Phylogenetic studies on European members of the family Scutoverticidae (Acari, Oribatida) and aspects to the character evolution within “Higher Oribatida“

Dissertation

Zur Erlangung des akademischen Grades Dr. rer. nat.

an der Naturwissenschaftlichen Fakultät der Karl-Franzens Universität Graz

vorgelegt von Mag. Sylvia Schäffer

Supervisor: Univ. Prof. Mag. Dr. Christian Sturmbauer Co-supervisor: ORat Dr. Günther Krisper

Graz, 2010

“A theory is something nobody believes, except the person who made it. An experiment is something everybody believes, except the person who made it.”

Albert Einstein

Science is wonderfully equipped to answer the question "How?" but it gets terribly confused when you ask the question "Why?"

Erwin Chargaff Many Thanks To…

Univ. Prof. Mag. Dr. Christian Sturmbauer

for his good supervision of my thesis ORat Dr. Günther Krisper for involving me in the FWF-project, sparking my interest in our “sweet “, little mites and being a

good supervisor Dr. Stephan Koblmüller for showing me the manifold fields in molecular phylogeny and helping me in all kinds of genetic

and writing problems Priv. Doz. Dr. Kristina Sefc & Priv. Doz. Dr. Steven Weiss

for support and advices my acarine working group

especially Dr. Tobias Pfingstl for the good cooperation in the project all my colleagues and friends here at the institute

in particular Dr. Kathrin Winkler, Mag. Michaela Maderbacher, Mag. Lisbeth Postl and Mag.

Julia Jagersbacher-Baumann for our common hours not only at the institute all of my closest friends

especially, I want to thank Dipl. Päd. Stefanie Pansi with the words „Amicitia vincit horas“ Ewald

for being by my side my parents

for supporting me in all kinds of things Contents

Summary ...... 1 Zusammenfassung ...... 4 Publications ...... 8

CHAPTER 1 General Introduction Oribatid mites ...... 10 Molecular genetic as tool for evolutionary questions...... 13 Objectives of the present study...... 15 References ...... 19

CHAPTER 2 Description of Scutovertex pileatus sp. nov. (Acari, Oribatida, Scutoverticidae) and molecular phylogenetic investigation of congeneric species in Austria Abstract...... 26 Introduction ...... 27 Material and Methods...... 27 Results ...... 30 Discussion...... 40 References ...... 43

CHAPTER 3 Contrasting mitochondrial DNA diversity estimates in Austrian Scutovertex minutus and S. sculptus (Acari, Oribatida, Brachypylina, Scutoverticidae) Abstract...... 46 Introduction ...... 47 Material and Methods...... 48 Results ...... 54 Discussion...... 58 Conclusions ...... 62 Appendix ...... 63 References ...... 63

CHAPTER 4 Phylogenetic analysis of European Scutovertex mites (Acari, Oribatida, Scutoverticidae) reveals paraphyly and cryptic diversity: A molecular genetic and morphological approach Abstract...... 72 Introduction ...... 73 Material and Methods...... 76 Results ...... 84 Discussion...... 88 Conclusions ...... 92 References ...... 93 Appendix A ...... 101 Appendix B...... 104

CHAPTER 5 Ancestral state reconstruction reveals multiple evolution of diagnostic characters in the “Higher Oribatida” (Acari), conflicting current classification schemes Abstract...... 105 Introduction ...... 107 Material and Methods...... 111 Results ...... 118 Discussion...... 130 Conclusions ...... 136 References ...... 137

Conclusions ...... 145 Curriculum Vitae...... 149 Summary

Summary

In mites species identification is normally based on morphological character sets but in the early nineties, molecular genetic studies found their ways into acarine research. Since that time, acarologists got important insights into inter- and intraspecific relationships, species diversity or evolutionary histories of different mite groups. Several phylogenetic studies on oribatid mites already confirmed the utility of different molecular markers for answering various evolutionary questions. For this reason, it was attempted to use molecular genetic analyses for a study on the evolutionary relationships within the oribatid mite family

Scutoverticidae and to trace morphological character evolution within the group

Circumdehiscentiae.

In general, many taxa of the family Scutoverticidae are morphologically very similar and thus hardly to distinguish. This is due to either short, inaccurate description of the species or limited knowledge of the amount of intraspecific variation and the diversity within this mite family. An often-stated problem in former literature is certainly the discrimination of the two most widespread Scutovertex species S. minutus and S. sculptus although both species possess clear diagnostic morphological characters differentiating them. For this reason, it was firstly attempted to solve these uncertainties by means of molecular genetic data. Additionally to the description of the new species Scutovertex pileatus sp. nov. in CHAPTER 2, sequence data of one mitochondrial gene were used to examine the taxonomic discreteness of the three last mentioned species. Phylogenetic analyses of DNA sequences of the COI gene (region 2, 567 bp) using Cymbaeremaeus cymba as outgroup, unambiguously support the discreteness of S. minutus, S. sculptus and S. pileatus by placing them reciprocally monophyletic. Interspecific distances among the three Scutovertex species amounted to 13.7% - 21.3%.

CHAPTER 3 focuses on inter-specific comparisons of patterns and degree of genetic diversity of the two species Scutovertex minutus and S. sculptus on the one hand and on the other hand

- 1 - Summary on the possibility of intra-specific sub-structuring reflecting a potential specialization to different habitat types (e.g. salt marshes, mosses of rocks or roofs) populated by S. sculptus.

Sequences of the COI gene (1,259 bp) were analyzed from a total of 83 specimens 83 specimens representing both species plus two individuals from S. pannonicus (outgroup), all collected from a total of 18 localities in Austria. Phylogenetic analyses based on maximum parsimony and Bayesian inference clearly revealed remarkable differences in mtDNA diversity between the two species. The results showed that diversity estimates in S. sculptus significantly exceed those in S. minutus. Concordant with this observed inter-specific differences of genetic diversity, the most common resent ancestor estimate of Austrian S. sculptus resulted in roughly four times the evolutionary age than for Austrian S. minutus (8-12 versus 2-3 Myr). The split between S. minutus and S. sculptus was estimated to an age of 48-

74 Myr which highlights their longstanding separate evolutionary history. Furthermore, the analyses revealed a clear geographic subdivision into samples north and south of the Central

Alps in S. minutus pointing to limited dispersal ability within this species. On the contrary, molecular data showed no phylogeographic structure in S. sculptus. Together with a high genetic diversity it is suggested that dispersal ability of last mentioned species is exceptional high, most probably facilitated by phoresy on birds. Finally, there was no intra-specific sub- structuring with regard to different habitat types in S. sculptus detectable, suggesting that this species can cope with a wide range of environmental conditions.

The family Scutoverticidae is considered to represent an assemblage of distantly related but morphologically similar genera which often led to some confusion in former literature. This is why there is urgent need for detailed studies on interspecific relationships, speciation processes or species diversity within this family. CHAPTER 4 represents the first molecular phylogeny of eleven nominal and two undescribed European mite species of the

Scutoverticidae, represented by four genera (Scutovertex, Provertex, Lamellovertex,

Exochocepheus) based on nucleotide sequences of one mitochondrial (COI) and two nuclear

- 2 - Summary

(28S rDNA, ef-1α) genes. The analyses on the COI and ef-1α genes clearly revealed the monophyly of the family and the monophyly of each investigated species excepting S. sculptus whose specimens clustered in two well supported clades. This last result suggests that S. sculptus represents a complex of several cryptic species which are morphologically indistinguishable, potentially caused by convergent evolution under harsh environmental conditions in similar habitats. Furthermore, the data unambiguously showed the paraphyly of the genus Scutovertex, resulting from the phylogenetic placement of S. pictus in a lineage together with E. hungaricus, L. caelatus and P. kuehnelti.

CHAPTER 5 deals with the systematics of the oribatid mites Circumdehiscentiae. There are several proposals in literature classifying this group. All of them (except one) base on different diagnostic morphological characters, for example the presence or absence of the secretory notogastral octotaxic system in adult mites and the development of scalps and centrodorsal setae both in nymphs. Tracing the character evolution of six morphological characters on a molecular phylogeny revealed several conflicts to the current morphological classification. Ancestral state reconstructions of the octotaxic system and the wrinkled gastronotic cuticle of nymphs yielded multiple independent evolution of the two traits corresponding to an explosive radiation in former time. However, ancestral character state reconstructions of the four remaining traits (scalps, centrodorsal setae, sclerits and pteromorphs) indicated a monophyletic origin of the single character states, highlighting their value as diagnostic characters within Circumdehiscentiae. However, this is only true when looking at a particular trait but not for a combination of traits. Despite all, the results of the present study showed that no current morphological classification scheme is appropriate as it is.

- 3 - Zusammenfassung

Zusammenfassung

Die Artunterscheidung bei Milben beruht hauptsächlich auf Untersuchungen verschiedenster morphologischer Merkmale. Seit Beginn der 90ziger Jahre werden auch molekulargenetische

Techniken in der Milbenforschung angewendet, die bereits wichtige Erkenntnisse über inter- und intraspezifische Verwandtschaftsbeziehungen, über Artdiversität und die Evolutionsgeschichte verschiedenster Milbengruppen geliefert haben. Auch innerhalb der großen Gruppe der

Hornmilben (Oribatida) wurden diverse genetische Untersuchungen getätigt, um ein besseres

Verständnis über deren evolutionäre Entwicklung zu bekommen. Mit Hilfe von molekularen

Markern konnten bereits eine Reihe von interessanten evolutionsbiologischen Fragestellungen innerhalb der Oribatida beantwortet werden. Im Rahmen meiner Dissertation, die sich hauptsächlich mit einer Familie dieser Gruppe, den Scutoverticidae, beschäftigt, sollten evolutionäre Fragestellungen mit Hilfe von genetischen Markern untersucht werden. Ein weiteres

Ziel dieser Arbeit bestand darin, die systematische Gliederung der Hornmilbengruppe

Circumdehiscentiae aufzuarbeiten. Dazu wurden einige besondere morphologische Merkmale, die innerhalb dieser Gruppe für diagnostische Zwecke verwendet werden, herangezogen um deren Entwicklung im Laufe der Evolution zu untersuchen.

Wenn man die Familie Scutoverticidae näher betrachtet, findet man einige Arten die sich morphologisch sehr ähnlich sind. Zusätzliche Probleme, wie kurze und ungenaue Beschreibungen der Arten oder das fehlende Wissen über die intraspezifische morphologische Variabilität, führten oftmals zu Schwierigkeiten bei der Unterscheidung einzelner Individuen. Ein sehr gutes

Beispiel dafür sind die zwei häufigsten Arten der Gattung Scutovertex, S. minutus und S. sculptus. Aus diesem Grund war das erste Ziel der Studie, eine klare Differenzierung dieser zwei

Arten zu erarbeiten, vor allem Mithilfe von molekularen Markern. Zusätzlich zur Beschreibung

- 4 - Zusammenfassung der neuen Art, Scutovertex pileatus sp. nov. werden in Kapitel 2 Sequenzdaten eines mitochondrialen Gens herangezogen, um die taxonomische Eigenständigkeit der drei erwähnten

Arten zu sichern. Die phylogenetische Analyse der DNA-Sequenzen des mitochondrialen COI-

Gens (Region 2, 567 bp), in der Cymbaeremaeus cymba als Außengruppe fungierte, bestätigte eindeutig die Eigenständigkeit von S. minutus, S. sculptus and S. pileatus, indem sie die Arten als monophyletisch auflöste. Die interspezifischen Distanzen zwischen den drei Scutovertex-Arten betragen 13.7 % - 21.3%.

Kapitel 3 umfasst einerseits Vergleiche genetischer Muster und der Diversität der zwei Arten

Scutovertex minutus and S. sculptus und andererseits die Möglichkeit einer innerartlichen

Strukturierung, welche die Besiedelung verschiedenster Habitattypen (z.B. Salzwiesen, von

Moosen bewachsene Felsen und Dächer) widerspiegeln könnte.

Insgesamt wurden für 83 Individuen der beiden Arten, plus 2 Individuen der Außengruppe S. pannonicus die Sequenzen des COI Gens (1259 Basenpaare) ermittelt. Die Proben stammten von

18 verschiedenen Sammelpunkten innerhalb von Österreich. Nach der Analyse der Sequenzen, mithilfe der Maximum Parsimony- und der Bayesian Inference-Methode, konnten erhebliche

Unterschiede in der Diversität der mitochondrialen DNA zwischen den zwei Arten aufgezeigt werden. Bei S. sculptus wurde sie weitaus höher eingeschätzt, als bei S. minutus. Mit diesem

Ergebnis geht auch die Tatsache einher, dass dem in Österreich vorkommenden S. sculptus ein viermal so hohes evolutionäres Alter zugeschrieben wird, wie jenes, welches für den

„österreichischen“ S. minutus ermittelt wurde (8-12 Mio. Jahre für S. sculptus im Gegensatz zu 2-

3 Mio. Jahre für S. minutus). Die Aufspaltung zwischen den beiden Arten wird vor 48-74

Millionen Jahren datiert, was deutlich für die eigenständige Evolutionsgeschichten der zwei

Arten spricht. Außerdem konnte für S. minutus eine klare geographische Auftrennung der Proben in „nördlich und südlich der Alpen“ gefunden werden, was auf eine eingeschränkte

- 5 - Zusammenfassung

Verbreitungsfähigkeit dieser Art hinweist. Im Gegensatz dazu, zeigten die molekularen Daten für

S. sculptus keine phylogeographischen Strukturen. Aufgrund der sehr hohen genetischen

Diversität kann man darauf schließen, dass S. sculptus die Fähigkeit besitzt, sich weiter zu verbreiten und das wahrscheinlich durch Phoresie auf Vögeln. Da keine habitats-spezifischen

Muster innerhalb von S. sculptus erkennbar sind, ist anzunehmen, dass diese Art mit verschiedensten Umweltbedingungen zurechtkommt.

Die Familie Scutoverticidae wird weitgehend als Ansammlung weitschichtig verwandter aber morphologisch sehr ähnlicher Gattungen angesehen, was immer wieder zu Verwirrungen in der bestehenden Literatur führte. Deshalb ist es von dringender Notwendigkeit, detaillierte und ausführliche Studien über zwischenartliche Verwandtschaftsbeziehungen, die Artentstehung und

–diversität innerhalb der Familie durchzuführen. In Kapitel 4 wird die erste molekulare

Phylogenie von elf anerkannten und zwei noch nicht beschriebenen europäischen Arten der

Scutoverticidae präsentiert. Diese Phylogenie umfasst vier Gattungen (Scutovertex, Provertex,

Lamellovertex, Exochocepheus) und beruht auf Nukleotidsequenzen des mitochondrialen COI

Gens und zwei nukleären Genen (28S rDNA, ef-1α). Die Ergebnisse zeigen eine ganz klare

Monophylie der Familie und auch die Monophylie aller untersuchten Arten, ausgenommen S. sculptus. Die Individuen der letztgenannten Art finden sich in zwei eindeutig getrennten Linien der Phylogenie wieder, was uns den Hinweis gibt, dass es sich bei S. sculptus um einen

„Komplex“ mehrerer kryptischer Arten handelt, die morphologisch nicht unterscheidbar sind; ausschlaggebend dafür könnte eine konvergente Evolution in ähnlichen Habitaten unter harten

Umweltbedingungen gewesen sein. Außerdem konnten wir die paraphyletische Stellung der

Gattung Scutovertex zeigen, da S. pictus zusammen mit E. hungaricus, L. caelatus und P. kuehnelti eine genetische Linie bildet.

- 6 - Zusammenfassung

Kapitel 5 beschäftigt sich mit der Systematik der Hornmilbengroßgruppe der

Circumdehiscentiae. Es gibt einige Arbeiten in der Literatur, die sich mit der Klassifikation dieser

Gruppe auseinandersetzen. Alle, bis auf eine, basieren auf diagnostischen morphologischen

Merkmalen, wie zum Beispiel dem Vorhandensein oder Fehlen des sektretorischen oktotaxischen

Systems bei adulten Milben und dem Tragen von Skalps sowie dem Vorhandensein oder Fehlen der centro-dorsalen Borsten im Nymphenstadium. Die Unkenntnis der möglichen homoplastischen Merkmalsentwicklung verlangt nach einer genaueren Studie darüber, wie diese diagnostischen Merkmale überhaupt evolutionär entstanden sein könnten. Indem die Entstehung von sechs ausgewählten Merkmalen auf die eruierte molekulare Phylogenie umlegt wurde, traten

Unterschiede zur gegenwärtigen morphologischen Klassifizierung dieser Gruppe zu Tage. Die

Rekonstruierung von Urzuständen des oktotaxischen Systems und der gefältelten

Kuticulastruktur der Nymphen, weist auf eine völlig unabhängige mehrfache Entstehung dieser zwei Merkmale, welche mit einer explosiven Radiation in früheren Zeiten einhergeht. Die

Analysen der vier übrigen Merkmale (Skalps, centro-dorsalen Borsten, Sklerite und

Pteromorphae) zeigen deren monophyletische Entstehung im Laufe der Evolution, was wiederum ihren Wert als mögliche diagnostische Merkmale innerhalb der Circumdehiscentiae aufzeigt.

Allerdings erscheinen sie nur dann wertvoll, wenn sie für sich alleine genutzt werden und nicht in

Kombination. Diese Studie zeigt, dass alle bestehenden Klassifizierungen innerhalb der

Circumdehiscentiae, basierend auf den genannten morphologischen Charakteren, in ihrer jetzigen

Weise keine Gliederung in natürliche Verwandtschaftsgruppen repräsentieren.

