Table Of Content2015. The Journal ofArachnology 43:40-53
Molecular phylogeny, biogeographic history, and evolution of cave-dwelling taxa in the European
harvestman genus Ischyropsalis (Opillones: Dyspnoi)
Axel L, Schonhofer1-2 Cristiano Vernesi3 Jochen Martens2 and Marshal Heelin': 'Department ofBiology, San Diego State
University, San Di,ego, CA 92182-461,4, USA. E-mail: [email protected]; institute ofZoology, Johannes
Gutenberg University Mainz, D-55G99 Mainz, Germany; department of Biodiversity and Molecular Ecology, Centre
—
for Research and Innovation Fondazione Edmund Mach, San Michele all’Adige 38010 (TN), Italy
Abstract. We estimated a multigenic molecular phylogeny and reconstructed biogeographic history for the European
harvestman genus Ischyropsalis C.L. Koch 1839 (Dyspnoi). To reconstruct historical biogeographic patterns we
conducted an algorithmic VIP analysis which revealed patterns consistent with a vicariance-dominated history. The
existing morphology-based systematic framework for Ischyropsalis is mostly inconsistent with molecular phylogenetic
results, and a new informal system is established that recognizes three main clades and several sub-clades. Species-level
analyses revealed two non-monophyletic species (I. pyrenaea Simon 1872 and I. luteipes Simon 1872); subspecies of I.
pyrenaea are distant relatives, and are formally elevated to species (I. pyrenaeapyrenaea to I. pyrenaea and I. pyrenaea
alpinulato I. alpinula). A preference forcryophilicmicrohabitats hasfavored thediversification ofhigh-altitudeandcave-
dwellingIschyropsalisspecies; molecularphylogenetic data suggest thatcave-dwellingspecies haveevolvedmultiple times
independently.
Keywords: Species tree, multilocus phylogeny, biogeographic modelling, convergence, ecological specialization, cave
evolution
Modern biogeographic studies ofharvestmen have revealed Studies of Ischryopsalis classification began with Hadzi
an interesting mixture of evidence for extremely limited (1931), who delineated a single Ischyropsalis subgenus,
dispersal (e.g., Thomas & Hedin 2008; Derkarabetian et al. Odontopalpa, based on apophysis characters on the male
2011; Boyer & Reuter 2012), combined with evidence for palpal patella (Fig. 1). Originally containing three species, two
occasional long-distance dispersal (e.g., Shultz & Regier 2009; species were synonymised (Martens 1969b) with I. kollariC.L.
Sharma & Giribet 2012; Schonhofer et al. 2013). As such, Koch 1839 (I. bosnica Roewer 1914, 1, triglavenis Hadzi 1931),
harvestmen are compelling systems for biogeographic re- the type species of Ischyropsalis. As it was not considered in
search, but as expected many harvestmen taxa remain to be Odontopalpa, the subgenus then became invalid. Hadzi
investigated using modern phylogenetic and biogeographic originally also placed I. dentipalpis Canestrini 1872 and later
methods. Within the European fauna, members of the genus I ravasinii Hadzi 1942 within Odontopalpa but had no males
Ischyropsalis C.L. Koch 1839 are among the most charismatic to confirm themale-specificcharacters ofthis subgenus forthe
harvestmen, with relatively large body sizes and massively latter. Roewer (1950) described numerous new Ischyropsalis
enlarged chelicerae used to catch prey (Fig. 1). Ecologically, species, but was suspected to have separated specimens of
members of this genus are often rare and found in difficult- common species to describe new species. Martens (1969b)
to-access habitats, including several obligate cave-dwelling detected these manipulations, later substantiated by v.
species. Helversen and Martens (1972), and synonymized 33 of 34 of
Ischyropsalis species limits have been investigated in the Roewer’s species.
context of the biological species concept (Mayr 1942), rarely Morphology-based phylogenetic hypotheses for Ischyropsa-
explicitly applied within arachnids. Martens (1969a) observed lis were published nearly simultaneously by Martens (1969b)
Ischyropsalis males offering females a secretion from the basal and Dresco (1970). Dresco drewhisconclusionsfromtheParis
cheliceral segment during courtship and copulation, and Museum collection, containing mostly Iberian Ischyropsalis,
demonstrated an association between the secretion offering while he had limited access to and knowledge ofthe remaining
and subsequent contact between this secretion area and the European fauna. Martens (1969b) considered most currently-
female mouthparts (Martens 1969b). The male cheliceral accepted species, and provided an extensive revision based on
secretion area includes bristle fields (Fig. 1) that are connected characters newly developed for Ischyropsalis systematics. The
by minute pores to massive glands (Martens & Schawaller systems of Martens and Dresco only agree in the isolated
1977). Martens (1969b) made thischeliceral secretion area and position ofI. hellwigii (Panzer 1794) while other hypothesised
the resulting associated male/female interaction a fundamental groupings show little congruence (Fig. 2). Authors after 1970
character to delineate Ischyropsalis species, and also found did not comment on the phylogenetic structure of the genus,
corresponding male genital characteristics. The presence of but added new species or further clarified species synonymies.
such charactercombinations has also been adopted for species Martens (1978) refined his system for some Alpine species.
