpp_0.1BETA2_%20for_-MAC10.6.tar.gz&can=2&q) was performed on the dated Bayesianconsensus phylogeny from BEAST. Utilizing phylogenetic and tem-poral information, Lagrange reconstructs the biogeographic historyof a set of discrete areas based on the distribution of extant taxa.Accounting for branch length information, Lagrange integratesthe probability of extinction and dispersal as stochastic events cal-culated over time. Nodes that showed ambiguous area reconstruc-tion results in the Bayesian consensus tree were furtherinvestigated by reconstructing over all 5000 retained BEAST trees,following Smith (2009) and Bendiksby et al. (2010). Lagrange out-put was manipulated using TextWrangler for input into ‘‘R’’ 2.13.1and Excel 2007, to calculate probabilities of all ancestral area sce-narios at each node, and to segregate the five best scoring scenariosby node age. Further confidence in reconstructions was attained byincluding the percentage of individual ancestral area reconstruc-tions per node (Smith, 2009). Lagrange C++ computation omits sce-narios scoring less than 5% probability in individualreconstructions, thereby ‘‘weeding out’’ weakly supported scenar-ios. No constraints on the maximal number of areas, or on thedirectionality and timing of dispersal, were used in our analyses.

3. Results

3.1. Dyspnoi 28S phylogeny

Tree topologies based on 28S MAFFT versus MAFFT + Gblocksalignments were similar (results not shown), so we used theMAFFT-only alignment. The 28S phylogeny including multipleDyspnoi and Eupnoi outgroup taxa (1268 aligned base pairs for82 terminals) strongly supports the superfamilies Ischyropsalidoi-dea and Troguloidea (1.00 pp; Fig. 2). The recently emended Saba-conidae (Giribet et al., 2010), including Hesperonemastoma,Sabacon and Taracus, receives low support (0.78 pp), but Hesperon-emastoma and Taracus are strongly supported as sister taxa (1.00pp). Sabacon is strongly supported as monophyletic (1.00 pp;Fig. 2) and shows high internal 28S sequence divergence, with sep-aration into four well supported primary clades (Fig. 2). Based on

28S phylogram branch lengths, sequence divergence within Saba-con exceeds that of any other Dyspnoi genus included in the anal-ysis, and is indeed greater than that of most Dyspnoi families(Fig. 2).

3.2. Phylogenetic relationships within Sabacon

Fifty-four Sabacon specimens plus 7 outgroup taxa specimenswere used to generate sequence data for 28S, CO1 and EF1a (59,50, and 42 sequences, respectively). Only 28S could be amplifiedfor some of the older samples and several EF1a PCR productsyielded no results in direct sequencing, possibly due to the closeproximity of the forward primer to additional introns reportedfor Sabacon (Hedin et al., 2010). The final alignments of CO1(968 bp) and EF1a (681 bp) were unambiguous. We excluded thedifficult-to-align (and sometimes partial) EF1a introns, as sug-gested by Gblocks. 28S was difficult to align and we compared treetopologies from the MAFFT and MAFFT + Gblocks alignments. Re-sults based on these alternative alignments were very similar, dif-fering only for nodes that showed low support in analyses derivedfrom both alignments. An exception involved S. occidentalis se-quence OP2591, which changed position within the occidentalis-clade with high support from alternative alignments. The place-ment of this particular sequence does not greatly impact the bio-geographic analyses presented below. We used the MAFFT-onlyalignment (1236 aligned bp) in subsequent concatenated analyses.For individual gene trees see Suppl. Figs. 1–3.

In agreement with results from the larger 28S dataset (Fig. 2),analysis of the concatenated alignment (2885 total aligned bp)recovers Sabacon as monophyletic with strong support (1.00 pp,Fig. 3). Within Sabacon, four primary clades emerged that we de-fine here for further discussion (Figs. 3–5): (1) western NorthAmerican occidentalis-clade, basal placement in Sabacon, unites S.briggsi (1a), S. occidentalis and S. siskiyou (1b); (2) mitchelli-Japan-clade, unites the eastern North American S. mitchelli and the Japa-nese S. akiyoshiensis and S. imamurai; (3) astoriensis-Asia-clade,unites the western North American S. astoriensis and S. sheari(3a) with S. jiriensis and Sabacon sp. from Nepal and sampled Chi-nese Sabacon (3b) – this clade is subdivided according to geo-graphic origin; (4) cavicolens-Europe-clade, unites the easternNorth American S. cavicolens (4a) and sampled European Sabaconspecies. These four clades are strongly supported in both individualgene and concatenated analysis (Fig. 2). A conspicuous implicationof this phylogenetic structuring is that three of four regional faunas(wNA, eNA, east Asia) are non-monophyletic, and that three cladesinclude intercontinental disjuncts.