- 7 - Publications

Publications

Parts of the present work are already published or submitted in scientific journals or presented at different congresses:

Schäffer, S., Krisper, G., Koblmüller, S., Sturmbauer, C. 2007. First molecular genetic analysis of

selected scutoverticid species (Oribatida, Scutoverticidae). 6. Milbenkundliches Kolloquium,

Kiel. (Poster)

Schäffer, S., Krisper, G., Pfingstl, T., Sturmbauer, C., 2008. Description of Scutovertex pileatus sp.

nov. (Acari, Oribatida, Scutoverticidae) and molecular phylogenetic investigation of

congeneric species in Austria. Zool. Anz. 247, 249-258.

Schäffer, S., Koblmüller, S., Sturmbauer, C., Krisper, G. 2008. Phylogenetic relationships among the

Austrian scutoverticid species (Oribatida, Scutoverticidae) – morphological versus molecular

genetic data. EURAAC, Montpellier. (Poster)

Schäffer, S., Koblmüller, S., Krisper, G. (2009). Zur Systematik einiger europäischer Scutoverticidae

– 7. Milbenkundliches Kolloquium, Poznan. (Oral presentation)

Schäffer, S., Koblmüller, S., Pfingstl, T., Sturmbauer, C., Krisper, G., in press. Contrasting

mitochondrial DNA diversity estimates in Austrian Scutovertex minutus and S. sculptus

(Acari, Oribatida, Brachypylina, Scutoverticidae). Pedobiologia. (2009) Doi:

10.1016/j.pedobi.2009.09.004

Schäffer, S., Pfingstl, T., Koblmüller, S., Winkler, K.A., Sturmbauer, C., Krisper, G., in press.

Phylogenetic analysis of European Scutovertex mites (Acari, Oribatida, Scutoverticidae)

reveals paraphyly and cryptic diversity: a molecular genetic and morphological approach.

Mol. Phylogenet. Evol. (2010). Doi: 10.1016/j.ympev.2009.11.025

Schäffer, S., Koblmüller, S., Pfingstl, T., Sturmbauer, C., Krisper, G. Ancestral state reconstructions

reveal multiple evolution of “diagnostic” characters in “Higher Oribatida” (Acari) conflicting

current classification schemes. submitted.

- 8 - Publications

Further publications:

Schäffer, S., Krisper, G., 2007. Morphological analysis of the adult and juvenile instars of Scutovertex

minutus (Acari, Oribatida, Scutoverticidae). Rev. Suisse Zool. 114, 663-683.

Pfingstl, T., Schäffer, S., Ebermann, E., Krisper, G., 2008. Intraspecific morphological variation of

Scutovertex sculptus Michael (Acari: Oribatida: Scutoverticidae) and description of its

juvenile stages. Zootaxa 1829, 31-51.

Pfingstl, T., Schäffer, S., Ebermann, E., Krisper, G., 2009. Differentiation between two epilittoral

species, Scutovertex arenocolus spec. nov. and Scutovertex pilosetosus Polderman (Acari:

Oribatida) from different European coasts. Zootaxa 2153, 35-54.

Pfingstl, T., Schäffer, S., Ebermann, E., Krisper, G., 2009. Morphological analysis of the juvenile

stages of Provertex kuehnelti Mihelčič (Acari, Oribatida, Scutoverticidae). Acta zool. Hung.

55, 365-379.

Pfingstl, T., Schäffer, S., Ebermann, E., Krisper, G., 2010. Scutovertex alpinus Willmann, 1953

(Acari: Oribatida: Scutoverticidae) – redescription and geographical distribution. J. Nat. Hist.

44, 379-388.

Pfingstl, T., Schäffer, S., Ebermann, E., Krisper, G. The discovery of Scutovertex ianus sp. n. (Acari,

Oribatida) – a combined approach of comparative morphology, morphometry and molecular

data. Contrib. Zool. (2010) In press

Pfingstl, T., Schäffer, S., Krisper, G. Re-evaluation of the synonymy of Latovertex Mahunka, 1987

and Exochocepheus Woolley & Higgins, 1968 (Acari, Oribatida, Scutoverticidae). Int. J.

Acarol. (2010) In press.

- 9 -

CHAPTER

1

General Introduction

Tectocepheus sarekensis

1. General Introduction

Oribatid mites

Oribatida (“moss mites”) are one the four main groups of the Acariformes

(=”Actinotrichida”) within Acari. First fossil records of oribatid mites are dated to an age of

380 mya located in Devonian sediments (Shear et al., 1984; Norton et al., 1988). The group comprises more than 9,000 described species (Subías, 2004) world wide but their total number is estimated up to 100,000 species (Schatz, 2002).

Oribatid mites are the most abundant group of arthropods in organic soil layer. For example up to 500,000 individuals per m2 can be found in forest litter, representing over 100 species (Schatz and Behan-Pelletier, 2008). A few species occur in aquatic or limnic habitats but the most can be found in all types of terrestrial ecosystems, e.g. any kind of forest soils, inundation meadows, mosses, salt marshes of saline soils etc. Species of the oribatid mites play an important role in decomposition of plant litter, in nutrient cycling and soil formation

(Schatz and Behan-Pelletier, 2008).

Specimens of the group are usually from very small size (only up to 2 mm) and show typically a strong sclerotized body referring to their synonymous names “beetle mites” or

“armoured mites”. They are "K-selected" organisms (Crossley, 1977; Norton, 1994) having low metabolic rates, slow development (1-3 generations per year; Norton, 1994) and also slow fecundity. The development duration from egg to adult can vary from several months

(Ermilov, 2008a-b) to several years (Solhøy, 1975). It has been shown that especially species living in cooler climates have a longer life cycle compared to species of the temperate zones.

For example in the Antarctic species Alaskozetes antarcticus, development duration may be more than three years (Burn, 1986).

Within Oribatida we can find both modes of reproduction, sex as well as parthenogenesis, with some families (e.g. Malaconothridae, Trhypochthoniidae) exclusively reproducing via parthenogenesis (Norton and Palmer, 1991).

- 10 - 1. General Introduction

There are several contradictory studies on the feeding biology of oribatid mites.

Schuster (1956) stated that oribatid mites have a wide feeding range and divided the mites according to their diet into three types: 1) macrophytophagous (feeding on macroflora, e.g. leaf litter, roots), 2) microphytophagous (feeding on microflora, e.g. lichens, mosses, algae) and 3) nonspecialists (feeding on micro- and macroflora). While most of the taxa are microphytophagous or nonspecialists, fewer species fall in the category macrophytophagous

(almost only specimens of the Phthiracaroidea). Moreover, Scheu and Setälä (2002) categorized the oribatid mites to the microbi-detritivorous soil organisms. However, other authors mentioned that Oribatida generally prefer dark pigmented microfungi (“Dematiacea”)

(Hartenstein, 1962; Maraun et al., 1998 & 2003a). In a more recent study Schneider et al.

(2005) could firstly show that oribatid mites also feed on certain ectomycorrhizal fungi, e.g. the ericoid mycorrhizal fungus Hymenoscyphus ericae and the ectomycorrhizal fungus

Boletus badius were preferentially consumed by each of the studied species.

The higher classification of oribatid mites is still largely unresolved, caused by discrepancies of different analytical approaches and the use of different characters and/or data sets (e.g. Norton, 1998; Domes et al., 2007a; Dabert et al., in press). One fundamental proposal to the phylogeny of Oribatida was set by Grandjean (1969) who subdivided the species based on several morphological characters into six major groups, the Palaeosomata,

Enarthronota, Parhyposomata, Mixonomata, Desmonomata and Circumdehiscentiae (=

Brachypylina, “Higher Oribatida”). Figure 1 shows a hypothetical phylogenetic relationship of the six groups obtained from 14 morphological characters after Wauthy (2006; available from www.naturalsciences.be/science/collections/mites/elements/14). Until now, the monophyly of Oribatida has been still very unclear. Norton (1998) suggested, based on a comprehensive study on apomorphic characters that a second group of Acariformes, the

Astigmata evolved within the oribatid Desmonomata, more precisely within the superfamily

Malaconothrioidea. Furthermore, data on the opisthonotal gland chemistry of several oribatid

- 11 - 1. General Introduction mites support Norton´s hypotheses (Sakata and Norton, 2001; Raspotnig, 2006). However, molecular studies by Maraun et al. (2004) who published the first molecular genetic phylogeny of Oribatida based on the 28S rDNA gene did not support Norton´s hypothesis.

Also phylogenetic analyses of two other markers (18S rDNA, ef-1α) by Domes et al. (2007a) placed the Astigmata outside monophyletic Oribatida supporting Maraun´s results. But the most recent proposal published by Dabert et al. (in press) revealed, again based on studies of the 18s rDNA gene, the origin of Astigmata within Oribatida as suggested by Norton (1998) with the morphological data.

Fig. 1. Phylogenetic reconstruction of the six major oribatid groups (green) obtained from 14 morphological characters. Modified from Wauthy (2006; available from www.naturalsciences.be/science/collections/mites/elements/14).

Beside the uncertain classification of Astigmata, there are also questions within the grouping of the Circumdehiscentiae which represents the largest and taxonomically richest group of the Oribatida. Looking in former and recent literature, acarologists stated several approaches to classify this group. The first and most cited proposal was set by Grandjean (1954) who divided the group based on three main morphological characters in five subdivisions: the

Opsiopheredermata, the Eupheredermata, the dorsodeficient Apheredermata, the normal

- 12 - 1. General Introduction pycnonotic Apheredermata and the Poronota. In later studies authors either adapted

Grandjean´s system as for example Wauthy (2006; www.naturalsciences.be/science/collections/mites/elements/) who included a sixth group, the

“Circumdehiscentiae (=Pycno- and Poronota) with wrinkled nymphs” to the

Circumdehiscentiae, or reduced the five groups into only two, the Pycno- and Poronota

(Subías, 2004). However, there were also some studies questioning Grandjean´s systematic, such as those from Woas (2002) who tried to address several phylogenetic relationships among circumdehiscent superfamilies. But up to now, none of the known classification schemes seems to be the right one.

Molecular genetic as tool for evolutionary questions

Most molecular genetic studies dealing with several questions within the Acari - starting in the early nineties - have addressed economically important or health-threatening mite taxa such as Varroa (Anderson and Trueman, 2000), Psoroptidae (Zahler et al., 1998;

Essig et al., 1999) Tetranychidae (Navajas et al., 1992; Navajas, 1998; Toda et al., 2000 etc.) and Ixodidae (Black and Piesman, 1994; Crampton et al., 1996; Norris et al., 1999 etc.). At the beginning of the ”molecular era”, only a few studies tackled evolutionary questions on rather “non-important” mite groups as for example Salomone et al. (1996) who investigated the taxonomic status of two Steganacarus species using the mitochondrial COI gene. But in the last decade, the interest on such groups had highly increased. Especially the Oribatida were under great aspect of various phylogenetic and evolutionary studies (Salomone et al.,

2002; Maraun et al., 2004; Heethoff et al., 2007; Laumann et al., 2007; Dabert et al., in press etc.).

- 13 - 1. General Introduction

Looking on the molecular markers used in acarine research [reviewed by Cruickshank

(2002)], we can find a broad spectrum depending on the particular goal of the studies. For phylogenetics at low taxonomic levels especially the second internal transcribed spacer of the nuclear ribosomal gene cluster (ITS2) and the mitochondrial protein-coding gene cytochrome oxidase I (COI) were used. While ITS2 was more often used in studies on economically important taxa (e.g. Navajas et al., 1998; Essig et al., 1999), the mitochondrial COI gene has been widely used within Acari (Navajas et al., 1998; Anderson and Trueman, 2000; Salomone et al., 2002, Tixier et al., 2006; Heethoff et al., 2007; Marangi et al., 2009). To resolve phylogenetic questions at very deep taxonomic levels, the nuclear ribosomal genes 18S (Otto and Wilson, 2001; Lekveishvili and Klompen, 2004; Dabert et al., in press) and 28S rDNA

(Cruickshank and Thomas, 1999; Maraun et al., 2003b & 2004) were commonly taken. Both markers are equally powerful because they contain conservative regions usable for the deep branch investigations. Furthermore, there are two other nuclear markers that are almost exclusively used in phylogenetic studies on oribatid mites, the single copy protein-coding gene, elongation factor-1alpha (ef-1α) and the heat shock protein 82 (hsp82) gene (Schaefer et al., 2006; Laumann et al., 2007; Domes et al., 2007 b). Only the ef-1α gene was also used for investigations on phylogenetic relationships among Mesostigmata (Klompen, 2000;

Lekveishvili and Klompen, 2004). The limited use of the ef-1α is certainly due to the problem of paralogy in this gene as a result of gene duplication in genome. This could lead to the trouble, if the same copy of the gene has been sequenced in all the taxa (Cruickshank et al.,

2001). However, ef-1α is a single copy gene in arthropods for which complete genomes (e.g.

Drosophila melanogaster, Apis mellifera) are available, preventing possible confusions between paralogs and orthologs (Ayoub et al., 2007).

- 14 - 1. General Introduction

Objectives of the present study

In mites species identification is normally based on morphological character sets. But it is not unusual that scientists have some problems in distinguishing morphologically similar species. This is why acarologists in former and recent time often solved these uncertainties by describing questionable specimens as new species. One of such examples represents the oribatid mite family Scutoverticidae.

In 1836 Koch described a species called “Cepheus minutus” which was later transferred by Sellnick (1928) to the genus Scutovertex. Hence, Scutovertex minutus represents the “oldest” (first) described species of the family. Almost 43 years later, Michael

(1879) described the “first” species of this family Scutovertex sculptus and presented a genus diagnosis of Scutovertex. Therein he only formulated the shape of lamellae, the sensillus and the lenticulus as diagnostic characters for the genus. Until Grandjean (1954) firstly mentioned the family name “Scutovertexidae” (name was not correctly declined according to the Latin grammar why it has been changed to Scutoverticidae lately) only a handful Scutovertex species were named. Most new species and genera were described in the sixties to the eighties of the last century, but at the same time many wrongly classified taxa were already transferred to other genera. It was a common practice to “put” species which could not be assigned to one genus or family, in the family Scutoverticidae. So the family was often considered as a conglomeration of distantly related but morphologically similar genera. There were still some acarologists who mentioned the urgent need for a comprehensive revision of the family

(Bernini, 1976) but on the other hand some authors caused more confusion by transferring several genera from one family to another (Woas, 2002). Furthermore, some authors tried to formulate a diagnostic key for the family (Giljarov and Krivolutsky, 1975; Sitnikova, 1980;

Pérez-Iñigo, 1993) but always with a limited set of morphological characters resulting in the fact that many specimens were wrongly distinguished. For example there are many various drawings of Scutovertex minutus in former literature representing definitively not always the

- 15 - 1. General Introduction same species (Balogh, 1972; Giljarov and Krivolutsky, 1975; Pérez-Iñigo, 1993; Schäffer and

Krisper, 2007). Of course, in this context the question arises if the variability within the species is that high or if the diversity within the genus is higher than expected.

But why is this family of interest for studying, besides their taxonomic uncertainties?

Their preference for extreme environments such as mosses or lichens on very sun-exposed rocks and roofs (Krisper and Schuster, 2001; Krisper et al., 2002; Smrž, 2006) as well as saline soils, salt marshes and inundation meadows (Schuster, 1958; Weigmann, 2004), indicates their potential to play a major role as pioneer organisms at the first steps of succession (Skubala, 1995). Furthermore, Cronberg et al. (2006) showed that mites are also an important factor fertilizing mosses. The authors found that fertile moss shoots attract mites

[amongst others two Scutovertex species (S. minutus and S. sculptus)] and also springtails, which in turn carry moss sperm and so enhance the fertilization process in mosses. Moreover, there is evidence from a few previous studies that scutoverticid mites might be important bioindicators of e.g. air pollution (Weigmann & Kratz 1987, Steiner 1995a-b) demonstrating the impact of various human activities on the environment. To sum up, species of this family are important soil organism in any kind of regard.

Another reason why Scutoverticidae are of interest is their so far assigned placement at the base of one circumdehiscent group, the Poronota. Members of this family are characterized by wrinkled nymphs and adults that bear sacculi on the notogaster homologous to the so called octotaxic organ consisting of four pairs of porose areas eponymous for poronotic mites (Grandjean, 1954). Because of the minute appearance and reduced number

(one to three pairs) of the sacculi some authors (Norton and Alberti, 1997) hypothesized that they represent the plesiomorphic state of the octotaxic system. All these facts would conclude that this licneremaeoid family could be the most recent common ancestor of the Poronota.

As above shown, molecular genetic approaches are good tools to solve several evolutionary questions also within mites. Thus, a variety of molecular markers was employed

- 16 - 1. General Introduction to elucidate the intra- and interspecific relationships within the Scutoverticidae on the one hand and on the other hand to get insights on speciation processes, species diversity and the evolutionary history of the family (CHAPTERS 2-4).

In the second part of my work (CHAPTER 5) I dealt with the uncertain systematic of the Circumdehiscentiae. Molecular genetic data should aid in obtaining a better knowledge on the utility of the current classification approaches basing on morphological data.

To the main goals of the further chapters…

One of the most often-stated difficulties in distinguishing species within the

Scutoverticidae concerns the two most widespread Scutovertex species, S. minutus and S. sculptus (Strenzke, 1943; Pérez-Iñigo, 1993). Although both species possess clear diagnostic characters differentiating them (cuticle structure, prodorsal ridges etc.), several authors have mentioned some problems. One reason therefore might be a possible wide range of intraspecific variation within the taxa (Strenzke, 1943). In the course of my studies I could identify a new undescribed species showing several distinct morphological characters, within this species complex. So the first attempt of the study, was to describe this new species as

Scutovertex pileatus sp. nov. Furthermore, I examined the taxonomic discreteness of the new species compared to S. minutus and S. sculptus by means of the mitochondrial gene COI

(CHAPTER 2).