delimitation and higher-level classification in other Opiliones, Luque (1991, 1992) and Prieto (1990a, b) described the
for example in the Nemastomatidae (Schonhofer & Martens Spanish fauna in more detail. Most recently, Schonhofer and
2012), Dicranolasmatidae (Gruber 1998) and Sabaconidae Martens (2010a) and Luque and Labrada (2012) added two
(Martens 1972; Suzuki 1974). new species and discussed regional species diversity in the
40
—
SCHONHOFER ET AL.—ISCHYROPSALIS PHYLOGENY 41
Figure 1. Ischyropsalis habitus and morphological features, a) /. hellwigii hellwigii male habitus, Austria, Soboth; b) I. pyrenaeapyrenaea
female habitus, France, Grotte de 1’Estelas; c) distal part of penis, /. pyrenaea alpinula (Martens 1978; Fig. 381); d) distal part of penis, /.
dentipalpis (as I. Helvetica-, from Martens 1978: Fig. 382); e-g) different types of glans sclerites (groups sensu Martens, 1969b; Fig. 32): e) I.
hellwigiigroup, f) /. kollari group, g) I. dentipalpis group; h, i) male cheliceral armament, h) I. hellwigii hellwigii (Martens 1978: Fig. 289), i) /.
dentipalpis(Schonhofer& Martens2010a);j)apophysison palpal patellaofmaleI. dentipalpis(Schonhofer& Martens2010a; Fig. 6);k-m)distal
part ofmalechelicerae (medialview), apophysesandcheliceral secretion-extruding-field indicated bybristlefield: k) I. lithoclasica(Schonhofer&
Martens 2010a; Fig. 13), 1) I. adamii, m) I. hellwigii (both Martens 1969b; Fig. 22).
Western Alps and the Cantabrian Mountains, respectively. groups are strictly allopatric. This broad-scale phylogenetic
Twenty-two species-level taxa are currently recognized in the structure and the proposed species inter-relationships imply
genus (Schonhofer 2013a). repeated dispersal and isolation between the major mountain
Based on the revision of Martens (1969b), distributional systems in Western and Central Europe. On the other hand,
patterns in Ischyropsalis suggest a complex biogeographic individual Ischyropsalis species have generally narrow distri-
history. The three species groups of Martens (1969b) are butions, and at least nine species are obligate cave endemics.
widespread and overlap in distribution, while species within Cave-dwelling species are unlikely to disperse over large
—
1 1
42 THE JOURNAL OF ARACHNOLOGY
jiMMiMMiiMMiMiM HIM
muellneri i i i*
Phylum IV " mi Si
.iniiiiii magdalenae kollari-
3imiir* group
iiiiiiiiniiiiiiiiiiiiii dispar iimi i
noltei strandi
petiginosa hadzii
espagnoli kollari
ipyrenaeai
« inodifera i
Phylum VI dentipalpis
i \
kollari manicata
Phylum
III
luteipes
Phylum IV denti-
gigantea ravasinn
paipis-
ssttrraanncdni lithoclasica group
llllIllllll
Phylum II InIIIIIIIIIII11111IIII1 robusta :
nun
- «iiiiiiiiiiiiiiim iiiiii adamii in*
3 1111 i 1111»
I IiiiiiiiiiiiiiiiiI iiiiiii carli iiiiiii 11IV
I Phylum f 4i 11 1 hellwigii lucantei iiiiiii. hellwigii-group ~
111!IIIIIIMIIIIMIIIIIL 2 11 11 i i i i>
hellwigii hellwigii iiiiiii-
—
Figure 2. Morphology-based phylogenetic hypotheses for Ischyropsalis according to Dresco (1970, left) and Martens (1969b, right). Some
synonymies/misinterpretationsused by Drescowerereplacedforeaseofcomparison(weuseI. muellneriforI. hellwigii,I. hellwigiihellwigiiforI.
taunica, /. adamii for /. apuanus, /. kollari for /. trigalvensis and I. hellwigii lucantei for I. superba). Phylogenetic lineages are compared and
matched iftheir taxa composition shows at least two matches agreed upon by both authors; matching taxa are indicated on dashed branches;
taxa corresponding to alternative lineages are indicated on black branches; taxa considered by only one author are designated in grey. Non-
matching species are separated by space between the species names. Species ofsubgenus Odontopalpa sensu Hadzi (1931) shown in italics.
distances, and may have originated from widespread epigean Martens (1969b. 1978), Luque (1991, 1992) and Schonhofer
species (Porter 2007). and Martens (2010a), or provided by C. Prieto for Spanish
We present here the first molecular phylogenetic analysis of samples. A few specimens were raised to adulthood incaptivity
Ischyropsalis to investigate the validity of alternative higher- to allow morphological species identification. We included as
level classification schemes as well as multiple species and many described species as possible, and conducted intraspecific
subspecies hypotheses (Martens 1969b; Dresco 1970). This samplingtoincludegeographicallyandmorphologicallydistinct
molecular phylogenetic framework also allows us to address lineages. Voucher specimen details and repository information
the phylogenetic value of currently used morphological are provided in the Appendix. Voucher specimens (with
—
characters because of their relatively simple organization associated collection numbers) are deposited in the collections
and structure, Martens (1969b) suggested the possibility of ofJ. Martens(CJM. Mainz, Germany), C. Prieto (CCP, Bilbao,
morphological convergence in male genitalia ofIschyropsalis. Spain), A. Schonhofer (AXLS, Mainz, Germany—), C. Vernesi
The distribution of all Ischyropsalis taxa is reconstructed (FEM: Centre for Research and Innovation Fondazione
based upon literature, museum collections and personal Edmund Mach, San Michele all’Adige, Italy) and the San
collection data, and these data are used in algorithmic Diego State University Terrestrial Arthropod collection (OP).