3.3. Divergence time estimation using BEAST

Rate heterogeneity as estimated by the random local clockmodel in BEAST (Drummond and Suchard, 2010) was generallylow throughout Sabacon and Sabaconidae, although we found sig-nificantly increased rate change in the astoriensis-Asia-clade withadditional increase within S. astoriensis. This result holds true forthe Dyspnoi, with minor rate changes between major groups (onlydecreasing in Dicranolasmatidae) but marked changes in individ-ual lower level taxa (Anelasmocephalus, Mitostoma). The uncali-brated log-normal analysis rejected clocklike evolution for EF1a(stdev: 2.1) and 28S (2.2), but not for CO1 (stdev: 0.3), justifying fi-nal BEAST settings as previously specified.

Unconstrained BEAST trees differed noticeably from MrBayestrees at deeper nodes, recovering a basal polytomy including Sab-acon and outgroups, and implied different interrelationships ofthe four major Sabacon clades. As these alternative BEAST recon-structions showed low support, and BEAST is known to convergeon local optima, we constrained basal nodes to force BEAST to



converge to the same topology as MrBayes, as suggested by Smith(2009). This resulted in faster convergence and higher ESS valuesfor all parameters and allowed setting of more complex substitu-tion models as estimated by jModelTest. The resulting BEAST chro-nogram is shown in Fig. 4 (see also Suppl. Fig. 4).

According to our reconstruction Sabacon originated about85 Ma ago (53–132 highest posterior density (HPD)), with subse-quent diversification into four primary clades occurring within arelative short time frame of �20 Ma (Fig. 4). The estimated agesof the most recent common ancestor (MRCA) of the primary cladesfalls within a time window of less than 10 Ma (mean times of 30–40 Ma), near the Eocene–Oligocene boundary. Intraspecific diver-gences within nominal taxa are estimated to be relatively old, oftenpredating the Pliocene. More recent divergences, partly dating tothe Pleistocene, were detected within the more densely sampledSabacon cavicolens (2.1–3.2 Ma) and S. occidentalis clades (1.5 Ma).

3.4. Ancestral area reconstruction using S-DIVA and Lagrange

S-DIVA analyses were conducted on a set of post-burnin treesresulting from Bayesian analysis of the full concatenated dataset.S-DIVA results vary depending upon whether the number of max-imal areas per ancestral node was constrained to 3 or 4, or left

unconstrained at 5. Nodes for which S-DIVA calculated severalequally likely scenarios if left unconstrained often tended to favora single area scenario in more constrained analyses (Fig. 5). Also,when optimal results could have yielded the same area scenario(e.g., a scenario including three areas), the reconstructions didnot agree. Finally, nodes with alternative scenarios showed a high-er number of possibilities that also differed considerably from theLagrange C++ results.

Lagrange C++ analyses were conducted on a set of trees resultingfrom BEAST analysis of the reduced concatenated dataset. Fig. 4 piecharts show the total proportion of each scenario at nodes with pos-sible alternatives, whereas histograms show frequencies of the fivebest scoring scenarios distributed over the 95% higher posterior den-sity of node age reconstructions (see also Suppl. Fig. 4). Pie chartsthereby depict the total likelihood of each scenario at the nodes,while the likelihood of timing of scenarios is shown by histograms.Large differences in the timing of alternative scenarios were generallynot detectable in our dataset. While the two expanded histograms forthe astoriensis-Asia and mitchelli-Japan clades, and for the most re-cent common ancestor of Sabacon show a shift of about 10–15 Maat the most likely time of the individual area reconstruction, theseare small differences in comparison to the overall 95% higher poster-ior densities and the total histogram coverage (Suppl. Fig. 4).