Important insights into the evolutionary and demographic history of species can be obtained from inter-specific comparisons of patterns and degree of genetic diversity. Most

Scutovertex species have high similarities in different life history traits hypothesizing similar patterns and degrees of genetic variation. Furthermore, they are small moss-dwellers with low dispersal ability which must have an influence on the phylogeographic structure of the species. To test these two hypotheses I analyzed sequences of the mitochondrial COI gene of the two most widespread species S. minutus and S. sculptus collected from a total of 18 - 17 - 1. General Introduction locations in Austria. Moreover, it was attempted to investigate a potential intraspecific sub- structuring resulting from a possible specialization to different habitat types populated by S. sculptus (CHAPTER 3).

As already mentioned above, the family Scutoverticidae is considered to represent an assemblage of distantly related but morphologically similar genera. This is why I tackled several evolutionary questions such as interspecific relationships, speciation processes or species diversity, on the Scutoverticidae. By using three molecular markers, one mitochondrial (COI) and two nuclear (28S rDNA, ef-1α) genes it was attempted to evaluate the taxonomy of 11 nominal and two undescribed European species of the family and to elucidate the phylogenetic relationships between the Scutovertex species. Because of the high potential for cryptic diversity in small-size and short generation time animals, it was further expected to uncover a possible cryptic diversity within members of the family (CHAPTER

4).

In a final study, I focused on the taxonomy of the oribatid mite group

Circumdehiscentiae. There are several proposals from literature (e.g. Grandjean, 1954;

Subías, 2004) to classify this group. All of them (except one) are based on different diagnostic characters. However, some authors have already mentioned that some of these characters might have evolved multiple times, potentially leading to conflicts with current morphological classifications. To test these hypotheses, I established a molecular phylogeny based on three nuclear markers (28S rDNA, ef-1α, hsp82) and traced the evolution of six morphological traits of interest over the molecular phylogeny using parsimony, likelihood, Bayesian and stochastic approaches. With the help of these methods, I tried to evaluate which of the proposed classification schemes is the most appropriate one for the Circumdehiscentiae

(CHAPTER 5).

- 18 - 1. General Introduction

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- 25 -

CHAPTER

2

Description of Scutovertex pileatus sp. nov. (Acari, Oribatida, Scutoverticidae) and molecular phylogenetic investigation of congeneric species in Austria

Sylvia Schäffer, Günther Krisper, Tobias Pfingstl & Christian Sturmbauer

Zoologischer Anzeiger 247 (2008), 249-258 2. Description of Scutovertex pileatus sp. nov.

Abstract

The oribatid mite Scutovertex pileatus sp. nov. is described on the basis of adult individuals originating from southern Austria (Carinthia). The new species shows the typical habitus of

Scutovertex and is distinguished by the cerotegument and cuticle forming irregular nodules and bars over the entire body; the rostrum with two visor-like projections, with the ventral projection larger and arched ventrally; short lamellar setae; two pairs of converging ridges between the lamellae; small notogastral setae which are not broadened distally; and a sclerotized rib across the mentum. Furthermore, DNA sequences of the COI gene (region 2,

567-bp) of S. pileatus were compared with those of S. minutus, S. sculptus, using

Cymbaeremaeus cymba as the outgroup. Molecular data unambiguously support the discreteness of all three species by placing them reciprocally monophyletic, as well as by large genetic divergences. Interspecific distances among C. cymba, S. minutus, S. pileatus and

S. sculptus amounted to 13.7% - 29.9%.

Key words: Oribatid mites, taxonomy, morphology, DNA sequences, cytochrome oxidase subunit I.

- 26 - 2. Description of Scutovertex pileatus sp. nov.

Introduction

To date, eight genera and about sixty species (Shtanchaeva and Netuzhilin 2003,

Subias 2004) of the oribatid mite family Scutoverticidae are known worldwide. Most of them can be found in lichens and moss of extreme environments (e.g. Krisper et al. 2002; Schuster

1958; Smrž 1992; Weigmann 1973, 2004). The taxonomic status of many species of this family is uncertain because of inadequate description of genera and species, often without any comparison to related taxa. (Fernandez and Cleva 2002). The often stated difficulty of identifying two of the most common species of the genus Scutovertex Michael 1879,

Scutovertex minutus (Koch, 1836) and S. sculptus Michael, 1879 (Strenzke 1943, Pérez-Iñigo

1993, Weigmann 2006), highlights this problem. Even as both species possess diagnostic characters such as prodorsal ridges, cerotegument layer, cuticle structure and the form of the lenticulus and notogastral setae, identification is problematic due to the wide range of intraspecific variation (Michael 1884, Strenzke 1943, Willmann 1953). In the course of the investigations on Austrian species, a new species was found showing several distinct morphological characters, within this species complex.

This new species is herein described as Scutovertex pileatus sp. nov. Further, the new species as well as two congenerics within the species complex are studied by means of the mitochondrial gene cytochrome oxidase I (COI).

Material and methods

Sampling

The mites investigated in this paper were collected from mosses and lichens on sun- exposed rocks in several Austrian locations. Specimens of S. pileatus sp. nov. were found at three localities in Carinthia: a) Laas, mosses on rocks near the ascend to the hospital, 900 m asl, 28.3.2005; b) Hochosterwitz, mosses from the SW-exposed wall of the castle, 690 m asl, - 27 - 2. Description of Scutovertex pileatus sp. nov.

26.6.2005; c) SW-orientated slope of the rockslide area “Schütt” at the bottom of the mountain Dobratsch near Villach; mosses on sun exposed boulders (two sub-samples), 570 m asl; 26.5.1996 and 8.8.2005.

Mites identified as S. minutus (according to Schäffer and Krisper 2007) were found in samples from Asparn/Zaya (Lower Austria; 230 m asl; 16.11.1996 and 29.3.2005) and Pogier

(Styria; 670 m asl; 4.10.2006).

Individuals of S. sculptus originated from Illmitz (Burgenland; “Oberer Stinkersee”;

120 m asl; 16.9.2005), Ernstbrunn (Lower Austria; Toter Hengst; 290 m asl; 29.3.2005) and

Ruin Falkenstein (Lower Austria; 420 m asl; 28.3.2006).

The species Cymbaeremaeus cymba (Nicolet, 1855) family Cymbaeremaeidae served as the outgroup; specimens were collected at Röthelstein; Styria; 1200 m asl; 13.10.2005.

Berlese-Tullgren funnels were used to extract the mites from the substrate.

Individuals for morphological analyses were preserved in 70% ethanol, those for molecular analyses in absolute ethanol.

Morphological analysis

Some mites were embedded in lactic acid (as clearing agent) using cavity slides for general view, and then washed in aqua dest. and mounted in Swan-medium (a mixture of arabic gum, aqua dest., glucose, chloral hydrate and glacial ethanoic acid) as permanent slides; other specimens were transferred directly from ethanol into Swan-medium. Light microscopical investigations were done with a differential interference contrast microscope

(Olympus BH- 2), equipped with a drawing tube. Micrographs were taken with a digital camera (Olympus Camedia C4040 zoom). For scanning electron microscopy the specimens were dehydrated in ascending ethanol concentrations, air-dried, mounted on aluminium-stubs with double sided adhesive tape and coated with gold. SEM-micrographs were taken at the

- 28 - 2. Description of Scutovertex pileatus sp. nov.

Research Institute for Electron Microscopy, Technical University Graz, with a Zeiss Leo

Gemini DSM 982.

Molecular phylogenetic analysis

DNA extraction

Total genomic DNA was extracted from single ethanol-preserved specimens using the modified CTAB (hexadecyltriethylammonium bromide) method after Boyce et al. (1989).

Tissues were digested in 500 µl CTAB buffer (1.4 M NaCl, 0.1 M Tris- HCl pH 8.0, 2%

CTAB, 0.02 M EDTA, 0.2% 2-mercaptoethanol) and incubated at 37°C and 300 rpm for about 18 hours. Proteins and carbohydrates were removed with 500 µl chloroform by centrifuging the mixture for 10 min. Supernatant was decanted and saved. Afterwards 250 µl fresh CTAB buffer was added to the chloroform phase, centrifuged for again 10 min and supernatant was combined with the first. The DNA was precipitated with cold isopropanol at -

20°C for 2 hours, centrifuged for 30 min, washed with 70% and 100% ethanol, dried at 45°C and re-suspended in 20 µl 0.1 M TE-buffer.

PCR and DNA sequencing

A fragment of the COI (region 2) gene was amplified using the primer pairs Mite COI-

2F (5´-TTYGAYCCIDYIGGRGGAGGAGATCC-3´) and Mite COI-2R (5´-

GGRTARTCWGARTAWCGNCGWGGTAT-3´) (Otto and Wilson 2001).

PCR amplification was performed in 20 µl reaction volume containing 0.09 units of Taq polymerase (5 units µl-1, BioTherm, GenXpress), 1.7 µl MgCl2 (25 mM), 2 µl 10x reaction buffer, 2 µl of each primer (10 mM), 2 µl 10x dNTP mix (10 mM), 7.4 µl aqua dest., and 3 µl of DNA template. The temperature profile of the PCR was 94°C for 30 s, 49-51°C for 30 s and 72°C for 30 s. Thirty-five cycles were performed followed by a final extension step of

72°C for 7 min. To verify the success of PCR, products were electrophorezed on a 2%

- 29 - 2. Description of Scutovertex pileatus sp. nov. agarose gel. After purification with the enzyme cleaner ExoSAP-IT (Amersham Biosciences), the final products were sequenced with the forward primer COI-2F using the BigDye

Sequence Terminator v3.1 Cylce Sequencing Kit (Applied Biosystems) following the sequence protocol described in Koblmüller et al. (2004). DNA fragments were purified with

Sephadex G-50 (Amersham Biosciences) and visualized on a 3120 capillary sequencer

(Applied Biosystems).

Data analysis

Sequences were aligned by eye in MEGA version 3.1 (Kumar et al. 2004). Pairwise distances were constructed with MEGA 3.1 and then analyzed in PAUP* 4.02a (Swofford

2002) using parsimony (not shown) and the neighbor-joining method (Saitou and Nei 1987) with C. cymba as the outgroup. Since alternative phylogenetic methods and models of molecular evolution did not affect topology, equal weights and uncorrected p-distances were used. All sequences are available at GenBank (accession numbers EU437746- EU437781).

Results

Description of S. pileatus sp. nov.

Diagnosis

Habitus corresponding to typical Scutovertex. Length 522 µm (mean), notogastral width 295 µm (mean). Cuticle dark brown to light brown. Cerotegument and cuticle form irregular nodules and bars over whole body. Rostrum with two visor-like projections, ventral one larger and arched ventrally. Lamellar setae short, only about a third of length of rostral setae. Between lamellae two pairs of converging ridges. Sensillus clavate, spinose. 10 to 11 pairs of small notogastral setae, distally not broadened. Across the mentum a clearly developed, sclerotized rib. 6 pairs of genital setae inserting in line, g1 about twice as long as

- 30 - 2. Description of Scutovertex pileatus sp. nov. others. Leg chaetome without solenidia: I (1-4-2-4-18), II (1-4-2-4-15), III (2-2-1-3-15), IV

(1-2-2-3-12).

Type series

Holotype and three paratypes are deposited at the Bavarian State Collection of

Zoology, Munich (ZSM; Nr. ZSMA20071403 and ZMSA200771404); additionally, three paratypes are preserved in the Berlin Museum of Natural History, Nr. ZMB 32688 (all types are stored in 70% ethanol). Permanent slides are in the collection of the authors. Type locality of holo- and paratypes: Austria, Carinthia, SW-orientated slope of the rockslide area “Schütt” at the bottom of the mountain Dobratsch near Villach; mosses on sun exposed boulders, 570m asl; 8.8.2005.

Description of the adult

Measurements (n = 23): Mean total length: 522 µm (range 481-575 µm). Mean notogastral width: 295 µm (256-338 µm); holotype 496 µm x 277 µm.

Habitus (Figs.1a-c; 2a; 3a; 4a): Body in dorsal view oval. Living specimens light brown to dark brown, in lactic acid and ethanol color changes towards ochre to yellow.

Integument: Cerotegument (Fig. 2a) strongly developed; around lenticulus thick nodes; remaining part of notogaster covered with irregular strong bars or fused nodes. Nodes and bars formed of cerotegument and cuticle (Figs. 2b, d); in some individuals only thick notogastral nodes. Cerotegument on large parts of the body thick and amorphous; a thinner and finely granulated layer in protected areas (e.g. bothridium, acetabulum), granules forming reticulate pattern (Fig. 2c). Cuticle without foveae.

- 31 - 2. Description of Scutovertex pileatus sp. nov.

Fig. 1. (a-f) S. pileatus, (a-c) habitus (legs omitted); (a) dorsal aspect; arrows indicate the position of lyrifissures im; (b) lateral aspect; thick, white arrows point to lyrifissures; thick, black arrow to lateral opisthosomal grand; thin arrows indicate saccules (S1-S3); (c) ventral aspect; (d) subcapitulum, without pedipalp; (e) pedipalp in lateral view; (f) chelicere in lateral view, paraxial.

- 32 - 2. Description of Scutovertex pileatus sp. nov.

Prodorsum: Rostrum visor-like, in dorsal view rounded (Fig. 2a); from lateral aspect

(Fig. 3b) two clear projections thereof ventral one larger and arched ventrally. Rostral setae inserting between the two projections and curving anteriorly towards each other. Lamellae ridge-like, ending in slim lamellar cusps. Lamellar setae (see arrow in Fig. 2e) only about a third of length of rostral setae, originating at top of cusps (Figs. 1b, 2e). Translamella narrow and weakly developed. Between bothridia two convergent ridges meeting in the first half of prodorsum originating from anterior border of notogaster; two additional ridges with irregular positions on the prodorsum running more or less parallel lateral to latter, never reaching each other or translamella (Figs. 2a, f). No interlamellar setae. Sensillus clavate and spinose (Fig.

3c). Bothridium wide, with a triangle-shaped opening; margin without incision, on antiaxial side a small apophysis with a short ridge running ventrally (Fig. 3c). A “V”-shaped ridge rostrad of leg I, in position of tutorium (Fig. 3e).

Notogaster (Figs. 1a, 2a): Oval; medially, anterior part of notogaster not clearly separated from prodorsum by a suture. Lateral borders of lenticulus concave. Humeral angle conspicuously developed. Elements of octotaxic system represented by three pairs of very small saccules (S1-S3) (Fig. 1b, thin arrows). Number of notogastral setae variable from 10 up to 11 pairs: c2, da, la, dm, lp, dp, h1-3 and ps1-3; variations concerning da and dp. Only in one case 11 pairs with one additional left seta dp were observed. All setae small, acuminate

(Figs. 2g, 3f). Lyrifissure ia (Figs. 3c-d) situated on a very small cuticular nodule under humeral angle of notogaster; lyrifissure im latero-median on notogaster; ih and ips on lateral border and ip dorsally of line ps1 and ps2 (Fig. 1b). Openings of lateral opisthosomal glands located ventrally of line lyrifissures im and setae h3 (Fig. 1b).

Camerostome: No genal incision; rostral margin composed of two structures; one apical lobe-like projection originating below the rostral setae and one lateral triangular longish lamella originating from the postero-lateral corner of the camerostome.

Rostrophragma forming the inner margin of camerostome (Figs. 3b, 4b).

- 33 - 2. Description of Scutovertex pileatus sp. nov.

Fig. 2. (a-g) S. pileatus, dorsal aspects; (a) habitus; arrows point to prodorsal ridges; (b) optical section through lateral margin of notogaster, nodules of cuticle (thick arrow) and cerotegument (thin arrow); (c) cuticle nodule (near bothridium) covered with reticulated cerotegument; (d) cuticle wrinkle of notogaster with broken cerotegument layer; (e) left part of rostrum, arrow points to lamellar seta; (f) lenticulus (Le), prodorsal convergent ridges (thin arrow) and two additional ridges (thick arrow); (g) Notogastral seta h3.

- 34 - 2. Description of Scutovertex pileatus sp. nov.

Fig. 3. (a-f) S. pileatus, lateral aspects; (a) habitus, ovipositor extruded; (b) rostrum, thick arrow indicates rostrophragma, thin arrows point to rostral projections; ro = rostral seta; (c) sensillus and bothridium, arrow points to nodule in the humeral region containing lyrifissure ia; (d) micrograph of lyrifissure ia (differential interference contrast); (e) left lateral part of prodorsum with V-shaped tutorium; (f) notogastral setae ps2 and ps3.

- 35 - 2. Description of Scutovertex pileatus sp. nov.

Fig. 4. (a-b) S. pileatus, ventral aspects; (a) habitus; (b) subcapitulum, ro = rostral seta, RU = rutellum, G = gena, M = mentum, a = anterior subcapitular seta, m = median subcapitular seta, h = hysterostomatic seta; arrow indicates sclerotized rib running across mentum.

Gnathosoma (Figs. 1d, 4b): Subcapitulum diarthric. Mentum with sclerotized rib running across its surface and forming a deep furrow together with the anterior margin of mentum (mentotectum); the simple hysterostomatic setae (h) inserting on this rib. Genae with sharp lateral edges; one pair of anterior (a) and median (m) subcapitular setae, both finely serrate. Rutellum pantelebasic, distal with three teeth, first one very strong, two others small and mostly covered by anterior margin of rutellum; rutellar brush well developed.

Pedipalp (Fig. 1e) pentamerous; chaetome: 0-2-1-3-9; solenidia: 0-0-1. The four tarsal eupathidia bacilliform with slightly broadened basis. Tip of solenidion reaching basis of eupathidium acm.

Chelicere (Fig. 1f) with two setae: cha and chb, more or less of same length.