analyses to reconstruct Ischyropsalis biogeographic history. Molecular methods and analyses. Genomic DNA was
Finally, molecular phylogenetic results provide insight into extracted from tissue using the Qiagen DNeasy kit (Valencia,
whethercave-dwellingtaxa originate from a singleancestor, or CA, USA). The polymerase chain reaction (PCR) was used to
have evolved independently multiple times. amplify the following gene fragments: 28S rRNA (28S), using
METHODS the primers ZXlelong (Schonhoferet al. 2013), ZX1, and ZR2
(Mallatt & Sullivan 1998); mitochondrial CytochromeOxidase
Sampling. —Ischyropsalis specimens were collected by hand, (COl), using C1-J-1718SPIDERA and C1-N-2776SPIDER
1
mainly during our own field excursions. Some specimens were (Vink et al. 2005); and nuclear Elongation Factor 1-alpha
also generously donated by colleagues (see Acknowledg- (EFla), using OP2BSAB and OPRC4 (Hedin et al. 2010).
ments). Specimens were preserved in 100% EtOH, with the PCR protocols followed Hedin et al. (2010) for EFloc, Thomas
majority being collected in 80% and transferred soon and Hedin (2008) for COl, and Hedin and Thomas (2010) for
thereafter. Morphological identifications were based on 28S, the latter using an annealing temperature of 56°C.
SCHONHOFER ET AL. -ISCHYROPSALIS PHYLOGENY 43
Amplicons were purified on Millipore plates and directly Biogeographic analysis.— We compiled geographic distribu-
Sanger sequenced at Macrogen USA in both directions. tion data for all known Ischyropsalis taxa. A total of 1,360
SEQUENCHER v4.5 was used to assemble and edit sequence distribution records were extracted from literature, museum
contigs, with ambiguous sites scored using standard ambiguity and personal collections, of which 1,220 could be georefer-
codes. enced at a minimum accuracy of two digital degrees. Over
COl and EFla exon sequences were aligned manually in 1,100 coordinates matched species and geographic lineages in
Mega 4.0 (Tamura et al. 2007) using amino acid translation, our dataset and were used for biogeographic reconstruction.
while 28S and EFla intron sequences were aligned with An interactive map with al! referenced localities is available
MAFFT vers. 6 (http://mafft.cbrc.jp/alignment/software/), online (Schonhofer 2013b).
using the Q-INS-i strategy as recommended by Katoh and Applying the extant distribution of species and the species
Toh (2008). To account for alignment uncertainty in the 28S tree derived from *BEAST analyses, the Vicariance Inference
and the EFla intron data, outgroup genera were removed and Program (VIP, Arias 2010; Arias et al. 201 1) was used to infer
the data were realigned using the same strategies as above. the distribution of clades and distributional barriers. This
Models of DNA sequence evolution were evaluated using program reconstructs the probability ofdistributional barriers
jModelTest 0.1.1 (Posada 2008) under three substitution using parsimony. For area reconstruction in VIP the grid was
schemes (JC, HKY, GTR) on a fixed BIONJ tree, allowing set to 0.3 without fill. To account for misidentifications, a
for unequal base frequencies and among-she rate variation. maximum area overlap of5% was allowed between species. A
Final model selection was based on the Akaike Information heuristic search was conducted using 1000 iterations, setting
Criterion (AIC) and individual models were applied to full sector search and otherwise using default settings.
respective partitions in all downstream phylogenetic analyses. RESULTS
Bayesian inference using MrBayes v3.2.0 (Huelsenbeck & —
Ronquist2001; Ronquistetal. 2011)wasapplied tosinglegenes Molecular phylogeny. A total of39 Ischyropsalisspecimens
as well as concatenated datasets. DNA sequences were were included in molecular phylogenetic analyses, with
partitioned by gene, EFla by exon and intron, and CO and samples available for all described subspecies, and 18 of 22
EFlaexons by codon position. Bayesian analyses were ruIn for currently described species. Previously published sequences
co5ofmnivslpellriitgoennfgcreeenqeudreianatcgiinoeonssst,ihcwashdewrederreoinpspaatlelidscfaibseedeslfotowhrea0ls.lt01asnadmaapnrlddeddsetvpaianardtaaimro-dn wS1ec8rh7eo2nihanoncfdleurdIe.d&nfordMiofamerrGtaeennSsBiamn2o0kn10fb1o;r87I9.Gil(ruSithebuiepltetsz,etI&. raloR.heugs2it0ea1r0S;2i0m0so1en;e
eters (ESS >200, PSRF = 1.00; Ronquist et al. 2011). Analyses lApapsemnadiGxo)o.dnOiugthgtro&upGsoeoqduneingchets f1o9r42theansdistAecrugcleanveerlalaCeSrhaetaor-
wererepeated tofurthercheckforconvergence.Thefirst40%of 1986 were obtained from Richart & Hedin (2013).