Fig. 2. Results of 28S Bayesian phylogenetic analysis of Dyspnoi including Eupnoi outgroups. GenBank accession numbers are given for taxa not listed in Table 1.



Lagrange (Fig. 4) and S-DIVA (Fig. 5) reconstructions supportintercontinental dispersal in two of three intercontinental disjunctclades. Within the cavicolens-Europe-clade, S-DIVA unambiguouslysupports Europe as the center of origin. While Lagrange also sup-ports a European center of origin with high likelihood, this recon-struction does not exclude a shared ancestral area of Europe andeastern North America (i.e., ambiguous dispersal directionality).The biogeographic connection between Europe and eastern NorthAmerica supports the assignment of the fossil Sabacon claviger toa lineage of European (+eastern North American) origin, as empha-sized here. Results for the mitchelli-Japan-clade differ between anal-yses. Lagrange shows higher support for an ancestral area

combining Japan with eastern North America, while S-DIVA favorsa Japanese center of origin. Neither reconstruction method providesunambiguous results for dispersal directionality in the astoriensis-Asia-clade. At deeper nodes, both methods weakly support westernNorth America as the ancestral area for Sabaconidae (Suppl. Fig. 4).

4. Discussion

4.1. Sabacon systematics

Sabacon is the nominative genus of the originally monotypicfamily Sabaconidae (Dresco, 1970). Subsequent evaluations of the

Fig. 3. Bayesian phylogram of Sabacon and Ischyropsalidoid outgroups based on concatenated data. Members of primary Sabacon clades (1–4) discussed in the text areindicated by different colors. Posterior probabilities given at each node with values in parentheses indicating support from single gene reconstructions of 28S, CO1 and EF1a(see Suppl. Figs. 1–3) respectively for primary Sabacon clades.



family first rejected its status (Gruber, 1978; Martens, 1972; Shear,1975) but then accepted an altered family diagnosis (Martens,1976, 1980, 1983; Shear, 1986). Martens et al., (1980), Martens(1983) based evidence on the peculiar and derived genital mor-phology, unique within the Dyspnoi. Later, Shear (1986) down-graded genital morphology as genus specific for Sabacon andincluded Taracus in the Sabaconidae on the basis of a cladistic anal-ysis. The latest emendation was by Giribet et al. (2010), addingHesperonemastoma to Sabaconidae based on molecular phyloge-netic affinities to Taracus. Our reassessment of Sabaconidae basedupon a more comprehensive taxon sample finds low support for

the relationship of Sabacon with Taracus plus Hesperonemastoma(Fig. 2; comparable to Giribet et al., 2010).

Compared to 28S rRNA divergence of other families within theDyspnoi (Fig. 2), Sabacon emerges as an isolated and highly diver-gent clade, and perhaps should be re-classified into multiple gen-era. While we do not attempt to taxonomically subdivideSabacon in this paper, we provide evidence for deep divergenceencouraging such endeavors. Support for a taxonomic split alsostems from morphology, where comparisons of Sabacon molecularlineages reveal clade-specific differences in male genital morphol-ogy. This situation is similar to that seen within the Dyspnoi family

Fig. 4. Consensus chronogram of Bayesian BEAST and Lagrange likelihood analysis based on 5000 trees. Scale at bottom shows time in millions of years. Letters at nodes, piecharts and specimen identifier correspond to area-legend top left. Indicators (1–4) at nodes indicate clades discussed in the text. Numbers at nodes report mean node ages,node bars the respective 95% posterior density based on the consensus tree. When more than one ancestral area was likely for the consensus tree percentage distributions ofscenarios were re-estimated over all BEAST trees and segregated by time shown in pie charts using Lagrange C++ and histogram bars at nodes. Dotted line, scaled on the left,indicates change of average temperature over time according to Zachos et al. (2001). Outgroup branches (dashed) have been shortened and posterior density and histogramsomitted to focus on Tertiary events. For a fully expanded analysis see Suppl. Fig. 4.



Fig. 5. S-DIVA reconstructions. Histograms on the left show differences of analyses constraint for 3, 4, and 5 areas per node. Colored circles and letters at nodes show identicalresults for all three constraints. Indicators (1–4) at nodes indicate clades discussed in the text.