Trägardh´s organ longer than mobile digit.

Epimeral region (Fig. 1c): Epimeral setal formula: 3-1-2-2; all setae acute, slim and smooth; seta 1c located on basis of pedotectum I. Pedotectum I large, escutcheon-like, hiding

- 36 - 2. Description of Scutovertex pileatus sp. nov. acetabulum I completely. Pedotectum II from dorsal view Y-shaped. Apodemata never reaching median axis. Apodem IV absent.

Anogenital region (Fig. 1c): Genital valves approximately trapezium-shaped; one groove running caudally of genital opening towards acetabulum IV (Fig. 4a). Six pairs of genital setae standing in line and inserting near the inner border of genital valves. Setae g1 in

75% of the investigated specimens more than twice as long as g2-6; in the remaining 25% g1 about one and a half longer as g2-6. Distance between genital and anal opening two-thirds of length of genital opening. One pair of aggenital setae, latero-posteriorly of genital opening.

Anal valves long, broadened posteriorly and surrounded by a shallow groove. Two pairs of pointed and slim anal setae. Lyrifissures iad situated laterally, near front edge of anal orifice.

Adanal setae ad1, ad2 located posterior of anal orifice; ad3 lateral to it.

Legs (Figs. 5a-d): leg-I and -IV longer than leg-II and -III. All femora with one dorsal stigma; from here on a trachea running to the distal part of tibia (leg I to II) or to the proximal part of tarsus (leg III to IV); additionally a second short branch of trachea in femora of legs I and II end within same leg segment. Also one trachea within trochanters of legs III and IV; stigma opening proximally and paraxially. Genua I and II with variations in setation (Figs. 5e- f); in some specimens ventral seta absent. In one specimen, ventral seta v´ on tibia IV doubled. Chaetome and solenidia of legs (numbers in [ ] = individual variation): I (1-4-2[3]-4-

18) (1-2- 2), II (1-4-2[3]-4-15) (1-1-2), III (2-2-1-3-15) (1-1-0), IV (1-2-2-3-12) (0-1-0).

- 37 - 2. Description of Scutovertex pileatus sp. nov.

Fig. 5. (a-f) S. pileatus, legs; (a) - I, right, antiaxial aspect; (b) - II, left, antiaxial aspect; (c) - III, left, antiaxial aspect; (d) - IV, left, antiaxial aspect; (e and f) - genu, dorsal aspect, (ventral seta v developed); (e) - I, right; (f) - II, left.

- 38 - 2. Description of Scutovertex pileatus sp. nov.

Molecular phylogenetic analysis

The neighbor joining tree (NJ) based upon a 567-bp segment of the COI gene (region

2) (36 individuals) supports reciprocal monophyly of the species analyzed, with bootstrap values of 99-100 for all clades, representing the species S. minutus, S. pileatus sp. nov., S. sculptus, and C. cymba as the outgroup (Fig. 6).

The interspecific distance between C. cymba and S. pileatus sp. nov. amounted to

24.0%, whilst S. sculptus and S. minutus differed by 26.0% and 29.9%, respectively. Pairwise genetic distances within and between the investigated Scutovertex-species are shown in Table

1.

Fig. 6. Genetic relationships among three Scutovertex species and Cymbaeremaeus cymba. NJ-Tree, uncorrected; bootstrap values are shown when higher than 50.

- 39 - 2. Description of Scutovertex pileatus sp. nov.

Table 1. Pairwise genetic distances.

dia dis (averaged) ni nh (maximum) S. pileatus S. minutus S. sculptus S. pileatus 14 7 0,01 S. minutus 9 3 0,03 0,205 S. sculptus 12 6 0,08 0,213 0,137 ni = number of analyzed individuals, nh = number of haplotypes, dia = intraspecific distances

(uncorrected), dis = interspecific distances (uncorrected).

Discussion

Our combined analysis of comparative morphological and molecular data resolved the status of S. pileatus sp. nov., with morphological features clearly placing this species within

Scutovertex. In table 2 six species of this genus occurring in Austria (S. pileatus sp. nov., S. minutus; S. sculptus; S. alpinus Willmann, 1953; S. pannonicus Schuster, 1958; and S. pictus

Kunst, 1959) are compared with two further species, S. bulgaricus Kunst, 1961 and S. siculus

(Berlese, 1887). The two latter ones exhibit most of similarities to the new species in regard to the habitus. Data for table 2 were taken from own investigations and from descriptions and figures in literature (Berlese 1887, Kunst 1959, 1961, Michael 1884, Schuster 1958,

Weigmann 2006, Willmann 1953, Woas 1998). Varying characters as length of cusps, width of lamellae and translamella, often used in identification keys, were excluded in this comparison. S. pileatus sp. nov. is clearly distinguishable from all others by its short lamellar setae, two pairs of converging parallel prodorsal ridges between lamellae, the conspicuous rostral lobes, and the continuous transverse ridge on mentum as well as the deep furrow between this latter ridge and the mentotectum. Other characters, e.g. shape of posterior notogastral setae or leg setation, are shared with the one or other species but their unique combination helps in the discrimination of species, additionally.

- 40 - 2. Description of Scutovertex pileatus sp. nov.

Table 2. Comparison of several morphological characters of selected Scutovertex species. = character developed; - = character not developed; ? = no data available: le = lamellar seta; ro = rostral seta; TL = translamella; ng = notogastral; g1, g2 = genital setae; GO = genital opening, AO = anal opening. ) ro s

TL p ≈ g

? ? ? ? ? ? ? ? ? – 450 le

≤ + bum reachin convergent, not irregularly wrinkled; wrinkled; irregularly )long ( )long ro TL g ? ? ? ? ? ? – – 10 wrinkled side by side by side reachin

densely finely finely densely convergent, not clavate, spinousclavate, spinous clavate, )short (1/2 of of (1/2 )short + ro ;

≈

? ? ? ? ? ? ? ? – le inous p s very shortvery irregularly irregularly road clavate, road clavate, wrinkled b small, acuminatesmall, acuminate small, broadened slightly 1/2 GO-lengthof as GO-length same 3/4 GO-length of )long ( )long es g icuspictus S. S. bulgaricus siculus S. ro TL > g th g ? ? ? ? ? ? ? le 10-11 len distinct slightly slightly roadened nodules + nodules b convergent, convergent, reachin same as GO- as same variable bul )long ( )long ous clavate, spinous ro

≈

? ? ? ? ? ? le visible weakly weakly not clearly clearly not discernable faintly wrinkled faintly small, acuminate small, )long ( )long ro

≈ th

spinous clavate, spin g ? ? ??? ? ? ? ? ? le 10 len roadened distinctly distinctly wrinkles? nodules + b reaching TL reaching same as GO- as same )long ( )long ro

TL ≈ g

– shallow weak, le 10-12 wrinkles? nodules + S. minutus S. S. sculptus S. alpinus S. pannon reachin convergent, not not convergent, ) long ( ro sp. TL x g – wrinkles nov. g 10-11 in linein side side by side side by ?side by side in line 481-575 550-659 590-660 550-630 773-800 445-525 470-535 lobe like narrow on noduleon nodule on 1-2-2-3-12 1-2-2-3-12 1-2-2-3-12 ? 2-2-1-3-15 2-2-1-3-15 1-4-3-4-15 1-4-3-4-15 1-4-3-4-18 1-4-3-4-18 ?-4-3-4-17 ? continuous interrupted interrupted ? 2 pairs, not not 2 pairs, reachin S. pileatus S. pileatus stron clavate, spinousclavate, clavate, spinous clavate, short (1/3 of small, acuminatesmall, broadened slightly 2/3 of GO-length2/3 of GO-length as same nodules + irregular Scutoverte ps1 , h3 g1-g2 ections j ro ng cuticle ng cuticle surface setation leg IV body length (µm) v-shaped tutorium rostral setation leg III ridge on mentum position Characters p sensillus ng cuticle ng cuticle foveae pairs ofng setae setation leg II setation leg I lamellar setae lamellar lyrifissure ia lyrifissure distance GO-AO distance ng setae prodorsal ridges

41 2. Description of Scutovertex pileatus sp. nov.

There are only few examples in oribatid literature dealing with a combination of morphological and genetic data for species discrimination. Salomone et al. (2003) described

Carabodes tyrrhenicus Salomone, Avanzati, Baratti and Bernini, 2003 and compared their new species with other members of the genus Carabodes C. L. Koch, 1836 by using comparative morphological data and genetic analyses based on allozymes. The COI gene was successfully used for resolving taxonomic and phylogenetic problems of Steganacarus

Ewing, 1917 based on morphological evidence by Salomone et al. (1996, 2002). In the present study, data of the COI gene support the validity of S. pileatus sp. nov. as a well- differentiated new taxon. Moreover, these results also show that the two most common species of the genus in Austria, Scutovertex minutus and S. sculptus, are separated by an equal genetic distance. These new facts confirm Strenzke´s statement on the status of these two species (1943, p. 67): “Die Unterschiede zwischen Sc. minutus C.L. Koch und sculptus Mich. sind so beträchtlich, daß sie wohl als zwei gut charakterisierte Arten aufgefaßt warden dürfen.”

We conclude that more extensive molecular phylogenetic investigations should help to give further insights into species assignments within the genus, and clarify the phylogeny and sister group relationships of the Scutoverticidae.

Acknowledgements

We want to thank the following persons and institutions: Jaroslav Smrž (Praha) for the loan of material from the acarological collection of M. Kunst, Peter Horak and Ernst Ebermann

(Graz) for collecting samples of mosses, and for the help of our molecular genetic lab team in some methological questions; furthermore the head of the Research Institute for Electron

Microscopy, Technical University Graz, and his staff for assistance with the SEM micrographs. This work is funded by the Austrian Science Fund (FWF), project number

P19544-B16.

42 2. Description of Scutovertex pileatus sp. nov.

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Weigmann, G., 2004. Recovery of the oribatid mite community in a floodplain after decline due to

long-term flooding. In: Weigmann, G., Alberti, G., Wohltmann, A., Ragusa, S. (Eds.)

Phytophaga XIV: Acarine Biodiversity in the Natural and Human Sphere: Proceedings of the

Vth Symposium of the European Association of Acarologists, Berlin, pp. 201-207.

Weigmann, G., 2006. Die Tierwelt Deutschlands, 76. Teil: Hornmilben (Oribatida). Goecke and

Evers, Keltern.

Willmann, C., 1953. Neue Milben aus den östlichen Alpen. Sitzberichte der Österreichischen

Akademie der Wissenschaften mathematisch-naturwissenschaftliche Klasse, Abteilung I 162,

449-519.

Woas, S., 1998. Mosaikverteilung der Merkmale basaler Höherer Oribatiden – Die Gattungen

Passalozetes und Scutovertex (Acari, Oribatei). In: Ebermann, E. (Ed.) Arthropod Biology:

Contributions to Morphology, Ecology and Systematics. Biosystematics and Ecology Series

14, pp. 291-313.

45

CHAPTER

3

Contrasting mitochondrial DNA diversity estimates in Austrian Scutovertex minutus and S. sculptus (Acari, Oribatida, Brachypylina, Scutoverticidae)

Sylvia Schäffer, Stephan Koblmüller, Tobias Pfingstl, Christian Sturmbauer & Günther Krisper

Pedobiologia (2009) in press 3. MtDNA diversity patterns in Austrian Scutovertex

Abstract

Important insights into the evolutionary and demographic history of species can be obtained from inter-specific comparisons of patterns and degree of genetic diversity. Analysis of sequences of the mitochondrial cytochrome oxidase I (COI) gene revealed remarkable differences in mtDNA diversity estimates and the distribution of mitochondrial lineages between the two closely related oribatid mite species Scutovertex sculptus and S. minutus in

Austria. Divergence time estimates revealed an age of approximately 48–74 Myr for the split between the two Scutovertex species and age estimates of about 2-3 and 8-12 Myr for the most recent common ancestors of Austrian S. minutus and S. sculptus, respectively. Genetic diversity was considerably lower in S. minutus than in S. sculptus. A clear geographic subdivision into samples north and south of the Central Alps became evident in S. minutus, whereas no phylogeographic structure was found in S. sculptus. Together with a high genetic diversity this is suggestive of a panmictic population and exceptional dispersal ability, most probably facilitated by phoresy on birds. The lack of sub-structure with regard to habitat types in S. sculptus suggests that this species can cope with a wide range of environmental conditions.

Key words. COI gene, intraspecific genetic diversity, phoresy, phylogeography, Scutovertex

- 46 - 3. MtDNA diversity patterns in Austrian Scutovertex

Introduction

Patterns and degree of intraspecific genetic diversity can provide important information for deducing the evolutionary history of species and are assumed to reflect population size, ecology, and the species’ ability to adapt to different environments. Several biological and demographic factors such as population structure, population bottlenecks, natural selection, life cycle, and reproductive mode affect genetic diversity (Caballero and

Hill, 1992; Charlesworth et al., 1993; O’Brien, 1994; Hedrick, 2005; Subramanian, 2009).

The oribatid mite family Scutoverticidae is assigned to a subgroup of the

Circumdehiscentiae (”Higher Oribatida”) at the base of the Poronota and is characterized by wrinkled nymphs and adults lacking pteromorphae (Grandjean, 1954). To date, the family comprises eight genera with more than 60 species worldwide, whereby Scutovertex is the most speciose. The two most common members of this genus are Scutovertex sculptus and S. minutus with a widespread Palaearctic distribution. These species have probably been confused in the past, but recent re-descriptions should prevent misidentification (Schäffer and

Krisper, 2007; Pfingstl et al., 2008). Both species have similar body size and an approximately six month-long generation time (Grafeneder pers. comm.). Furthermore, similar to S. sculptus, S. minutus seems to be panphytophagous (Smrž, 2006). Their preferred habitats are sun-exposed mosses and lichens, implying extreme living conditions for the mites. Thus, it is important for these species to be able to tolerate rapid environmental changes such as desiccation, inundation and temperature fluctuation (Smrž, 1992). Their adaptation to such extreme environmental conditions allows them to play a major role as pioneer organisms. For example, S. sculptus has been recorded on different kinds of dumps

(Skubała, 1999, 2004), on saline soils and in salt marshes (Seniczak et al., 1985; Schuster,

1959) or other dry habitats (Schatz, 1995; Migliorini and Bernini, 1999). Scutovertex minutus is commonly found on rocks and artificial habitats such as roofs (Smrž, 1992), walls (Steiner,

- 47 - 3. MtDNA diversity patterns in Austrian Scutovertex

1995a) or roadside slopes (Eitminaviciute, 2006), and sometimes on tree trunks (Pschorn-

Walcher and Gunhold, 1957). It has been also reported to be a pioneer species in a chalk quarry in England (Parr, 1978). Moreover, there is evidence that scutoverticid mites, and in particular the species included in the present study, might be important bioindicators, e.g. of air pollution (Weigmann and Kratz, 1987; Steiner, 1995a-b).

The present study sets out to achieve two goals. First, it aims to test for potential differences in the patterns of genetic diversity in S. minutus and S. sculptus in Austria. Given their high similarity in most life history traits we hypothesize that, albeit both species occur in different types of habitat, they do show similar patterns and degrees of genetic variation and that both species show a clear phylogeographic structure, assuming that they are small moss- dwellers with low dispersal ability. Second, this study investigated the possibility of intra- specific sub-structuring reflecting a potential specialization to different habitat types populated in S. sculptus. We hypothesize that S. sculptus populations living in mosses on rocks are differentiated from those inhabiting mosses on saline soils due to a potential adaptation to the markedly different environmental conditions in these habitats. To test these hypotheses we analyzed sequences of the mitochondrial cytochrome oxidase I (COI) gene, which has been widely used for resolving phylogenetic and evolutionary questions in mites

(Salomone et al., 2002; Heethoff et al., 2007; Ros and Breeuwer, 2007; Marangi et al., 2009).

Material and Methods

Sampling and DNA extraction

In this study we investigated 48 specimens of S. sculptus and 35 of S. minutus, collected from a total of 18 localities in Austria between 2005 and 2008 (Fig. 1 and Table 1). Specimens were extracted from mosses and lichens with Berlese-Tullgren funnels and preserved in absolute ethanol. Species identification was done using the criteria defined in Schäffer and

Krisper (2007) and Pfingstl et al. (2008). Total genomic DNA was extracted from single

- 48 - 3. MtDNA diversity patterns in Austrian Scutovertex individuals applying a modified CTAB (hexadecyltriethylammonium bromide) method

(Boyce et al. 1989) (for details see Appendix). After DNA extraction, the sclerotized body remnants were mounted on permanent slides and used for sex determination.

PCR and DNA sequencing

The mitochondrial COI gene was amplified using the primers COI_Fsy (5´-

GNTCAACAAWTCATWAAG-3´) and COI_Rsy (5´-

TAAACTTCNGGYTGNCCAAAAAATCA-3´) for COI-region1 (modified after Heethoff et al., 2007), Mite COI-2F and Mite COI-2R (Otto and Wilson, 2001) for COI-region2.

Polymerase chain reaction (PCR), purification of PCR products, and sequencing followed the protocol described in Schäffer et al. (2008). Sequences are available from GenBank under the accession numbers listed in Table 1.

Fig. 1. (a) Map of Austria (modified from www.lib.utexas.edu/maps/europe/austria.gif) with sampling sites. (b) Lakes of the “Seewinkel” in the surroundings of Lake Neusiedl (Burgenland). z = Scutovertex sculptus, ‹ = S. minutus, „ = S. pannonicus. Number code of sampling sites same as in Table 1. Each Austrian province is marked by one color.