trees were discarded as burn-in, with remaining trees used to The protein alignments of COl and the EFla exon
wmreeacrxoenismitnrutumecrtpraleitmkeeadlxiahisomodupomst(ceMlraLido)ercprcreoodnbicbaaiblitiletinytaitteresede.(pSptpr)leietoffurcseliqanudgeensc.itehAse ceEtoFnlatlaa.ipn2ae0rd1a3ln)o,ogsgwaehpasvfeaonubdnedennondosettoeepvcitcdeoeddnoicnnesm.fiotWreehpaanrroatvleeostgthsmaetinnal(ItCshlcoohuuygsrhe-
pthaertirtaixomnilnGgUIsc(hSetmaemaotuatkliisne2d006a;boSvielvewsatrso r&ecMoincsthraulcatked20w1i2t)h, opsalis. PCR products directly Sanger sequenced showed no
evidence for multiple overlapping sequences as would be
with support assessed using 1000 rapid bootstrap replications. expected if paralogs were common. However, this result is
As an alternative to concatenation, a species tree was potentially biased by the PCR primers that we utilized. As
rmeecnotnesdtriunct*edBEusAiSnTg t(hHeelmeudlt&ispDecriuesmmcooanldesc2e0n1t0;moDdreulmimmoplned- osufcoht,hwereiaslcshorsyeoaprscahlieddofiodrtEaFxlaa(Hpeadrialnoegtsailn. t2h0e12t;raunnspcurbilpitsohmeeds
et al. 2012). Models of molecular evolution suggested by data). Specifically, we blasted Ischyropsalis EFla sequences (I.
jModelTest were implemented, with codon partitioning carli Lessert 1905, AXLS 145) against the transcriptomes of
applied to COl and EFla exons ((1+2), 3). Priors were set Sabacon Simon 1879, Acuclavella and Hesperonemastoma
to gamma if modified, otherwise default priors were used. Gruber 1970 (tblastx, e value le-20) in Geneious 7.0.6. These
Analyses were run until ESS values exceeded 200 for most searches recovered a single EFla sequence for each of these
priors, which was after 200,000,000 generations. Analyses ischryopsalidoid taxa, suggesting a lack of paralogy in this
were replicated twice and checked for convergence using clade. We emphasize that Opiliones is an old taxon with
Tracer vl.5 (Rambaut & Drummond 2007). ancient internal divergences (Hedin et al. 2012), and that gene
Species trees were estimated using two different sets of duplication in one lineage does not imply similar molecular
operational taxonomic units (OTUs). The first set included evolutionary patterns in other, distantly related taxa. Similar
only described and accepted species and subspecies {— dynamics of EFla paralogy gain and loss are observed, for
conservative species set), while the second set was extended example, in insects (Djernaes & Damgaard 2006).
to include additional genetic lineages within the species /. Models of sequence evolution were selected as follows:
adamii Canestrini 1873, I. dentipalpis and I. luteipes Simon 28SrRNA: GTR+I+G; COl codon position 1: SYM+G; COl
1872 recovered in concatenated analyses (= diversified species codon position 2: F81+G; COl codon position 3: GTR+G;
set). Preference for one of these OTU settings was evaluated EFla codon position 1: HKY+I+G; EFla codon position 2:
by comparing marginal likelihoods using Bayes Factor HKY; EFla codon position 3: GTR+G; EFla intron:
analysis (Grummer et al. 2014), as implemented in Tracer HKY+G. In *BEAST settings were modified for EFIa-exon
vl.5 (Rambaut & Drummond 2007). to SYM+I+G (in *BEAST set to TN93+I+G, base frequencies
44 THE JOURNAL OF ARACHNOLOGY
equal) and for COl to GTR+I+G (same in *BEAST), both group arenot strictly allopatric but overlap in manycases, and
separated by codon. in the Iberian distributional area at least two sympatric sister
Ischyropsalis is clearly monophyletic with respect to the species are present.
North American Acuclavella and Ceratolasma allowing
, DISCUSSION
removal of these outgroups for further reconstructions.
—
Results of the concatenated Bayesian and ML analyses, both Higher-level phvlogeny. Comparing phylogenetic hypoth-
with and without outgroups, were generally congruent eses (Fig. 2) of Martens (1969b) and Dresco (1970) with
(Fig. 3), with the few topological differences observed at molecular-based concatenated (Fig. 3) or *BEAST trees
weakly supported nodes. Alternative topologies recovered (Fig. 4) reveals little congruence. These alternative hypotheses
different arrangements between Ischyropsalis litlioclasica agree only in the separate (Martens 1969b; Dresco 1970) and
Schonhofer & Martens 2010a, 7. kollari and 7. ravasinii, and basal placement (Martens 1969b) of the 7. hellwigii group.