Nemastomatidae, members of which exhibit relative stasis of bodyform but display greater variation in male genital and/or secondarysexual characteristics (Schönhofer and Martens, 2012).

Sabacon is separated into four well-defined molecular clades(Figs. 3–5); here we discuss morphological support for theseclades.

(1) Occidentalis-clade: Sabacon briggsi is sister to a group com-prising S. occidentalis and S. siskiyou. The latter are very sim-ilar but unique within Sabacon in exhibiting inflated penisglandes with a firmly attached, three-dimensional and partlydivided stylus (Shear, 1975). From the single male S. briggsiavailable for our analysis we can confirm stylus modifica-tions, including a regular row of backwards pointing teeth.All three species share large conical cheliceral apophysesvery similar in shape and size. Within the occidentalis andsiskiyou group OP2591 is genetically unique and needs tobe further investigated. Specimens collected from this loca-tion are smaller than other members of this clade, but onlyfemales are available. We expect at least stylus-specificdifferences for males from this potentially undescribedspecies.

(2) Mitchelli-Japan-clade: The eastern North American S. mitch-elli is phylogenetically related to Japanese taxa, a result sug-gested by Cokendolpher (1984) based on comparativemorphology. All three species in the recovered mitchelli-Japan-clade lack male cheliceral glands, but differ in genitalmorphology. The relatively simple penis of S. mitchelli(Shear, 1975) differs from the three-dimensional glandespenis with distinct spination, found in the two Japanese spe-cies (Suzuki, 1974). For the nine described Japanese Sabaconspecies, Suzuki (1974) recognized the following three spe-cies groups based on external morphology (penis, ovipositor,armaments of male chelicera and palpal patella, scutum, andbody size): pygmaeus-group, dentipalpis-group, and akiyoshi-ensis-group. It is interesting that both S. akiyoshiensis of theakiyoshiensis-group, which is the smallest Japanese species(ca. 1.5 mm in male body length) and S. imamurai of thedentipalpis-group, which is the largest Japanese species (ca.4 mm), are placed in the same clade despite their large dif-ference in external morphology and body size. The phyloge-netic position of the pygmaeus-group, not sampled in thisstudy, remains to be determined. Also, there are Sabaconspecies from the Chinese Sichuan that appear morphologi-cally-allied to Japanese taxa (Martens, 1972; Tsurusaki andSong, 1993a, J.M. unpublished results); the phylogeneticposition of these taxa remains to be determined.

(3) Astoriensis-Asia-clade: This group is subdivided according togeographic origin. The western North American cladeincludes S. sheari and S. astoriensis. Cokendolpher (1984)considered S. sheari to be closely related to S. mitchelli basedupon external morphology, but this relationship is not sup-ported by our data. There is considerable sequence diver-gence between the Oregon/Washington versus Idahopopulations of S. astoriensis. Investigation of specimens fromthese disjunct areas reveals differences in male cheliceraeand penis morphology (pers. obs.), perhaps consistent withspecies-level differentiation. There remains uncertainty inthe correct phylogenetic placement of taxa in this clade. Dif-ferent analyses swapped the position of S. sheari (Figs. 2 and3), and basal relationships in the astoriensis-sheari-clade ofthe full dataset (Fig. 3) are partly unresolved. We also foundconsiderable rate change within this clade suggesting align-ment or systematic errors, which might be overcome byincorporating additional Asian taxa. Our current sparse sam-pling prevents strong conclusions regarding the phyloge-

netic structuring of mainland Asian taxa. According to ourresults and morphological evidence we argue that most ofthe Chinese and Nepalese divergence is relatively recent,but we acknowledge that this fauna is morphologically dis-parate, and that our current sample does not capture mostof this morphological divergence.