- 49 - 3. MtDNA diversity patterns in Austrian Scutovertex

Table 1. List of studied haplotypes (h) with specimen identification, sex, sampling locality, habitat type and GenBank Accession Numbers. M = mosses, L = lichens, T = tussocks

h Species Sex Sampling locality with Habitat types Accession identification number code No. 1 SmPo1+3 X, ♂ Pogier - m roof of an old house (M) GQ890361/ GQ890362 1 SmBach1-6 1-3 ♀;5-6 X Bachsdorf - p roof of a stable (M) GQ890379 - GQ890384 1 SmBach9-13 ♀,♀,♀,♂,♀ Bachsdorf - p roof of a stable (M) GQ890385 - GQ890389 2 SmOe1, 5+6 X, ♀, ♂ Öblarn - k stone wall by riverbank GQ890363 - (M) GQ890365 3 SmA1, 5-7 ♀,♂,♀,♂ Asparn - e tiled roof (M, L) GQ890366 - GQ890369 3 SmUsb4 ♀ Unterstinkenbrunn - d roof of an old house (M) GQ890395 4 SmE3 X Ernstbrunn - f rocks (M, L, T) GQ890370 5 SmKal1-4 X, ♀,♀,♀ Graz - o rocks (M) GQ890371 - GQ890374 5 SmLi1 ♂ Ligist - n stone wall (M) GQ890391 Scutovertex minutus minutus Scutovertex 5 SmGlb4 ♀ Bad Gleichenberg - q rocky platforms (M) GQ890376 6 SmGlb1 ♀ Bad Gleichenberg - q rocky platforms (M) GQ890375 7 SmGlb7 X Bad Gleichenberg - q rocky platforms (M) GQ890377 8 SmUsb1 X Unterstinkenbrunn - d roof of an old house (M) GQ890392 8 SmGlb3 ♀ Bad Gleichenberg - q rocky platforms (M) GQ890378 9 SmIb1 ♀ Innsbruck - b rocks (M) GQ890390 10 SmUsb2 ♀ Unterstinkenbrunn - d roof of an old house (M) GQ890393 10 SmUsb3 ♀ Unterstinkenbrunn - d roof of an old house (M) GQ890394 11 SsI_C1 ♀ Illmitz - Zicklacke - i saline soil (M) GQ890396 12 SsI_C5 X Illmitz - Zicklacke - i saline soil (M) GQ890397 13 SsI_C7 X Illmitz - Zicklacke - i saline soil (M) GQ890398 14 SsI_C8 ♂ Illmitz - Zicklacke - i saline soil (M) GQ890399 15 SsI_C9+11 ♀, ♀ Illmitz - Zicklacke - i saline soil (M) GQ890400 - GQ890433 16 SsI_C10 ♀ Illmitz - Zicklacke - i saline soil (M) GQ890401 17 SsI_D2 X Illmitz - Lange Lacke - j saline soil (M) GQ890402 18 SsI_D7+16 ♀, ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890403 - GQ890410 19 SsI_D9 X Illmitz - Lange Lacke - j saline soil (M) GQ890404

tus 20 SsI_D10 ♂ Illmitz - Lange Lacke - j saline soil (M) GQ890405 lp 21 SsI_D11 ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890406 cu s 22 SsI_D12 X Illmitz - Lange Lacke - j saline soil (M) GQ890407 23 SsI_D14 ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890408 vertex

o 24 SsI_D15 ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890409 t u

c 25 SsE4+7 ♀, ♂ Ernstbrunn - f rocks (M, L, T) GQ890411 - S GQ890414 26 SsE5+6 ♂, X Ernstbrunn - f rocks (M, L, T) GQ890412 - GQ890413 27 SsRF1 ♀ Ruin Falkenstein - c rocks (M) GQ890415 28 SsRF2+4+6 ♀, X, ♀ Ruin Falkenstein - c rocks (M) GQ890416 - GQ890418 29 SsFer1 ♂ Ferlach - r rocks (M) GQ890419 30 SsFer2 ♀ Ferlach - r rocks (M) GQ890420 30 SsI_D13 X Illmitz - Lange Lacke - j saline soil (M) GQ890432 31 SsFer3 ♂ Ferlach - r rocks (M) GQ890421 32 SsFer4 ♀ Ferlach - r rocks (M) GQ890422

- 50 - 3. MtDNA diversity patterns in Austrian Scutovertex

33 SsI_A5 ♀ Illmitz - copse - h fine-grained forest soil GQ890423 (M) 34 SsI_A6 ♀ Illmitz - copse - h fine-grained forest soil GQ890424 (M) 35 SsI_A8 ♂ Illmitz - copse - h fine-grained forest soil GQ890425 (M) 36 SsI_C16+17 ♂, ♀ Illmitz - Zicklacke - i saline soil (M) GQ890436 - GQ890437 36 SsI_B5 ♀ Illmitz - "Hölle" - g saline soil (M, T) GQ890426 37 SsI_B6 ♀ Illmitz - "Hölle" - g saline soil (M, T) GQ890427 38 SsI_B7 ♀ Illmitz - "Hölle" - g saline soil (M, T) GQ890428 39 SsI_C15 ♀ Illmitz - Zicklacke - i saline soil (M) GQ890435 39 SsI_B8 ♂ Illmitz - "Hölle" - g saline soil (M, T) GQ890429 40 SsI_D4 ♂ Illmitz - Lange Lacke - j saline soil (M) GQ890430 41 SsI_D5 ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890431 42 SsI_C13 X Illmitz - Zicklacke - i saline soil (M) GQ890434 43 SsI_C18 ♀ Illmitz - Zicklacke - i saline soil (M) GQ890438 44 SsHlb1 X Häuslberg - l free-standing boulder (M) GQ890439 45 SsHlb2 ♂ Häuslberg - l free-standing boulder (M) GQ890440 46 SsFliess3 ♀ Fließ - a arid grassland (M, T) GQ890441 47 SsFliess4 X Fließ - a arid grassland (M, T) GQ890442 48 SsFliess1 ♂ Fließ - a arid grassland (M, T) GQ890443 out- SpaI_C6 X Illmitz - Zicklacke - i saline soil (M) GQ890444 group SpaI_B8 ♀ Illmitz - Lange Lacke - j saline soil (M) GQ890445

♀ = female, ♂ = male, X = unidentified

Data analysis

Sequence verification was performed by comparisons with known mite COI sequences in GenBank. Sequences were aligned by eye in MEGA 3.1 (Kumar et al., 2004) and number of haplotypes, haplotype (Hd) and nucleotide diversity (π), as well as rates of synonymous

(KS) and non-synonymous (KA) substitutions were calculated in DnaSP v4.50.3 (Rozas et al.,

2003). To assess their statistical significance, inter-specific differences in Hd and π were tested against a null distribution obtained by randomizing sequences between species (1000 permutations) and recalculating these indices (Muñoz-Fuentes et al., 2005). To test for selection based on the KA/KS ratio, we applied the Kumar method (Nei and Kumar, 2000;

Kumar et al., 2001) as implemented in MEGA, assessing the statistical significance by means of 1000 bootstrap replicates.

- 51 - 3. MtDNA diversity patterns in Austrian Scutovertex

Phylogenetic inference was based on maximum parsimony (MP) and Bayesian inference (BI), conducted in PAUP* 4.02a (Swofford, 2002) and MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003), respectively. Preliminary phylogenetic studies (Schäffer et al., unpublished) showed that S. pannonicus - another species of the genus Scutovertex – is closely related to the two studied species and therefore served as outgroup. Heuristic tree search under MP criteria applied random addition of taxa and TBR branch swapping (1000 replicates). Statistical support for the resulting topologies was assessed by bootstrapping

(1000 pseudo-replicates). For Bayesian inference of phylogeny the data were partitioned by codon position with the GTR+I+G model (Rodríguez et al., 1990) applied to each partition.

Posterior probabilities were obtained from a Metropolis-coupled Markov chain Monte Carlo simulation (2 independent runs; 8 chains with 7 million generations each; chain temperature:

0.2; trees sampled every 100 generations). Chain stationarity and run parameter convergence were checked using Tracer v1.4 (Rambaut and Drummond, 2007; available at http:// beast.bio.ed.ac.uk/Tracer/). The first 10% of all sampled trees were discarded as burn-in before calculating a 50% majority rule consensus tree from the remaining trees.

To test for rate constancy among all taxa, and hence, to justify the use of a molecular- clock model for estimating divergence times, we performed a Bayesian relative rates test according to the method described by Wilcox et al. (2004). The posterior probability distribution of branch lengths for all branches was obtained by saving branch lengths for every 100th sampled tree (after burn-in) of the MrBayes run. For each tree the distance from the most recent common ancestor (MRCA) of the ingroup to each of the terminal taxa was calculated with Cadence v.1.0.1 (Wilcox et al., 2004; available at http://www.biosci.utexas.edu/antisense/). The distribution of branch lengths was plotted in the program SPSS ver. 16.0. Since all distance estimates from the MRCA to the terminal taxa had broadly overlapping distributions (Fig. 2), we concluded that there are no significant substitution rate differences between the taxa (see Wilcox et al., 2004), justifying the

- 52 - 3. MtDNA diversity patterns in Austrian Scutovertex application of a strict molecular clock. Divergence time estimates were based on ML- corrected pairwise distances calculated in PAUP*, applying the best-fitting substitution model

HKY+I+G (Hasegawa et al., 1985; base frequencies: A = 0.2239, C = 0.1603, G = 0.1972, T

= 0.4185; ti/tv ratio = 10.7349; proportion of invariable sites: I = 0.5802; gamma shape parameter: α = 0.6969) selected by the hierarchical likelihood ratio test (hLRT) implemented in Modeltest 3.06 (Posada and Crandall, 1998). To translate genetic distances into absolute age estimates we applied a divergence rate of 1.5-2.3 % Myr-1, which is commonly used in arthropods (Brower, 1994; Juan et al., 1996; Quek et al., 2004) and has been also shown to be applicable for divergence time estimations in oribatid mites (Salomone et al., 2002; Heethoff et al., 2007).

Nucleotide sequences were translated into amino acid sequences in DAMBE (Xia and

Xie, 2001) using the invertebrate mitochondrial genetic code. An amino acid BIONJ tree was calculated in PALM (Rice et al., 2000; Guindon and Gascuel, 2003; online version at http://palm.iis.sinica.edu.tw/contact.html), applying MtArt (Abascal et al., 2007) as the best- fit substitution model selected by the Akaike Information Criterion (AIC) implemented in

ProtTest (Abascal et al., 2005). Nodal support was assessed via bootstrapping (100 pseudo- replicates).

Fig. 2. Distribution of branch lengths from the most recent common ancestor (MRCA) to the terminal taxa (outgroup was excluded). ‹ = 95 % confidence interval.

- 53 - 3. MtDNA diversity patterns in Austrian Scutovertex

Results DNA polymorphism

We amplified two fragments of the COI gene - region 1 with 659 bp and region 2 with

622 bp - overlapping at 22 bp. The resulting fragment had a length of 1259 bp corresponding to the positions 37-1296 of the Steganacarus magnus COI gene and 13-432 of the S. magnus

COI protein (Domes et al., 2008). The sequences were free of gaps and no internal stop codons were detected, thus, we can exclude the possibility of pseudogene amplification. In total, 38 Scutovertex sculptus, ten S. minutus and two S. pannonicus (outgroup) haplotypes were identified. Average nucleotide frequencies of S. minutus haplotypes were 41.4% for T,

16.6% for C, 21.4% for A, 20.7% for G and of S. sculptus 40.7% for T, 18.6% for C, 21.1% for A, 19.7% for G. The high AT bias in the COI gene is a common feature for insects (Simon et al., 1994) and is similar to that observed in other studies on mites (Salomone et al., 2002;

Mortimer and Jansen van Vuuren, 2007; Marangi et al., 2009). Both Hd (0.989 ± 0.007 versus

0.818 ± 0.048 in S. sculptus and S. minutus, respectively) and π (0.05173 ± 0.00447 and

0.01854 ± 0.00256 in S. sculptus and S. minutus, respectively) differed significantly between the two Scutovertex species (P < 0.001). Pairwise sequence divergence (uncorrected p- distance) within S. minutus and S. sculptus ranged from 0 to 4 % and 0 to 11 %, respectively.

ML corrected distances ranged from 0 to 4 % within S. minutus and from 0 to 25 % within S. sculptus.

The relationship between KS and KA was very similar in both species (Fig. 3a-c), indicating that there are no inter-specific differences in selection on the COI gene. The test for selection significantly rejected the null-hypothesis of neutral evolution in favor of purifying selection (KA < KS; P < 0.001).

- 54 - 3. MtDNA diversity patterns in Austrian Scutovertex

Fig. 3. Relationship between synonymous (KS) and non-synonymous (KA) substitutions of the COI gene for all pairwise comparisons of (a) S. minutus (b) S. sculptus (c) the whole dataset.

Phylogenetic analysis

MP analyses yielded 12 equally most parsimonious trees with a length of 876 steps (CI excluding uninformative characters = 0.5645; RI = 0.8950; RC = 0.5119). The strict consensus tree of the 12 most parsimonious trees is shown in Fig. 4a. Despite some topological disagreements which exclusively concerned nodes that received only low statistical support (in at least one of the two analyses), the BI tree (Fig. 4b) shows an overall pattern congruent with the MP tree: within S. minutus a clear indication for a sub-division into northern and southern populations became evident, with three exceptions [haplotypes 7

(specimen from Bad Gleichenberg) and 8 (shared between individuals from Bad Gleichenberg and Unterstinkenbrunn) form an own clade and haplotype 9 (from Innsbruck) clusters with the specimens of the northern populations], suggesting that lineage sorting has not yet been completed. In contrast, there was no indication of geographic sub-structure within S. sculptus, although there were two well supported and highly divergent lineages, whereby one was represented by only four haplotypes of three populations (Illmitz-Zicklacke, Illmitz-Lange

Lacke, Ferlach) whereas the second S. sculptus lineage comprised the majority of samples.

- 55 - 3. MtDNA diversity patterns in Austrian Scutovertex

Fig. 4. Phylogenetic relationships among the Scutovertex sculptus and S. minutus haplotypes identified in this study, using S. pannonicus as outgroup, based on sequences of the mitochondrial COI gene: (a) MP tree; strict consensus tree of 12 most parsimonious trees. (b) BI tree. Bold numbers at nodes represent bootstrap values for MP (> 50 are shown) and posterior probabilities for BI (≥ 70 are shown). Italic numbers indicate the age (Myr) for the split of S. minutus and S. sculptus and for the MRCAs of the two species. Each symbol (same as in Fig. 1) represents one specimen. Colors and number code of populations refer to sampling localities in Fig. 1.

- 56 - 3. MtDNA diversity patterns in Austrian Scutovertex

Divergence time estimates revealed an age of 48.03 – 73.64 Myr (corresponding to a

ML corrected pairwise distance of 110.46 %) for the split between S. minutus and S. sculptus and age estimates of 1.93 – 2.96 Myr (ML distance, 4.44 %) and 7.84 – 12.02 Myr (ML distance, 18.03 %) for the MRCA of Austrian S. minutus and S. sculptus, respectively.

The BIONJ tree obtained from amino acid sequences is shown in Fig. 5. As is expected for amino acid sequences of closely related taxa, the tree was poorly resolved as compared to the trees based on nucleotide sequences. Nevertheless, again a clear subdivision into northern and southern populations within S. minutus became evident, whereas S. sculptus lacked any clear phylogeographic structure. The four S. sculptus samples of the highly divergent mtDNA lineage were again recovered as a distinct monophylum.

Fig. 5. BIONJ tree based on amino acid sequences. Bootstrap values > 50 are shown. Colors and number code of populations refer to sampling localities in Fig. 1.

- 57 - 3. MtDNA diversity patterns in Austrian Scutovertex

Discussion

Inter-specific differences in genetic diversity

Examining intraspecific diversity of congeneric species can reveal important insights into their evolutionary and demographic history (Baker et al., 1999; Fedorov, 1999; Yu et al.,

2004). This study detected a remarkable difference in mtDNA diversity between two closely related scutoverticid mite species on a regional scale, with diversity estimates in Austrian

Scutovertex sculptus significantly exceeding those in S. minutus. Given that population size varies considerably between taxa, abundant species are expected to be on average more polymorphic than rare species, regardless of the “noise” introduced by other evolutionary forces (Amos and Harwood, 1998). But with our sampling design a quantitative analysis of the population sizes was not possible and so we can not say which species is more abundant.

Concordant with the observed inter-specific differences of genetic diversity, the

MRCA estimate of Austrian S. sculptus resulted in roughly four times the evolutionary age than for Austrian S. minutus (7.84 – 12.02 versus 1.93 – 2.96 Myr). The split between S. minutus and S. sculptus was estimated to an age of 44.84 ± 6.03 Myr, highlighting their longstanding separate evolutionary history. Recent data on the phylogenetic relationships among European scutoverticid mites showed that S. minutus and S. sculptus do not represent sister taxa but that several recently described and potentially undescribed species branch off in between (Schäffer et al. submitted).