grouped I. navarrensis Roewer 1950 with I. robusta in the ML Further similarities only partly coincide with para- and
analyses. The same was true for the position of terminals polyphyletic groups, except for Hadzi’s Odontopalpa that
,
within the two I. luteipes clades and I. carli (Fig. 3). receives weak support in our analyses. Hadzi (1931) included
Bayesian and ML molecular phylogenetic results indicate the species 7. kollari (sub 7. triglavensis) and 7. dentipalpis
that Ischyropsalis is divided into three primary clades, defined (presently split into 7. lithoclasica and /. dentipalpis sensu
here for further discussion: 1) the 7. hellwigii group (compris- stricto; Schonhofer & Martens 2010a) in this subgenus and
ing the two described subspecies), 2) the I. manicata group assumed 7. ravasinii to be included, although he had no males
(including I. adamii, I. carli, I. manicata L. Koch 1869 and I. to confirm this placement. Martens (1969b) considered the
pyrenaea alpinula Martens 1978), and 3) the Iberian-Alpine genus Ischyropsalis too small to justify establishment of
group, including all remaining sampled species. The compo- subgenera. Accepting Odontopalpa would require the defini-
sition of these groups is further detailed in the Discussion. tion ofseveral ad—ditional subgenera, which he felt carried little
Molecular phylogenetic support for theseclades is strong (e.g., taxonomic value we generally agree with this recommenda-
Bayesian posterior probability values > 0.95), although tion.
slightly lower for the I. manicata group, which includes several While the detailed studies of previous authors refined the
species that are missing data for multiple genes (Appendix). characters for species-level taxonomy, higher-level groups (as
Bayesian and ML concatenated analyses (Fig. 3) recovered implied by molecular phylogeny) now lack morphologically
most of the currently accepted species as well-supported diagnostic characters. This situation is similar to other
lineages, with some exceptions. The subspecies ofIschyropsalis harvestmen groups, for example Trogu/us Latreille 1802,
pyrenaea are not recovered as monophyletic. but instead fall where characters delineating species appear clearer than those
into different higher-level groups. Ischyropsalis luteipes was characterizing higher-level groups (Schonhofer & Martens
found to be paraphyletic with 7. pyrenaea pyrenaea nested 2010b). The molecular results for Ischyropsalis suggest that
most of the presently used morphological systematic charac-
within 7. luteipes. Intraspecific genetic divergences were
generally low, except for 7. adamii and 7. dentipalpis, both ters are randomly distributed throughout the phylogeny. Even
male-specific characters of the genitalia and cheliceral
including divergent genetic lineages. Distinct lineages within
the non-monophyletic or internally-divergent taxa were apophyses seem problematic for use as phylogenetic charac-
defined as distinct OTUs in the *BEAST analysis of the ters. This is not completely unexpected as Martens (1969b)
diversified species set, and all received strong support (Fig. 4). mentioned the relatively simple organization of the penis in
However, the Bayes Factor analysis did not conclusively favor Ischyropsalis, perhaps prone to homoplasy or morphological
one species-set hypothesis over the alternative hypothesis (In stasis.
Bayes Factor: 3.635). Character conflict is well-illustrated by the “subspecies” of
Biogeographic analysis.—VIP results are visualised in 7. pyrenaea, previously believed to be closely related based on
morphology, but apparently belonging to different groups
Figs. 5 and 6, including distributions of all higher clades
(Fig. 5) and the Iberian-Alpine-group (Fig. 6). VIP recognized within Ischyropsalis (Fig. 3). The proposed shared characters
thirteen barriers corresponding to phylogenetic splits. Barriers ofthe 7. pyrenaea subspecies (Martens 1969b, 1978), including
cheliceral spination and apophyses, the male cheliceral bristle
were found in consistent positions over all reconstructions,
except for a few cases when individual VIP reconstructions field and the overall similarity ofthe male genital morphology
dropped taxa, which shifted the barrier in one direction (e.g., un-lobed and keeled sclerites of the penial glans), are
likely convergent. Indeed, the structure ofthesesclerites differs
towards one of the included groups but never changed its
considerably in detail, particularly in the connection towards
directionality or general position.
Biogeographic analyses indicate few putative long-range the sclerotised part ofthe penial stylus (Fig. 1). In 7. pyrenaea
dispersal events between different European mountain systems pyrenaea this sclerite is rather broad, while in 7. pyrenaea
within the 7. hellwigii and 7. manicata groups. The Iberian- alpAinnulaoivteirsvieexwtremoeflychnaarrarcotwe,rsappfeoarridnigaganlomsoisntgpetrieocloatvee.red
Alpine group showed a general pattern of diversification
molecular clades is given below. Ischyropsalis hadzii Roewer
cdoeicnrceiadsiinngg dwiistthribtuhteiodnivairseiaosnwoifthadnceecsrteraaslinagrepahsy,lorgeesnuelttiicngagien c1a9n5t0a,b7r.icgaigLanutqeuaeDr&esLcaobr1a96d8a,27.01p2e,tingiontosiancSliumdeodni1n91o3uranddat7a.
of the clades, with evidence for long-range dispersal generally
set, are also assigned tentatively to these larger groups.
absent. Thus geographic proximity generally coincides with
closer phylogenetic relationship within the Iberian-Alpine I) 7. hellwigii group (two described subspecies 7. h. hellwigii,
group (Fig. 6). Species distributions within the Iberian-Alpine 7. h. lucantei Simon 1879): Chelicerae massive, males with
SCHONHOFER ET AL—ISCHYROPSALIS PHYLOGENY 45
.