(4) Cavicolens-Europe-clade: Based on comparative morphology,Shear (1975) and Martens (1983) suggested a close relation-ship between S. cavicolens and European Sabacon, corrobo-rated in this study. Also, speciation in this clade is clearlyolder than the Pleistocene, consistent with the hypothesisof Martens (1983). Basal nodes within the group receive onlylow support, placing S. simoni closest to S. cavicolens.Strongly supported European relationships include S. para-doxus with S. viscayanus (as expected by Martens, 1983),and S. pasonianus with the Sabacon juvenile from the Cev-ennes in southern France (MCZDNA100711). These lattertaxa are not in geographic proximity and are geneticallydivergent. Sabacon cavicolens from eastern North Americaprobably comprises several cryptic species (estimated diver-gence mean between 2 and 12 Ma). Shear (1975) mentionedmorphological divergence within S. cavicolens, a matter thatrequires further investigation.

4.2. Historical biogeography

While the northern latitudes climate of the Eocene was moretropical, a sharp decrease in average global mean temperaturesat the Eocene–Oligocene boundary (�33 Ma; Zachos et al., 2001)marks the almost simultaneous intercontinental biogeographic ex-change observed in all Sabacon clades (Fig. 4). Most extant Sabacontaxa show preferences to permanently cool and moist habitats,even to include subtropical cloud forest taxa occurring in Nepal(some included in our sample; Martens, 1972, 1983). We hypoth-esize that the onset of global cooling enabled cryophilic Sabaconto disperse into more suitable habitats, and to diversify into themany lineages we see today, a causal connection also found forcold adapted snakes (Lynch, 2009) and salamanders (Vieiteset al., 2007; Zhang et al., 2008). Biotic conditions during connectionof the trans-Atlantic de Geer land bridge provide an illustrativeexample. At the Eocene–Oligocene boundary the climate is recon-structed as becoming more seasonal with cooler winters but mod-erate annual temperatures (Eldrett et al., 2009; Ivany et al., 2000).In Greenland, as part of the de Geer Bridge, these conditions in-cluded high annual precipitation promoting a humid coniferousforest (Eldrett et al., 2009). Modern humid coniferous forests houserich Sabacon faunas, as for example, in western North America.Fennoscandian Sabacon fossils from Baltic amber (Dunlop, 2006),also from the de Geer Bridge, most likely originated from Sciado-pityaceae (Wolfe et al., 2009), which together with other dominantconifers, went almost globally extinct in the late Oligocene. Thisclimatic and fossil evidence suggests high latitudinal dispersal con-ditions likely suitable for Sabacon. We hypothesize that globalcooling created large and maybe continuous suitable habitats, en-abling a general expansion of cryophilic Sabacon and connectinghabitat patches from the warmer Eocene.

Phylogenetic relationships within the cavicolens-Europe-cladeclearly favor Europe as the center of origin for this clade. A possiblescenario is that the complex and repeated fragmentation of Europe(Smith et al., 1994) caused early and deep divergence of EuropeanSabacon, while the trans-Atlantic de Geer land bridge facilitateddispersal to eastern North America. Further speciation within S.cavicolens is considerably younger and must have happened longafter the land bridge had been severed.

For the mitchelli-Japan-clade, ancestral areas are either Japan ora combination of Japan and eastern North America; the former fa-



vored by S-DIVA and the latter by Lagrange. Major events disrupt-ing a connection between Asia and eastern North America includethe uplift of the Rocky Mountains during the Eocene (45–36 Ma)followed by a sharp decrease in average temperatures during theOligocene (�33 Ma) to the present (Zachos et al., 2001). Whiledecreasing temperatures might have facilitated dispersal of coldadapted Sabacon, the increasing aridification of mid-continentalNorth America could have prohibited subsequent faunisticinterchange.

According to the MrBayes reconstruction of the full dataset(Fig. 3), the astoriensis-Asia-clade comprises two well supportedgeographic lineages, with western North American S. astoriensisand S. sheari as sister to the Asian Sabacon. The separating split,dating to 32 Ma (17–53 HPD) falls in the time frame for increasedfaunistic interchange between western Nearctic and eastern Pale-arctic (Sanmartín et al., 2001), while no recent Pleistocene ex-change is apparent. While our data does not allow an inferenceof dispersal directionality in this clade, high morphological diver-sity of recent Asian Sabacon indicates continental Asia as the po-tential source area.