Our results indicate a fine-scale geographic sub-structuring with little intra-population genetic variation in Austrian S. minutus and a large panmictic population of S. sculptus with very divergent haplotypes at single sampling sites. Differential selective pressure on the COI gene can be excluded as potential cause for the observed differences given similar relationships between KA and KS in both species (Fig. 3). The inferred negative selection (KA <

KS) is congruent to the patterns reported from other arthropods (DeSalle et al., 1987; Brower,

1994; Salomone et al., 2002; Heethoff et al., 2007). The observed differences in genetic

- 58 - 3. MtDNA diversity patterns in Austrian Scutovertex diversity might be explained by differences in the reproductive mode and/or different dispersal abilities and colonizing capacities. Grandjean (1941) reported on geographic variation in the sex ratio in S. minutus and stated the possibility of different reproductive modes within the genus Scutovertex (thelytokous parthenogenesis versus sexual reproduction). Evidence from various studies on other organisms suggests that parthenogenetic populations predominantly occur in marginal habitats such as high latitudes and altitudes or in island-like habitats, and in environments classified as stressful, transient or disturbed (Cuellar, 1977; Glesener and Tilman, 1978; Bierzychudek, 1985; Haag and Ebert,

2004; Kearney, 2005) and that asexual or self-fertilizing populations or species are typically characterized by a reduced genetic diversity (Jensen et al., 2002; Sweigart and Willis, 2003;

Kearney, 2005; Kearney and Blacket, 2008; Zierold et al., 2009). The majority of S. minutus samples included in the present study originated from artificial structures such as roofs or man-made stonewalls (Table 1), both of which represent rather recent structures and might be classified as marginal habitats. Albeit the sex-ratio of our S. minutus samples was clearly female biased (Table 1), we do not think that differences in the reproductive mode are primarily responsible for the different diversity measures in S. minutus and S. sculptus, because quite a few male individuals were identified in most populations. Thus we hypothesize that the observed pattern might be caused by differences in dispersal ability and/or colonizing ability.

Oribatid mites are considered to be poor dispersers (Weigmann, 1982) supporting our a priori hypothesis of low dispersal ability and hence low genetic diversity. However, this seems to be only true for S. minutus. Low overall genetic diversity is accompanied by clear phylogeographic structure, indicative of a limited potential to disperse. Scutovertex minutus populations show a subdivision into samples north and south of the Central Alps, which apparently curb gene flow in this species, whereas no phylogeographic structure was found in

S. sculptus. Furthermore, more than one haplotype was observed in only two out of the ten

- 59 - 3. MtDNA diversity patterns in Austrian Scutovertex populations of S. minutus included in our study (Unterstinkenbrunn and Bad Gleichenberg), corroborating limited dispersal. However, we note that two populations (Ligist and Innsbruck) only comprise one individual, indicating that species density varies considerably among sampling sites. The obvious lack of phylogeographic structure and the significantly higher genetic diversity in S. sculptus point to an exceptionally high dispersal ability, which seems quite surprising given the consideration of Weigmann (1982) and contrasts the high degrees of population sub-structuring typically found in small terrestrial flightless arthropods

(Salomone et al., 2002; Timmermans et al., 2005; Stevens and Hogg, 2006; McGaughran et al., 2008; Caterino and Chatzimanolis, 2009).

Although the possibility of dispersal by wind or water might seem obvious, considering the small size and little weight of the mites, such modes of dispersal are not common in Oribatida (Seyd, 1962). Oribatid mites might inadvertently attach to nest-dwelling vertebrates (Miko and Stanko, 1991), and a few taxa were shown to actively disperse by phoresy on arthropods (e.g. Norton, 1980; Townsend et al., 2008). There is some indication that scutoverticid mites disperse by phoresy on arthropods (Niemi, 1995). However, this dispersal mechanism is insufficient for dispersal over longer distances. Thus, we hypothesize that the most likely way of dispersal in S. sculptus is by phoresy on birds. Several studies revealed that Arctic birds (Lebedeva and Krivolutsky, 2003) and many non-passerines birds and Passeriformes (Krivolutsky and Lebedeva, 2004a, 2004b) carry oribatid mites: Amongst other species, S. minutus was found sporadically in feathers of the common eider (Somateria mollissima) and great black-backed gull (Larus marinus), both marine birds, and of waxwing, house and tree sparrows or nests of bluethroat (Luscinia svecica) and chaffinch (Fringilla coelebs). These data point to the possibility that S. sculptus might be distributed in this way, especially when considering that it is unfortunately always questionable if the specimens found in the above mentioned studies and classified as S. minutus were identified correctly

(see introduction).

- 60 - 3. MtDNA diversity patterns in Austrian Scutovertex

Additionally, differences in colonizing capacity might be responsible for the observed phylogeographic patterns. Colonizing success depends on reproductive mode, mobility, fertility, resource availability and habitat characteristics (Debouzie et al., 2002). In laboratory microcosms, Domes et al. (2007) investigated re-colonization of defaunated soil and litter by sexual and parthenogenetic oribatid mites. Contradicting general theory, they showed that parthenogenetic species are not faster colonizers. In general, sexual species react better on resource depletion and can adapt more easily on unstable conditions. Not surprisingly, generalists such as panphytophages and eurytopic species are dominant in colonizing new habitats (Wallwork, 1983; Skubala, 1995). The high colonizing capacity of S. sculptus was further shown by Skubala (1999) who showed that this species can occur in high numbers on dolomitic and industrial dumps, representing early successional soils.

Influence of different habitat types on population structure in S. sculptus

Since S. sculptus is found in various, often extreme types of habitats we expected to find some degree of genetic differentiation among populations from different habitat types, in particular in light of recent evidence for the existence of several newly recognized Scutovertex species (Schäffer et al, 2008; Pfingstl et al., 2009; Schäffer et al., unpublished data) in

Europe. Even if there is no obvious morphological variation among S. sculptus specimens from different habitats (Pfingstl et al., 2008), markedly different environmental conditions might imply physiological adaptation to a particular environment, potentially resulting in a pronounced genetic structure (Pilot et al., 2006; Bahrndorff et al., 2008; Maraldo et al., 2008).

However, no clear phylogeographic structure was observed in S. sculptus. Specimens originating from extreme habitat types, such as the saline soils from Illmitz, were not grouped together. Indeed, every major phylogenetic lineage contains individuals from Illmitz.

Furthermore, it should be noted that all major mitochondrial lineages of European S. sculptus

- 61 - 3. MtDNA diversity patterns in Austrian Scutovertex were present in Austria and in particular in Illmitz (Schäffer et al., unpublished data) indicating that this species is able to cope with a wide range of environmental conditions.

Conclusions

This study demonstrates a remarkable difference in the patterns and degree of genetic diversity in two closely related scutoverticid mites in Austria. Low levels of diversity and high degrees of population sub-structuring in S. minutus point to a limited dispersal ability, whereas high genetic diversity and the lack of phylogeographic structure in S. sculptus are suggestive of a panmictic population with high colonizing capacity and dispersal ability, which is attributed to a potential phoresy on birds. However, more data are needed to confirm or reject this hypothesis with confidence. Within S. sculptus no sub-structuring with regard to different habitat types was evident, suggesting a high physiological potency of this species.

Acknowledgements

Financial support was provided by the Austrian Science Fund (FWF, project number P19544-

B16). We are grateful to E. Ebermann, G. Raspotnig, H. Schatz, J. Jagersbacher-Baumann and E. McCullough for providing moss samples for our study and we are indebted to the administration of the National Park “Neusiedler See-Seewinkel” for the permission to collect moss and soil samples. N. Grafeneder gave helpful information on the generation time. K.M.

Sefc kindly provided a perl-script, originally developed for Sefc et al. (2007), to assess the significance of inter-specific differences in haplotype and nucleotide diversity. We also want to thank S. Weiss for the helpful comments and linguistic correction of this manuscript.

Finally, we thank the anonymous reviewers for their helpful comments.

- 62 - 3. MtDNA diversity patterns in Austrian Scutovertex

Appendix

Extraction protocol

Individuals were collected alive and preserved in absolute ethanol until use. Single specimens were placed in an Eppendorf tube containing 500 µl CTAB buffer [1.4 M NaCl, 0.1 M Tris-

HCl, 0.02 M EDTA, 2 % hexadecyltriethylammonium bromide, 0.2 % 2-mercaptoethanol (pH

8)] and gently crushed with a fine metal probe. Then 5 µl proteinase K (1mg/ml-1) was added to the samples. The tubes were incubated at 55 °C for three hours. 500 µl of choroform was added to the sample and this mixture was shaken several times. Following centrifugation (13

200 rpm) for 10 min, the supernatant was decanted and stored. 250 µl fresh CTAB buffer was added to the chloroform phase and, after re-centrifugation, the supernatant was pooled with the supernatant produced in the previous step. Precipitation was carried out with 600 µl cold isopropanol at -20 °C for 2-12 hours. The precipitated DNA was spinned down, the supernatant was discarded and the pellet was washed first with 70 % and in a second step with

100 % ethanol. Finally the pellet was dried and re-suspended in 25 µl TE buffer. The remaining body remnants were transferred from chloroform, washed in ethanol and fixed in a microscopic slide for further morphological analyses.

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CHAPTER

4

Phylogenetic analysis of European Scutovertex

mites (Acari, Oribatida, Scutoverticidae)

reveals paraphyly and cryptic diversity:

A molecular genetic and morphological approach

Sylvia Schäffer, Tobias Pfingstl, Stephan Koblmüller, Kathrin A. Winkler, Christian Sturmbauer & Günther Krisper

Molecular Phylogenetics and Evolution (2010) in press 4. Phylogeny of European Scutovertex mites

Abstract

The soil and moss dwelling oribatid mite family Scutoverticidae is considered to represent an assemblage of distantly related but morphologically similar genera. We used nucleotide sequences of one mitochondrial (COI) and two nuclear (28S rDNA, ef-1α) genes, and 79 morphological characters to elucidate the phylogenetic relationships among eleven nominal plus two undescribed European mite species of the family Scutoverticidae with a particular focus on the genus Scutovertex. Both molecular genetic and morphological data revealed a paraphyletic genus Scutovertex, with S. pictus probably representing a distinct genus, and Provertex kuehnelti was confirmed as member of the family Scutoverticidae. Molecular genetic data confirmed several recently described Scutovertex species and thus the high species diversity within this genus in Europe and suggest that S. sculptus represents a complex of several cryptic species exhibiting marked genetic, but hardly any morphological divergence.

Key words. Oribatid mites, Scutovertex, multidisciplinary approach, phylogeny, paraphylum, cryptic species

- 72 - 4. Phylogeny of European Scutovertex mites

Introduction

In recent years, genetic studies have highlighted cryptic diversity in various groups of organisms, indicated by large genetic distances within traditionally recognized, sometimes even well-known, taxa (Edwards and Dimock, 1997; Hebert et al., 2004; Katongo et al., 2005; Kon et al., 2007; Mayer et al., 2007; Metzger et al., 2009). These cryptic species are so similar morphologically that they are almost or entirely indistinguishable based on morphological characters alone, albeit many cryptic species have been subsequently supported by subtle morphological differences found in post-hoc analyses of morphological data (Mathews et al.,

2008; Padial and de la Riva, 2009). The potential for cryptic diversity seems particularly high in small-size and short generation time animals (Marzluff and Dial, 1991; Kon et al., 2007).

Moreover, some biomes seem to home more cryptic species than others, and particularly in tropical and marine habitats it appears to be a widespread phenomenon (Baric and Sturmbauer,

1997; Wilcox et al., 1997; Bond and Sierwald, 2002; Hebert et al., 2004; Sáez and Lozano, 2005;

Chan et al., 2007), whereas the number of cryptic species in temperate terrestrial biomes seems to be smaller (Schlick-Steiner et al., 2006; King et al., 2008; Murray et al,. 2008).

In mites, species identification is typically based on morphological character sets. There are many species which are morphologically very similar and thus hardly to distinguish. One example for such a group including morphologically very similar taxa is the oribatid mite family

Scutoverticidae which is assigned to a subgroup of the Circumdehiscentiae (”Higher Oribatida”) at the base of the Poronota. This subgroup shows wrinkled nymphs and adults which bear sacculi on the notogaster homologous to the so called octotaxic system consisting of four pairs of porose areas characterizing the Poronota (Grandjean, 1954; 1969).

Despite their systematic position the Scutoverticidae are often considered as a conglomeration of distantly related but morphologically similar genera. Bernini (1976) already

- 73 - 4. Phylogeny of European Scutovertex mites pointed out the urgent need for a comprehensive and detailed revision of this family to check the membership of the different genera to this taxon, whereby Scutovertex as the eponymous genus should serve as reference. Meanwhile, several new taxa were described and Shtanchaeva and

Netuzhilin (2003) published a revision of the Scutoverticidae, describing some new species and summing up the knowledge without any attempt to include additional characters to eliminate the taxonomic uncertainties. Up to now the systematic classification within the Scutoverticidae has been suffering from two major problems: the short, fragmentary and often inaccurate descriptions of species and genera, and the limited knowledge of the amount of intraspecific variation and the diversity of this mite family. These two factors led to the description of new taxa (some of them may represent synonyms) and caused some taxonomical confusion, e.g. in the genus Provertex

(Krisper and Schuster, 2009).

To date, this family comprises eight genera with about 60 species worldwide (Subías,

2004; resp. 2008) whereof only one-third occurs in Europe. Most species can be found in the very south-western (Spain) or eastern (Russia) European part. They are adapted to extreme environmental conditions such as regular desiccation, inundation and temperature fluctuation because their preferred habitats are mosses, lichens or tussocks on sun exposed rocks and roofs

(Krisper et al., 2002; Smrž, 2006), as well as saline soils, salt marshes or inundation meadows

(Schuster, 1958; Weigmann, 2004). Due to their adaptation to extreme environmental conditions, scutoverticid mites play an important ecological role as pioneer species at the first steps of succession (Skubala, 1995). General information on life span, population size etc. is more or less lacking but most members of the family reproduce sexually and generation times vary from two to six months (Ermilov, 2008; personal observations). Recent data on the genetic diversity in two

Austrian species revealed marked inter-specific differences suggestive of differences in population size and dispersal ability (Schäffer et al., in press).

- 74 - 4. Phylogeny of European Scutovertex mites

Especially in the genus Scutovertex species diversity is very high. Problems to distinguish between the two most widespread species S. minutus and S. sculptus (e.g. mentioned by

Weigmann, 2006) were solved recently by detailed re-descriptions of both species (Schäffer and

Krisper, 2007; Pfingstl et al., 2008) and their taxonomic discreteness and different population structure was also confirmed by molecular genetic data (Schäffer et al., 2008). In Central,

Northern and Western Europe eight Scutovertex-species are known (S. alpinus, S. arenocolus, S. ianus, S. minutus, S. pictus, S. pileatus, S. pannonicus, and S. sculptus), some of which have been described only recently (Schäffer et al., 2008; Pfingstl et al., 2009, in press, submitted), plus five additional species representing three different genera (Lamellovertex caelatus; Exochocepheus hungaricus; Provertex delamarei, P. kuehnelti, P. mailloli).

In the present study we attempt to i) evaluate the taxonomic status of eleven nominal and two undescribed European species of the family Scutoverticidae ii) elucidate the phylogenetic relationships between the Scutovertex species and iii) uncover a potential cryptic diversity within the genus Scutovertex. To achieve our aims, we used three molecular markers (one mitochondrial and two nuclear genes) which allow us to resolve both ancient and recent nodes in a phylogenetic tree. Additionally 79 well defined characters and character states were used to obtain a morphology-based phylogeny for comparative purpose.

- 75 - 4. Phylogeny of European Scutovertex mites

Material and Methods

Sample collection

This study includes eleven nominal plus two undescribed species of the family

Scutoverticidae, collected from different localities in Central, Northern and Western Europe between 2005 and 2009. Based on Grandjean (1969) we chose Unduloribates undulatus

(Unduloribatidae) and three specimens of Cymbaeremaeus cymba (Cymbaeremaeidae) as outgroup taxa because both families also belong to the subgroup of Circumdehiscentiae with wrinkled nymphs. Information on sampling localities is given in Table1 and Fig. 1. Specimens were extracted from mosses and lichens collected on sun exposed rocks and roofs or salt marshes with Berlese-Tullgren funnels. Individuals for morphological analyses were preserved in 70% ethanol, those for molecular genetic analyses in absolute ethanol.

Morphological data

79 morphological characters or character states, respectively, (see Fig. 2) were recorded for five individuals per species. Character coding was based on unordered multistate characters

(Appendix A+B).

Phylogenetic reconstruction was carried out by MP using PAUP* (search options: heuristic search; random addition of taxa; TBR branch swapping with 1,000 replicates), using one specimen per species, since there were no intraspecific differences. Statistical support was assessed by bootstrapping (1,000 pseudo-replicates).

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Table 1. Specimens, sample ID, sampling location and GenBank accession numbers for the samples analyzed in this study. Car. = Carinthia.