0,94(0.94)/86(90J lithoclasicaAXLS409, Italy, Roncobello
(-) lithoclasicaAXLS429, Italy, Stalle Moeschel
* i— ravasinii 143, Italy, Treviso, Cansiglio
Odontopalpa sensu Hadzi, 1931 ravasinii58, Italy, Treviso, Cansiglio
%X
kollariAXLS296, Austria, Koralpe
kollariAXLS914, Austria, Tschiernock
0,92(-)l57(68)
*i dentipalpisCJM5918, Italy, Aosta
dentipalpisAXLS1187, Switzerland
dentipalpis,CJM5920, Italy, Torino
strandi22, Italy, Verona, S. Anna dAlfaedo
strandi25, Italy, Verona, Camposilvano
strandi63, Italy, Verona, Monte Baldo
muellneriCJM6752, Italy, Villanova
luteipesAXLS645, France, Col de Port
-(-)i-(95) I
luteipesAXLS707, France, Donzenac
Pyrenean clade
% luteipes shultzreg, no locality
pyrenaeapyrenaea AXLS620, France, I'Estelas
pyrenaeapyrenaea AXLS655, France, Betharram
luteipesAXLS549, France, Lamanere
luteipes MCZDNA100705, Spain, Nuria
luteipesCJM5336, France, Taulis
Cantabrian clade
—
*i disparCCP4188, Spain, Berberana
magdalenae CCP4568, Spain, Galmades
0.97(1)169(100) nodifera CJM5570, Spain, Urkiola
navarrensisCCPIn, Spain, Iribas
robusta CJM5474, Portugal, Sintres
carli72, Italy, Vercelli, Valle Cervo
carli69, Italy, Vercelli, Borgosesia
carliAXLS145, Switzerland, Lungern
L carliCJM6251 Switzerland, Speikkogel
Ischyropsalis ,
manicataAXLS1188, Romania, Closani
pyrenaea alpinula AXLS3, Italy, Cuneo, Gr. dell'Qmo
pyrenaea alpinula CJM6377, Italy, Cuneo, Balmoura
adamii38, Italy, Roma, Vallepietera
adamiiCMI, Italy, Roma, Bellegra
adamii41, Italy, Salerno, Valle dell'Angelo
adamii44, Italy, Salerno, Campania Rofrano
hellwigiihellwigiiAXLS240, Slovenia, Olseva hellwigii
-group
chellwigiilucanteiAXLS654, France, Gr. Betharram
Acuclavella merickeliOP2237, USA
0.04 C Acuclavella sheariOP2720, USA
—
Acuclavella makah OP2715, USA
Ceratolasma tricantha USA
,
—
Figure 3. Bayesian consensus phylogram based on concatenated molecular data. Numbers at nodes show support values resulting from
different analyses: 1) Bayesian posterior probabilities; numbers in parentheses are Bayesian posterior probabilities after removal ofoutgroup
genera and realignment of28S and EF13 intron, 2) maximum likelihood bootstrap values (in italics); in parentheses after removal ofoutgroup
and realignment of28S and EFloc intron. Nodeswith pp = 1.00 and bootstrap values >94 indicated by asterisks. Support values below0.94 pp
and 70 bootstrap not shown.
46 THE JOURNAL OF ARACHNOLOGY
-Okollari
0.59
Hi lithoclasica
0.81 -m ravasiniica
dentipalpis-N Q)
j
—
—B dentipalpis-S\N
Cftl
O
muellneri Q)
S
Iberian- strand's
Alpine
luteipes-NW
group
a pyr. pyrenaea ra
B luteipes-E
0.99 magdalenae
[71
disparQ]
a nodifera
a robusta
Q
0.68
-7navarrensis
B
0.88 carli
0.84 B manicata
manicata B pyr. alpinula Q)
group
adamii-C
B adamii-S
hel. hellwigii
hellwigii
group hel. lucantei
A. sheari
A. merickeli
A. makah
HI-
Ceratolasma
—
Figure 4. *BEASTspeciestree. Basal nodesgraphicallyshortened toallowexpanded viewofingroup. Posteriorprobabilitiesgiven at nodes;
pp of 1.00 indicated by an asterisk. Distribution ofspecies group taxa according to Martens (1969b) is symbolized at tips: dentipalpis group
indicated by black squares, kollari group indicated by open diamonds. /. lithoclasica was formerly considered 1. dentipalpis, /. dentipalpis was
considered I. Helvetica (Martens 1969b; 1978), and /. pyrenaea alpinula was described as a subspecies by Martens (1978). Predominantly cave-
dwelling species indicated by a cave icon.
a large dorsal cheliceral glandular bristle field. Sclerite of The following groups share long bristles on the penial
penial gland small, with irregularly spaced small spines. glandes:
Ischyropsalis li. lucantei is restricted to the central II) /. manicata group (I. adamii, /. carli, I. manicata, I.
Pyrenees, while I. h. hellwigii occupies a large area from pyrenaea alpinula)-. Penial sclerites narrow-stalked,
Germany to Southern Poland. appearing almost narrow leaf- to broad heart-shaped.
SCHONHOFER ET AL.—ISCHYROPSALIS PHYLOGENY 47
—
Figure 5. Distribution ofprimary Ischyropsalis groups in Europe, with VIP barriers shown as lines ofcrosses. Major groups include the /.
helhvigii (green squares), I. manicata (yellow triangles), and Iberian-Alpine (red circles) groups.