Although some biogeographic patterns seen in Sabacon are alsofound in other taxa, Sabacon is peculiar in several respects. Forexample, one connection for which Donoghue and Smith (2004)recovered the highest proportion of disjunct distributions, Asiaand Europe, is completely absent from our biogeographic scenar-ios. Also, there is no apparent phylogenetic affinity between spe-cies in eastern versus western North America. Donoghue andSmith (2004) re-analyzed the animal data of Sanmartín et al.(2001) and found few animal disjuncts between eastern NorthAmerica and Europe (7%), with a low proportion (4%) of thesereconstructed dispersal events occurring from Europe to easternNorth America. The eastern North America to Japan connectionis rare for animal disjuncts and dispersal corridors (Breweret al., 2012; Donoghue and Smith, 2004; Sanmartín et al., 2001).Another peculiarity concerning the mitchelli-Japan-clade is theestimated divergence time for the geographical groups(mean = 32 Ma, 16–53 HPD). Xiang et al. (2000) dated splits be-tween eleven species pairs of plants, and found most estimatesin the last 10 Ma (max. 12.6 Ma) in the late Miocene and Pliocene.These estimates probably correspond to other Opiliones taxa, e.g.,Crosbycus dasycnemus (Shear, 1986), Acropsopilio boopis (Suzuki,1976), and two Caddo species (Suzuki, 1976; Shultz and Regier,2009) with disjunct distributions in Japan (and China for Crosby-cus; Tsurusaki and Song, 1993b) and eastern North America.Divergence in these harvestmen taxa is likely relatively young,as evidenced by morphological similarity and low genetic diver-gence (when estimated).

Our molecular clock-derived divergence date estimates differconsiderably from similar molecular clock estimates reported byGiribet et al. (2010). They estimated the split between S. cavico-lens and European Sabacon at �75 Ma, which greatly exceedsour estimates (mean of 33 Ma, 24–46 HPD). Similarly, the Giribetet al. (2010) estimate for the MRCA of Ceratolasma and Ischyrop-salis (�160 Ma) again exceeds our estimate (65 Ma, 31–109HPD), as does the overall age of the Ischyropsalidoidea (243 ver-sus 137 Ma). These differences may stem from the predominantlyancient calibration points used by Giribet et al. (2010), which maynot account for fluctuations along the calibrated regression andinfluence younger nodes (Conroy and van Tuinen, 2003; Rutsch-mann et al., 2007). Applying their root age of 243 Ma to our anal-ysis caused considerable increase in overall HPD ranges (resultsnot shown), and we therefore refrained from using this possiblecalibration. Ultimately, incorporation of additional reliable cali-bration points and more DNA sequence data will be needed to re-solve this discrepancy.

To summarize, the onset of diversification within the four majorSabacon clades, as well as intercontinental exchange in threeclades, is nearly simultaneous and seems connected to global cool-ing, allowing dispersal across northern land bridges. Our data sup-ports the perspective that the study of arthropods provides morefine-scaled views on Holarctic biogeography. For example, whilecryophilic Sabacon were able to survive in suitable microhabitats,the aforementioned coniferous megaflora went mostly extinctand is no longer available for molecular based reconstructions.

Acknowledgements

We thank Jared Grummer, Dean Leavitt, Casey Richart and Jor-dan Satler for help with the phylogenetic methods and for generaldiscussion. Richard Ree and Stephen Smith provided help with theLagrange analysis and methods used in Smith (2009). Fred Coyle,Shahan Derkarabetian, Lucia Labrada, Dean Leavitt, Steven Thomas,Robin Keith, Wolfgang Schawaller, Joachim Schmidt, Jim Starrett,Jordan Satler, Adrienne Richart, Dale Richart, Dan Richart, Casey Ri-chart and Peter Scott helped to collect specimens. Research effortsof M. McCormack were funded by the American ArachnologicalSociety, and a U.S. Fish and Wildlife Service contract to M.H.A.L.S. was funded by a postdoc grant of the Deutsche Forschungs-gemeinschaft (DFG) to work on systematics and biogeography ofHolarctic Opiliones, and collected Sabacon in the southwesternAlps with the help of an EDIT/ATBI+M travel Grant (2008). Field re-search in Asia was funded by Feldbausch Stiftung and Wagner Stif-tung, both at Fachbereich Biologie, Mainz University, as annualgrants to of J.M.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2012.10.001.

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