GenBank Accession No. Sample Species COI 28S ef-1α Sampling locality ID Scutoverticidae Scutovertex S. alpinus SalpHT1 GU208673# GU208524 GU208619 Fuscherkarkopf/Großglockner/Car. - A SalpHT2 GU208674# GU208525 GU208620 Fuscherkarkopf/Großglockner/Car. - A SalpHT3 GU208675# GU208526 GU208621 Fuscherkarkopf /Großglockner/Car.- A S.arenocolus SarCoast3 GU208578 GU208527 GU208622 Darss-Zingst/Baltic Coast - D SarCoast7 GU208579 GU208528 GU208623 Darss-Zingst/Baltic Coast - D S. ianus SianSt5 GU208580 GU208529 GU208624 Stiwoll/Styria - A SianSch8 GU208581 GU208530 GU208625 Schladming/Styria - A SianAdm1 GU208582 GU208531 GU208626 Admont/Styria - A SianAu4 GU208583 GU208532 GU208627 Floodplain of Traun/Upper Astria - A SianAu5 GU208584 GU208533 GU208628 Floodplain of Traun/Upper Astria - A SianMos1 GU208585 GU208534 GU208629 Mosbach near Heidelberg - D S. minutus SmBach3 GQ890381* GU208535 GU208630 Bachsdorf/Styria - A SmPo3 GQ890362* GU208536 GU208631 Pogier/Styria - A SmKal3 GQ890373* GU208537 GU208632 Graz/Styria - A SmUsb4 GQ890395* GU208538 GU208633 Unterstinkenbrunn/Lower Austria - A S. pannonicus SpaI_B8 GQ890445* GU208539 GU208634 Lake " Zicklacke"/Burgenland - A SpaI_C6 GQ890444* GU208540 GU208635 Lake "Oberer Stinker"/Burgenland - A S. pictus SpKal6 GU208586 GU208541 GU208636 Graz/Styria - A SpBH9 GU208587 GU208542 GU208637 Castle Hochosterwitz/Carinthia - A S. pileatus SpilBH5 GU208588 GU208543 GU208638 Castle Hochosterwitz/Carinthia - A SpilL3 GU208589 GU208544 GU208639 Laas/Carinthia - A S. species1 SspHu5 GU208590 GU208545 GU208640 Fülöpháza/Kiskunság National Park - H SspHu6 GU208591 GU208546 GU208641 Fülöpháza/Kiskunság National Park - H S. species2 SspWa1 GU208592 GU208547 GU208642 Wangen am Ritten/South Tyrol - I SspWa2 GU208593 GU208548 GU208643 Wangen am Ritten/South Tyrol - I S. sculptus SsHlb2 GQ890440* GU208549 GU208644 Häuslberg/Styria - A SsI_B6 GQ890427* GU208550 GU208645 Lake "Oberer Stinker"/Burgenland - A SsI_C13 GQ890434* GU208551 GU208646 Lake "Zicklacke"/Burgenland - A SsFliess3 GQ890441* GU208552 GU208647 Fliess/Tyrol - A SsRu1 GU208594 GU208553 GU208648 Nizhniy Novgorod - RUS SsRu2 GU208595 GU208554 GU208649 Nizhniy Novgorod - RUS SsRu3 GU208596 GU208555 GU208650 Nizhniy Novgorod - RUS SsSW1 GU208597 GU208556 GU208651 Endeby near Uppsala - S SsSW2 GU208598 GU208557 GU208652 Endeby near Uppsala - S SsMos2 GU208599 GU208558 GU208653 Mosbach near Heidelberg - D SsIRL1 GU208600 GU208559 GU208654 Ceide-Fields - IRL SsF1 GU208601 GU208560 GU208655 Seignosse/Les Bourdaines - F SsF3 GU208602 GU208561 GU208656 Seignosse/Les Bourdaines - F SsXa1 GU208603 GU208562 GU208657 Xanten - D SsXa2 GU208604 GU208563 GU208658 Xanten - D

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Lamellovertex L. caelatus LcE3 GU208605 GU208564 GU208659 Ernstbrunn/Lower Austria - A LcE6 GU208606 GU208565 GU208660 Ernstbrunn/Lower Austria - A Provertex P. kuehnelti PkHT1 GU208607 GU208566 GU208661 F. Josef Höhe/Großglockner/Car. - A PkGe1 GU208608 GU208567 GU208662 Gesäuse/Styria - A PkIRL1 GU208609 GU208568 GU208663 Galway - IRL PkRom2 GU208610 GU208569 GU208664 Braşov-Bucegi mountains - RO Exochocepheus E. hungaricus EhungHu1 GU208611 GU208570 GU208665 Fülöpháza/Kiskunság National Park - H EhungHu4 GU208612 GU208571 GU208666 Fülöpháza/Kiskunság National Park - H EhungHu5 GU208613 GU208572 GU208667 Fülöpháza/Kiskunság National Park - H Unduloribatidae Unduloribates U. undulatus UuRom1 GU208614 GU208573 GU208668 Braşov-Bucegi mountains - RO UuRom3 GU208615 GU208574 GU208669 Braşov-Bucegi mountains - RO Cymbaeremaeidae Cymbaeremaeus C. cymba CcRoe3 GU208616 GU208575 GU208670 Röthelstein/Styria - A CcRoe5 GU208617 GU208576 GU208671 Röthelstein/Styria - A CcPlatte1 GU208618 GU208577 GU208672 Graz/Styria - A

#, Sequences from COI region2 fragment alone. *, Sequences not generated in the framework of this study were obtained from Schäffer et al. (in press).

Fig. 1. (a-b) Sampling localities of the specimens used in this study: (a) Map of Europe. (b) Map of Austria. Species are marked by different colors or symbols. z = Scutovertex alpinus z = S. arenocolus z = S. ianus z = S. minutus z = S. pannonicus z = S. pictus z = S. pileatus z = S. sp.1 z = S. sp.2 z = S. sculptus z = Lamellovertex caelatus z = Exochocepheus hungaricus z = Provertex kuehnelti

X = Cymbaeremaeus cymba „ = Unduloribates undulatus

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Fig. 2. (a-e) Schematic representation of morphological characters investigated in this study: (a) prodorsum (dorsal view); (b) leg with setae (lateral view); (c) lateral body view; (d) subcapitulum (ventral view); (e) ventral body view. Abbreviations: a, anterior subcapitular seta; AG, anogenital region; ag, aggenital seta; an, anal seta; Ap, apodem; bo, bothridium; bS, sensillus; C, claw; cl, lamella; cu, cusp; E, epimeral region; ex, exobothridial seta; GV, genital valve; g, genital seta; h, hysterostomatic seta; in, interlamellar seta; ia, im, ip, lyrifissures; le, lamellar seta; Le, lenticulus; LS, leg surface; m, median subcapitular seta; MR, rib on mentum; N, notogastral surface; P, prodorsal surface; PL, prodorsum lateral; Pp, pedipalp; PR, prodorsal ridges; PtI, PtII, pedotectum I/II; R, rostrum; RO, respiratory organ; ro, rostral seta; RU, rutellar teeth; S, saccule of the octotaxic system; TL, translamella; TU, tutorium; c1-3, da, dm, dp, la, lm, lp, h1-3, ps1-3, notogastral setae.

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Molecular genetic analyses

Total genomic DNA was extracted from single individuals applying the CTAB

(hexadecyltriethylammonium bromide) method described in Schäffer et al. (in press) or the

DNeasy Blood & Tissue Kit (Qiagen, Vienna, Austria).

Fragments of COI, ef-1α and 28S rDNA genes were amplified by polymerase chain reaction (PCR) using the following primers: COI_1fwd (5´-GNTCAACAAWTCATWAAG-3´) and COI_2rev (5´-TAAACTTCNGGYTGNCCAAAAAATCA-3´) for COI region1 (modified after Heethoff et al., 2007), Mite COI-2F and Mite COI-2R (Otto and Wilson, 2001) for COI region 2, D3A and D3B (Litvaitis et al., 1994) for the D3 fragment of the 28S rDNA, and 40.71F and 52.RC (Regier and Shultz, 1997) for ef-1α. Since last-mentioned primer pair did not work well in all specimens we designed new ones: EF-SyFwd (5´- GGACAAACTGAAGGHW

GAGMG-3´) and EF-SyRev (5´- RKNGGTCKTGAGGGCGGTTCC-3´). Purification of PCR products, and sequencing reaction followed the protocol described in Schäffer et al. (2008). DNA fragments were purified with SephadexTM G-50 (Amersham Biosciences) following the manufacturer’s instruction and visualized on a 3130xl capillary sequencer (Applied Biosystems).

Sequences are available from GenBank under the accession numbers listed in Table 1.

We sequenced 1,259 bp of the mitochondrial COI gene, 316-324 bp of the D3 region of the nuclear 28S rDNA and 504 bp of the nuclear ef-1α gene in 54 specimens (in the three S. alpinus individuals the fragment of the COI-region1 could not be amplified). Sequences were verified by comparisons with known oribatid sequences from GenBank and aligned by eye in

MEGA 3.1 (Kumar et al., 2004). One 18-26 bp fragment of the 28S D3 region could not be aligned unambiguously and was excluded (also base position 153 from SpKal6) from the analyses. For the further phylogenetic analyses we always used all available sequences for each gene fragment.

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In a first step, for testing the performance of the single genes, we constructed separate phylogenies for the three genes plus a phylogeny for the COI region2 (because of S. alpinus, see above) using Bayesian inference (BI) as implemented in MrBayes 3.1.2 (Ronquist and

Huelsenbeck, 2003). For all Bayesian analyses genes (except 28S rDNA) were partitioned by codon position. Rate heterogeneity was set according to a gamma distribution with six rate categories (GTR model) for each data partition. Posterior probabilities were obtained from a

Metropolis-coupled Markov chain Monte Carlo simulation (2 independent runs; 4 chains with 3 million generations each; chain temperature: 0.2; trees sampled every 100 generations), with parameters estimated from the data set. Depending on the data set we applied different burn-ins to allow likelihood values to reach stationarity, so that the average standard deviation of split frequencies was <0.01.

In a second step, the fragments of all three genes were combined for further analyses

(length of concatenated data set = 2,059 bp). We analyzed two data sets, one with all available sequences, entitled full set (FS) and one without COI region1, entitled reduced set (RS). The RS data set served to get information on the phylogenetic placement of S. alpinus. Phylogenetic reconstructions by neighbor joining (NJ) and maximum parsimony (MP) were conducted in

PAUP* (Swofford, 2002), maximum likelihood (ML) in RAxML-7.0.3-WIN (Stamatakis, 2006) and BI in MrBayes. For NJ of FS and RS, the best-fit substitution model selected by the hierarchical likelihood ratio test (hLRT) implemented in Modeltest 3.06 (Posada and Crandall,

1998) was GTR+I+G (parameters of FS/RS: base frequencies: A = 0.3057/0.3112, C =

0.1742/0.1749, G = 0.1604/0.1790, T = 0.3597/0.3349; R-matrix: A↔C = 0.6075/0.9541; A↔G

= 10.6077/8.1492; A↔T = 1.0908/1.1455; C↔G = 1.3283/1.3145; C↔T = 12.0869/12.9849;

G↔T = 1.0000; proportion of invariable sites: I = 0.6165/0.5742; gamma shape parameter: α =

0.6548/0.3888). Heuristic tree searches under MP criteria applied random addition of taxa and

- 81 - 4. Phylogeny of European Scutovertex mites

TBR branch swapping (1,000 replicates). Statistical support for the resulting NJ and MP topologies was assessed by bootstrapping (1,000 pseudo-replicates). To find the best-scoring ML tree we used the default algorithm with 40 distinct rate categories, the GTR+I+G substitution model and COI and ef-1α were partitioned by gene and by codon position. Nodes were supported by bootstrapping (500 replicates). Settings for Bayesian Inference were same as mentioned above and the partitioning of COI and ef-1α was the same as for ML analysis.

To assess whether the topologies obtained by the different tree building algorithms differed significantly, we performed Kishino-Hasegawa (KH; Kishino and Hasegawa, 1989) and

Shimodaira-Hasegawa (SH) tests (Shimodaira and Hasegawa, 1999) in PAUP*. Alternative phylogenetic hypotheses were compared to a strict consensus topology of the NJ, MP, ML and BI trees also by means of KH and SH tests.

A Bayesian relative rates test according to the method describe by Wilcox et al. (2004) was conducted to test for significant differences in branch lengths and hence substitution rates at the COI gene, which is commonly used to estimated divergence times in arthropods (Brower,

1994; Juan et al., 1996; Quek et al., 2004), and also in oribatid mites (Salomone et al., 2002;

Heethoff et al., 2007). The posterior probability distribution of branch lengths for all branches was obtained by saving branch lengths for every 100th sampled tree (after burn-in) of the

MrBayes run. For each tree the distance from the most recent common ancestor (MRCA) of the ingroup to each of the terminal taxa was calculated with Cadence v.1.0.1 (Wilcox et al., 2004; available at http://www.biosci.utexas.edu/antisense/). Scutovertex alpinus was excluded from this analysis because of the lacking COI region1 sequence. The distribution of branch lengths was plotted in the program SPSS ver. 16.0. Because of considerable variations in relative rates among the ingroup taxa (Fig. 3), we refrained from applying a molecular clock to estimate divergence

- 82 - 4. Phylogeny of European Scutovertex mites times (Wilcox et al. 2004). Given a lack of possible calibration points we were not able use relaxed clock models on our data to reliably estimate divergence times either.

Fig. 3. Results of the Bayesian relative rates test. The distribution of branch lengths from the most recent common ancestor (MRCA) to the terminal taxa (outgroup was excluded) is shown. Colors are the same as in Fig. 1.

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Results

Pairwise sequence divergence (uncorrected p-distance) between scutoverticid species ranged from 15 to 24 % in the COI gene, from 0 to 7.4 % in the D3 fragment of 28S rDNA, and from 0.6 to 9.6 % in the ef-1α gene. In the combined data set, pairwise differences ranged from 8 to 19 %.

Phylogenetic analyses based on Bayesian inference of the whole COI gene, COI region 2 and ef-1α (Figs. 3a, 3b and 3d) yielded similar results and revealed well resolved topologies with high statistical support for the monophyly of the family Scutoverticidae and the monophyly of each species except S. sculptus, whose specimens clustered in two well supported clades: one included individuals from Russia, Sweden, Germany and Austria (“sculptus1”) and one comprised individuals from France, Ireland and Germany (“sculptus2”). By contrast, the 28S rDNA gene showed only a poorly resolved phylogeny (Fig. 4c). Only the monophyly of the genus Scutovertex and of the remaining genera was well supported. Single gene analyses resulted in different relative phylogenetic positions of the “sculptus1” and “sculptus2” clades.

All analyses with the combined data sets revealed highly consistent topologies (Figs. 4a- d). Only slight differences were observed with respect to the tree building algorithm used. MP of the FS/RS yielded 12/2 most parsimonious trees with a length of 3,519/1,916 steps (CI excluding uninformative characters = 0.3224/0.3421; RI = 0.7372/0.7576; RC = 0.2413/0.2633). An evaluation of the phylogenetic hypotheses obtained from NJ, MP, ML and BI by means of KH and SH tests revealed no significant differences between the alternative topologies except for the

MP tree in the SH test (Table 2).

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Fig. 4. Phylogeny of eleven nominal plus two undescribed European species of the family Scutoverticidae based on single gene analyses. Bayesian inference (BI) tree of three studied genes: (a) mitochondrial COI; (b) COI region2; (c) nuclear 28S rDNA; (d) nuclear ef-1α. Only posterior probabilities >50 are shown. Colors are the same as in Fig. 1.

- 85 - 4. Phylogeny of European Scutovertex mites

Fig. 5. Phylogeny of eleven nominal plus two undescribed European species of the family Scutoverticidae based on the concatenated data set of all available fragments of the COI, 28S rDNA and ef-1α genes. (a) NJ tree using the GTR+I+G model; (b) strict consensus of 34 most parsimonious trees; (c) ML tree using the GTR+I+G; (d) Bayesian 50% majority rule consensus tree. Bootstrap values (for NJ, MP and ML), and posterior probabilities (for BI) are shown when >50. Colors are the same as in Fig. 1.

- 86 - 4. Phylogeny of European Scutovertex mites

Table 2. Comparison of alternative phylogenetic hypotheses.

Tree KH test SH test

tree s.d. (diff) t P -lnL Δ-lnL P NJ 9 11.87557 0.7579 0.4486 16875.99005 19.70894 0.105 BI best 16856.81429 0.53318 0.832 MP 20 13.48681 1.4829 0.1382 16886.10722 29.82611 0.026* ML 5 7.68222 0.6509 0.5152 16856.28111 best

Kishino-Hasegawa (KH; Kishino and Hasegawa, 1989) and Shimodaira-Hasegawa tests (SH; Shimodaira and Hasegawa, 1998) were used to assess whether the topologies of NJ, MP, BI and ML differed significantly. * P<0.05.

Compared to the other three tree topologies the MP tree showed a slightly different branching order (branching order among P. kuehnelti, L. caelatus, E. hungaricus and S. pictus; placement of S. pannonicus and S. sp. 2 (specimens from Wangen/South Tyrol) albeit with low bootstrap support. A strict consensus tree of NJ, MP, ML and Bayesian Inference is shown in

Figure 5a. Scutovertex alpinus was added manually based on its position in the analyses of the

RS data (data not shown). Within the family Scutoverticidae two main clusters became evident, one with species of the genus Scutovertex and one with the members of the three other genera P. kuehnelti, L. caelatus, E. hungaricus plus S. pictus, rendering the genus Scutovertex paraphyletic.

This result conforms to the morphology-based phylogeny (21 constant, 16 parsimony- uninformative and 42 parsimony-informative characters; 34 most parsimonious trees; tree length

= 139; CI excluding uninformative characters = 0.7080; RI = 0.7027; RC = 0.5359), in which S. pictus clusters between L. caelatus and E. hungaricus with P. kuehnelti as sister taxon (Fig. 6b).

The test enforcing a monophyletic genus Scutovertex in the molecular phylogeny with S. pictus

- 87 - 4. Phylogeny of European Scutovertex mites representing the most ancestral split, resulted in a significantly worse fit to the data (KH-test: tree length difference = 64 steps, s.d. = 10.77344, t = 5.9405, P = <0.0001; SH-test: Δ-lnL =

130.98973, P = 0.000). With the exception of S. sculptus, all species within the genus Scutovertex were recovered, with high statistical support, as monophyletic. The two “sculptus” clades resulted as sister taxa in all methods, except MP (Fig. 5b), which showed no resolution between S. sculptus, S. ianus and S. sp.1 specimens. Scutovertex pileatus and S. alpinus resulted as the most basal representatives of the genus Scutovertex. The phylogenetic relationship of the remaining species S. minutus, S. pannonicus, S. arenocolus and the undescribed species S. sp.2 with individuals from Wangen differs slightly depending on the method used. Unlike the molecular phylogeny, the morphological data (Fig. 6b) revealed, with good to high statistical support, the monophyly of all species but lacked resolution of the phylogenetic relationships among the

Scutovertex species (with exception of S. pictus as already mentioned).