Sclerites always keeled and never bilobed, males with Illb) Iberian-clade (including two subclades): many
small distinctly circumscribed distolateral chelieeral species have keeled penial sclerites, otherwise
glandular bristle field. Individual species have a rather sclerites are always deeply bi-lobed. Males have
widespread distribution. Two species in the western large triangular dorsodistal chelieeral apophyses.
Alps (I. carli, /. pyrenaea alpinula), and one species in Members of the Pyrenean clade (I. luteipes [two
the Carpathians (I. mcmicata) and Apennines (/. lineages], I. pyrenaea pyrenaea) share no perceiv-
adamii), respectively; in the latter two areas these able characters, which highlights that most ob-
represent the only Ischyropsalis taxa known. servedcharacterstatescan evolve rapidly. Uniting
Ill) Iberian-Alpine group: Including the remaining species. features include distribution in the Pyrenees and
Penial sclerites broad-stalked or not narrowed towards molecular synapomorphies. The Cantabrian clade
intersection with stylus. Otherwise morphologically (/. dispar Simon 1872, I. magdalenae Simon 1881,
heterogeneous and best separated by geographic I. navarrensis, I. nodifera, I. rohustci, I. petiginosa,
distribution. likely /. cantahrica and I. gigantea) has a primary
Ilia) Alpine-clade: the group is separated into two uniting character, the bristle field on the basal
morphologically separable clades that share few chelieeral segment, situated on the distomedial
characteristics, maybe the bilobed penial sclerite, side. The chelieeral apophysis is generally very
which is not split in I. dentipalpis. Most species of large, often triangular and pointed, viewed later-
tluihnteihqocufliearsstipcaatg,erlIo.luamrpuela(lpI.noeprhdieynsHtiaispmaalopnnins,t1h8/e.98)mkaoellxlehairbipi,atl/pa. isalpnlecycl.iueTdshiniagsndgsrtmououdrpyeiorsefifnimnneoedreegdeoopgforpafuuplrhatithcieorannsea,vlaylsmueiasst.siionng,
—
(missing in I. hadzii that likely belongs to this Species-level systematics. Our molecular phylogenetic re-
group as well) and have a large plateau-like sults fail to recover two described species as monophyletic, or
bristle-field with sparse and scattered setation on recover species as monophyletic but with high internal genetic
the basal chelieeral segment (also in /. hadzii). divergence, perhaps consistent with cryptic or incipient
Members ofthe second group within this clade (I. specialion. As previously discussed the two subspecies of
ravasinii and /. strandi Kratochvil 1936) lack the Ischyropsalis pyrenaea are distantly related (Figs. 3 & 4).
palpal apophyses and bristle field and apophyses Considering their phylogenetic distance, they are hereby
are reduced or missing. Both groups are distrib- formally elevated to species (I. pyrenaea pyrenaea to I.
uted mainly in the Central to Eastern Alps, with pyrenaea and I. pyrenaea alpinula to /. alpinula). A careful
only I. dentipalpis in the Western Alps. redescription is warranted and may reveal further phyloge-
48 THE JOURNAL OF ARACHNOLOGY
Germany
France
-Wj
Austria
kollart-lithociasica+ra
Switzerland
Slovenia.
xxxx-x;^ Croatia
^tmindlS
dentipafpis standi-muellneri
wmm—m
France
robusta- restofCantabrian Clade
pymnaea
IkkpmjM
luteipes-Boutheast
Portugal
Figure 6. -a. Distribution of the Ischyropsalis Alpine clade in the Alps showing biogeographic reconstructions within major clades;
b. Distribution map ofthe Ischyropsalis Iberian cladeon the northern Iberian Peninsula and southern France. Thedistribution ofmicroendemic
species in the Cantabrian Mountains ofnorthern Spain was not further resolved. For both a & b, colors correspond to individual species and
lineages as supported by *BEAST and other analyses, and VIP barriers are shown as lines ofcrosses. Question marks indicate populations or
species where phylogenetic information is lacking.
SCHONHOFER ET AL.—ISCHYROPSALIS PHYLOGENY 49
netically useful characters. Also in need offurther study is the opsalis, which may be considered extreme ecological special-
paraphyletic I. luteipes, with the morphologically different I ists. Molecular phylogenetic trees include no large clades of
pyrenaea nested within this lineage (Fig. 3). Dresco (1970) exclusively cave-dwelling taxa (Fig. 4). Rather, it appears that
discussed three “ecoforms” ofI. luteipes but did not formally cave species have evolved multiple times in clades of mainly
describe these or provide characters for delineation. Character epigean species, a pattern reported from many other cave
reinvestigation and additional comprehensive geographic radiations (Porter2007), including harvestmen (Derkarabetian
sampling is required. et al. 2010; Hedin & Thomas 2010; Derkarabetian & Hedin
The central and southern lineages of the Italian Ischyr- 2014). However, the degree of cave specialization in Ischyr-
opsalis adamii are genetically clearly separable (Fig. 3), while opsalis is variable, with many surface-dwelling species
material from the isolated northern population was not regularly entering caves. Caves are the only available habitats
available for this study. Individual names for ail three at low altitudes, with surface habitats such as alpine gravel or
populations have been proposed by earlier authors and forests increasing towards high altitudes. Fully troglomorphic
require reassessment (southern Italy: I. adamii; northern Italy: taxa are rare, as most species retain eyes. The tendency
I. apuanus Caporiacco 1930; central Italy: I. apuanus nanus towards troglomorphism correlates with a preference for
Dresco 1968). Similarly, our data indicate that the southwest- cooler and more stable temperatures. For example, tempera-
ern-most population of I. dentipalpis is genetically distinct. ture preferences for the surface-dwelling I. luteipes were
This species has been recently re-evaluated based on mor- recorded at 11.5 °C with a range of 10 °C (Juberthie 1964).
phology (Schonhofer& Martens 2010a), and further studies of In the troglomorphic I. pyrenaea pyrenaea the mean temper-
its fragmented distribution were suggested. Our results ature is comparable, but the range does not exceed 5 °C. A
indicate differentiation within I. dentipalpis but refute a close similar preference range is exhibited by eastern Alpine cave
relationship to /. lithoclasica as hypothesized by Schonhofer endemics but with mean preferences shifted to even lower
and Martens (2010a). temperatures (I. strandi: 3.5-7.2 °C; I. muellneri: 5.2-7.8 °C;
Historical biogeography.—While Martens (1969b) assumed Juberthie 1964). The frequent evolution ofcave specialization
widespread species groups with strictly ailopatric species, our enables the regional co-occurrence of epigean and predomi-
phylogenetic results indicate a clearly different pattern. nantly troglomorphic species. The preference for low temper-
Herein, diversification and speciation is generally restricted atures might also explain the general absence ofIschyropsalis
to the same mountain range, specifically the Alps, the in more southern cavesystems as on the Balkan, as thesecaves
generally show higher mean temperatures.
Cantabrian Mountains, the Pyrenees, and, to a lesser extent,
the Apennines. Only in the monotypic Ischyropsalis hellwigii Members ofthe Iberian-Alpine group and all cave endemic
and I. manicata groups are species and lineages distantly species may be vulnerable to climate change given their
geographically restricted ranges and narrow ecological pref-
ailopatric, consistent with old vicariance or long-range
dispersal (Fig. 5). The remaining Ischyropsalis species belong- erences. Considering these factors, it will be important to
ing to the Iberian-Alpine group are frequently sympatric, protect relevant cave habitats as comprehensively as possible.
sometimes with completely overlapping distributions of ACKNOWLEDGMENTS
cblioosgeeloygrraeplhatiecdpattatxea,rnew.ig.t,hiIn tlhueteIibpeersiaann-dAlpI.inpeyrgernoauepa.shTohwes We are grateful to all colleagues who provided material for
molecular studies, including Siegfried Huber (Unteruhldingen,
a general trend ofdecreasing distribution area with decreasing
Germany), Marco Isaia (Turin, Italy), Enrico Lana (Verolengo,
taxonomic level, with the distribution of descendants con-
Italy), Tone Novak (Slovenj Gradec, Slovenia), Rodica Plaiasu
tained within the distributional area of reconstructed ances-
(Bucharest, Romania), Matteo Girardi (FEM, Trento, Italy)
tors. Therefore, dispersal seems not to be the main cause of
Claudio Chemini (Trento, Italy), Leonardo Latella (Verona,
diversification within Ischyropsalis which remains difficult to
, Italy),Valeria Lencioni(Trento, Italy)andCarlosPrieto(Bilbao,
test without reliably assignable fossils. The lack of Ischyr- Spain). Clemens Brandstaedter(Buers, Austria)and theGerman
opsalis fossils also means that we cannot rule out the presence =
Spider-Forum (http://spinnen-forum.de/smf/index.php7board
of now extinct representatives in areas outside the known
3.0) are thanked for their help with reconstructing problematic
distribution of the genus.
— localities. Casey Richart (San Diego, USA) provided outgroup
Evolution ofhabitat preferences. Most extant Ischyropsalis
sequences. Salvador Arias (Tucuman, Argentina) provided help
species are confined to montane habitats, preferring micro- with running the VIP analysis. A.L.S. was funded by a
habitats with moderate to cold temperatures and high and
postdoctoral grant of the Deutsche Forschungsgemeinschaft
constant humidity. On the Iberian Peninsula Ischyropsalis (DFG) to work on systematics and biogeography of Holarctic
species are confined to mountain regions situated along the Opiliones. J.M. was regularly sponsored by Feldbausch Foun-
northern and western coast, characterised by high and dation and Wagner Foundation, both at Fachbereich Biologic,
consistent rainfall and moderate variation in annual temper- Mainz University to carry out field work. Molecular research in
atures (Immerzeel et al. 2009). Precipitation in the Alps is the Hedin lab was supported by a California State University
generally high, and low temperature microhabitats are Program for Education and Research in Biotechnology (CSU-
abundant at high altitudes in this mountain chain. PERB) grant to M. Hedin. Comments of two anonymous
ItisnoticeablethatIschyropsalisshows ageneral trend from reviewers helped to improve the manuscript.
more generalist and widespread species (I. hellwigii and /.
manicata groups) towards specialized short-range endemics LITERATURE CITED
(most members of the Iberian-Alpine group). This trend Arias,J.S.2010.VIP.Onlineathttp://www.zmuc.dk/public/phylogeny/
manifests in the high number of cave endemics in Ischyr- vip/(last accessed 03.12.2014).