Discussion

Molecular genetic and morphological data revealed well resolved phylogenies, demonstrating the monophyly of all species, with the exception of Scutovertex sculptus. Despite only minor morphological differences to congeneric species, all recently described Scutovertex species included in this study appeared as genetically distinct. Thus, morphological differentiation in the studied European scutoverticid mites is accompanied by high degrees of genetic differentiation, which is not necessarily the case in other oribatid mite families (e.g.,

Avanzati et al., 1994).

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Fig. 6. Phylogeny of eleven nominal plus two undescribed European species of the family Scutoverticidae. (a) Strict consensus of NJ, MP, ML and BI trees of the concatenated data set of all available fragments of the COI, 28S rDNA and ef-1α genes. Bootstrap values of NJ and MP are shown above the branches, bootstrap values for ML and posterior probabilities for BI below (only values >50 are shown). (b) Strict consensus tree of 34 most parsimonious trees based on 79 external morphological characters or character states. Bootstrap values >50 are shown. Stippled lines highlight investigated members of the genus Scutovertex. * = Support values from analyses of the RS data. Colors are the same as in Fig. 1.

The most important congruence between morphology and molecular genetic data concerned the phylogenetic reconstruction of the genus Scutovertex itself. Both trees revealed a paraphylum Scutovertex, supporting the morphology-based hypothesis of Sitnikova (1980) that S. pictus might not belong to the genus Scutovertex. Moreover, several morphological

- 89 - 4. Phylogeny of European Scutovertex mites characteristics – e.g., the shape of the bothridium, the absence of the lenticulus and the type of respiratory organs in the legs - clearly separate S. pictus from all other members of the genus. Its definite position is still unclear because in the molecular tree it clusters, depending on the tree building algorithm used, with different members of the three other European scutoverticid genera. However, we hypothesize, that S. pictus does not belong to any other known genus of the family Scutoverticidae but rather constitutes a new genus pursuant to its distinct morphological characters mentioned above.

A further important finding concerns the phylogenetic placement of P. kuehnelti. In the molecular tree it was placed in a lineage together with E. hungaricus, L. caelatus and S. pictus, a grouping strongly supported by high bootstrap and posterior probability values. This result contradicts Woas’ statement (2002) that the genus Provertex would belong to the

Cymbaeremaeidae because of sharing some morphological characters. With regard to our investigations, his argument seems not to be substantive as there is no close relationship between

P. kuehnelti and Cymbaeremaeus cymba in any of the phylogenetic trees. Even in our morphology-based phylogeny P. kuehnelti does not occupy the most ancestral branch within the

Scutoverticidae, further rejecting a close affinity to C. cymba.

Despite well resolved phylogenies and congruencies between both data sets, unexpected results emerged from the molecular genetic data. The most obvious one was the high genetic divergence among samples classified as S. sculptus: morphologically indistinguishable individuals were separated into two well supported clades, “sculptus1” and “sculptus2” (Figs. 5a- b). This separation became evident in both the mitochondrial COI gene and the nuclear ef-1α gene, whereas the 28S rDNA lacked resolution at this divergence level. These two clades are allopatrically distributed with “sculptus1” in Eastern and “sculptus2” in Western Europe (Fig. 1), pointing to an ancient geographic separation of these two clades. Moreover, despite their well

- 90 - 4. Phylogeny of European Scutovertex mites supported genetic distinctness, two unidentified specimens from Hungary (S. sp.1) showed close morphological resemblance to S. sculptus - e.g., cuticle and cerotegument structure of notogaster, shape of notogastral setae. Given the congruence among the different molecular markers, incomplete lineage sorting could be eliminated as possible cause for the patterns observed within

S. sculptus. Instead, our findings are consistent with the possibility that S. sculptus actually represents a complex of cryptic species. Which one is representing the “real” S. sculptus can not be answered in this study since neither samples from the holo- or paratypes of this species

(described by Michael, 1879) nor specimens from a location site in England (locus typicus) were available for our analyses.

There are several reasons why morphological characters might be not useful in discriminating species, but there appear to be two general and recurrent frames for cryptic species

(Bickford et al., 2007): they are either differentiated by nonvisual mating signals (Byers and

Struble, 1990; Henry, 1998; Feulner et al., 2006; Stuart et al., 2006) and/or appear to be under selection promoting morphological stasis (Vrijenhoek et al., 1994; Rothschild and Mancinelli,

2001; Lefébure et al., 2006; Finston et al., 2007). Regarding the first point, nonvisual mating signals could also be important in differentiating among the different S. sculptus lineages because within oribatid mites indirect sperm transfer occurs by means of spermatophores. For S. sculptus, the “completely dissociated transfer” after Proctor (1998) is applicable (Pfingstl, pers. observations), where males and females never meet, and chemical cues induce the uptake of spermatophores by the female. Moreover, since S. sculptus occurs in extreme environments such as saline soils, salt marshes and other very dry habitats, convergent evolution under harsh conditions in similar habitats likely produced similar morphologies in genetically distinct lineages (also see Vrijenhoek et al., 1994; Rothschild and Mancinelli, 2001; Lefébure et al.,

2006; Finston et al., 2007). However, this raises the question ´what is really “extreme”`?

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Therefore we want to conform to Rothschild and Mancinelli (2001) who stated ´all physical factors are on a continuum, and extremes in the conditions that make it difficult for organisms to function are ´extreme`` (p. 1093, lines 5-7). The main habitats of our investigated mite species are mosses and lichens on sun-exposed places. Considering that these habitats can both dry up and be flooded completely it is obvious that they are extreme for the specimens living in.

We emphasize that many European Scutovertex species are morphologically very similar and several species have been recognized only recently (Schäffer et al., 2008; Pfingstl et al.,

2009; Weigmann, 2009). A good example is the new species S. ianus (Pfingstl et al., submitted) which exhibits morphological character states similar to either S. minutus or S. sculptus. Taking only a short look at S. ianus would certainly lead to wrong species identification. We note, that in the older literature species seem to have been mixed up, in particular S. minutus and S. sculptus, and clearly different morphological depictions have been referred to as one and the same species

(Balogh, 1972; Giljarov and Krivolutsky, 1975; Pérez-Iñigo, 1993; Woas, 1998).

Furthermore, it should be noted that S. minutus is possibly not as common as it has been stated in literature. We received samples from many European countries but the “real” S. minutus could be identified hitherto only in Austria and in Germany (samples not included in this study).

This suggests that the often-cited statement of the Palaearctic distribution of S. minutus (Subías,

2004, resp.2006; Weigmann, 2006) is not true. Scutovertex sculptus (or members of this cryptic species complex), on the other hand, seem to be very abundant throughout its Palaearctic distribution.

Conclusions

Molecular genetic and morphological data revealed a paraphyletic genus Scutovertex, with S. pictus likely representing a distinct genus, and confirmed the taxonomic placement of

- 92 - 4. Phylogeny of European Scutovertex mites

Provertex kuehnelti within the family Scutoverticidae. Furthermore, molecular genetic data confirmed several recently described Scutovertex species and thus the high species diversity within this genus in Europe and suggest that S. sculptus is a complex of several cryptic species showing marked genetic, but little (if any) morphological divergence.

Acknowledgements

Financial support was provided by the Austrian Science Fund (FWF, project number P19544-

B16). We are grateful to K. Brandl, E. Ebermann, S. Ermilov, C. Hellig, P. Horak, J.

Jagersbacher-Baumann, J. Knapp, I. Kulterer, E. McCullough and H. Schatz for providing moss samples for our study and we are indebted to the administration of the National Park “Neusiedler

See-Seewinkel” for the permission to collect moss and soil samples. Furthermore, the authors thank Prof. Dr. F. Hofer and his team at the Research Institute for electron Microscopy (FELMI) for the cooperation in making SEM-micrographs.

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Appendix A. Morphological characters and character states. 1. Prodorsal surface (P): smooth, no foveae (0); smooth, foveae (1); granular, no foveae, no wrinkles (2); granular, no foveae, wrinkles (3); granular, foveae, no wrinkles (4); granular, foveae, wrinkles (5). 2. Bothridium (bo): closed border, roundly shaped (0); closed border, longish shaped (1); open border, roundly shaped (2); open border, longish shaped (3). 3. Sensillus (bS) dimension: short, slim (0); short, thick (1); long, slim (2); long, thick (3). 4. Sensillus shape: spinose, broad, clavate and flattened (0); spinose, broad, clavate and spherical (1); spinose, slender, clavate and flattened (2); spinose, slender, clavate and spherical (3). 5. Lamella (cl): absent (0); short, collateral (1); short, convergent (2); long, collateral (3), long, convergent (4); broad, laterally overhanging (5). 6. Lamellar seta (le): short, slim, smooth (0); short, slim, spinose (1); short, thick, smooth (2); short, thick, spinose (3); long, slim, smooth (4); long, slim, spinose (5); long, thick, smooth (6); long, thick, spinose (7). 7. Interlamellar seta (in): absent (0); short, slim (1); short, thick (2); long, slim (3); long, thick (4). 8. Rostral seta (ro) dimension: short, thick (0); short, slim (1); long, thick (2); long, slim (3). 9. Rostral seta shape: smooth, spiniform (0); smooth, lanceolate (1); spinose, spiniform (2); spinose, lanceolate (3). 10. Exobothridial seta (ex): absent (0); short (1); long (2). 11. Rostrum (R): with one ridge (0); with two ridges (1); with two clear projections (2). 12. Lenticulus (Le): absent (0); lateral borders bend inward (1); oval (2); rectangular (3). 13. Translamella (TL): absent (0); narrow, straight (1); narrow, bent (2); broad, straight (3); broad, bent (4). 14. Cusps (cu): absent (0); small (1); large (2); broad, overhanging (3). 15. Prodorsal ridges (PR): absent (0); collateral, reaching TL (1); collateral, not reaching TL (2); converging, not fused, reaching TL (3); converging, not fused, not reaching TL (4); converging, fused, reaching TL (5); converging, fused, not reaching TL (6). 16. Notogastral surface (N): foveae, no blocs, no granules, no bars, not netlike (0); foveae, blocs, no granules, no bars, not netlike (1); foveae, blocs, granules, no bars, not netlike (2); foveae, no blocs, granules, no bars, not netlike (3); no foveae, no blocs, granules, no bars, not netlike (4); no foveae, no blocs, no granules, bars, not netlike (5); no foveae, no blocs, no granules, bars, netlike (6); no foveae, no blocs, granules, bars, not netlike (7); almost smooth (8). 17. Foveae on notogaster: absent (0); indistinct borders (1); distinct borders (2). 18. Lyrifissure ia: inconspicuous, not on a nodule (0); on a nodule (1). 19. Lyrifissure im: inconspicuous (0); very long (1). 20. Lyrifissure ip: inconspicuous (0); on a protuberance (1). 21. Pairs of notogastral setae: 10 (0); 12 (1); 13 (2); 14 (3); 15 (4). 22. Saccules (S) of the octotaxic system: absent (0); 1 pair (1); 2 pairs (2); 3 pairs (3); 4 pairs (4).

23.-37. Notogastral setae c1-3, da, dm, dp, la, lm, lp, h1-3, ps1-3: absent (0); slim, not spinose (1); slim, spinose (2); broadened, not spinose (3); broadened, spinose (4); thick, not spinose (5); thick, spinose (6). 38. Lateral prodorsum (PL) surface: smooth (0); granular (1). 39. Tutorium (TU): absent (0); not V-shaped (1); V-shaped (2). 40. Pedotectum I (PtI): small, not triangular (0); small, triangular (1); large, not triangular (2); large, triangular (3). - 101 - 4. Phylogeny of European Scutovertex mites

41. Pedotectum II (PtII): small, Y-shaped (0); small, triangular (1); large, Y-shaped (2); large, triangular (3). 42. Dens tutorius: absent (0); small (1); large (2). 43. Subcapitulum (S) surface: smooth (0); granular (1). 44.-46. Subcapitular setae (m, a, h): smooth (0); spinose (1). 47. Rutellar teeth (RU): 2 (0); 3 (1); 4 (2). 48. Rib on mentum (MR): absent (0); slender, straight (1); slender, V-shaped (2); broad, straight (3); broad, V- shaped (4); massive, reaching anterior border (5). 49. Pedipalp (Pp) “corne double”: absent (0); incomplete (1); complete (2). 50. Chaetome pedipalp (solenidion excluded): 0-2-1-3-9 (0); alternative (1). 51. Apophysis on palptarsus: absent (0); present (1). 52. Epimeral region (E) surface: smooth (0); granular (1). 53. Apodemata (Ap) III + IV: both absent (0); III present, IV absent (1); both present (2). 54. Epimeral setal formula: 3-1-2-2 (0); 3-1-3-2 (1); 3-1-3-3 (2); 3-1-3-4 (3); 3-1-3-1 (4). 55. Anogenital region (AG) surface: smooth (0); granular (1). 56. Genital valves (GV) shape: rounded, anteriorly broadened (0); rounded, posteriorly broadened (1); rectangular, anteriorly broadened (2); rectangular, posteriorly broadened (3). 57. Genital setal (g) formula: 5+5 (0); 6+6 (1); 9+9 (2); >9<12 (3); >12 (4). 58. Aggenital setal (ag) formula: setae absent (0); 1+1 (1); 2+2 (2). 59. Anal setal (an) formula: 2+2 (0); 3+3 (1).

60. Placement from g1 to g2: in a row (0); side by side (1).

61. Placement from g3 to g6: in a row (0); displaced laterally (1). 62. Placement of anal setae: medially on anal valve (0); next to inner border of anal valve (1). 63. Leg surface (LS): smooth (0); granular, no ridges (1); granular, ridges (2). 64. Respiratory organs (RO) in legs: absent (0); planar areae porosae (1); saccules (2); platytracheae (3), brachytracheae (4); tracheae (5). 65. Claw (C) number: monodactylous (0); bidactylous (2); tridactylous (3) 66. Claw shape: homodactylous (0); heterodactylous (1). 67. Dorsal setae on legs coupled with solenidia: lost in adult stage (1); present in all stages (2); lost in all stages (3). 68. Apophysis on tibia I: absent (0); small (1); large (2). 69. Lateral setae on legs: slender, smooth (0); slender, dentate (1); broadened, smooth (2); broadened, dentate (3); extremely broadened (4). 70. Position of the respiratory organs in leg I and II: femur (0); femur, tibia (1); femur, tibia, tarsus (2); femur, tarsus (3). 71. Position of the respiratory organs in leg III and IV: trochanter, femur (0); trochanter, femur, tibia (1), trochanter, femur, tibia, tarsus (2); trochanter, femur, tarsus (3); femur, tarsus (4) 72. Chaetome leg I: 1-3-3-4-16 (0); 1-4-2-4-18 (1); 1-4-3-4-18 (2); 1-4-3-4-19 (3); 1-5-3-4-18 (4). 73. Chaetome leg II: 1-4-2-4-15 (0); 1-4-3-4-15 (1); 1-3-3-5-14 (2); 1-5-3-4-15 (3). 74. Chaetome leg III: 2-2-1-3-15 (0); 2-3-1-3-15 (1); 2-2-2-3-14 (2); 2-4-1-3-15 (3).

- 102 - 4. Phylogeny of European Scutovertex mites

75. Chaetome leg IV: 1-2-2-3-12 (0); alternative (1). 76. Solenidia leg I: 1-2-2 (0); 1-2-3 (1). 77. Solenidia leg II: 1-1-2 (0); 1-1-1 (1). 78. Solenidia leg III: 0-1-0 (0); 1-1-0 (1). 79. Solenidia leg IV: 0-1-0 (0); alternative (1).

- 103 - 4. Phylogeny of European Scutovertex mites

Appendix B. Matrix for morphological characters and character states.

1 2 3 4 5

0 0 0 0 0 S. alpinus 22324 40200 11126 02101 04010 11010 11111 11123 20100 01310 S. arenocolus 22324 70200 11415 31100 03010 03010 33344 11123 20111 01410 S. ianus 22324 70200 11425 21100 03010 01010 11144 11123 20111 01310 S. minutus 22324 70200 11426 40100 13010 13310 33344 11123 20111 01410 S. pannonicus 22324 70200 11425 02100 03010 03010 33324 11123 20111 01010 S. pictus 32104 60200 00320 50000 00010 01010 11111 11103 20111 01400 S. pileatus 22323 00200 11324 70100 03010 33010 33331 11123 20111 01310 S. sculptus 22324 70200 11425 11100 03010 04010 66666 11123 20100 01410 L. caelatus 33304 70200 20120 60000 00101 11111 11111 10113 20100 01100 P. kuehnelti 22112 00220 00104 40000 30110 11111 11111 11101 20111 00010 E. hungaricus 22325 70220 00034 60001 01010 01010 11111 11113 20111 00010 C. cymba 30110 00101 00000 60000 20110 11111 11111 10103 30100 02300

6 7 8

0 0 0 S. alpinus 01201 01101 00253 11230 02100 0010 S. arenocolus 01201 01101 10253 11230 02100 0010 S. ianus 01201 01101 10253 11230 02100 0010 S. minutus 01201 01101 10253 11230 02100 0010 S. pannonicus 01201 01101 10253 11230 02100 0010 S. pictus 11201 01100 01243 11231 13010 0010 S. pileatus 01201 01100 00253 11230 01000 0010 S. sculptus 01201 01101 10253 11230 02100 0010 L. caelatus 01201 01100 01240 01221 12110 0010 P. kuehnelti 01201 01100 01233 11131 12100 0010 E. hungaricus 01241 01100 01233 11241 12000 0010 C. cymba 11001 01100 01243 01202 20220 0110

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CHAPTER

5

Ancestral state reconstruction reveals multiple evolution of diagnostic characters in the “Higher Oribatida” (Acari), conflicting current classification schemes

Sylvia Schäffer, Stephan Koblmüller, Tobias Pfingstl, Christian Sturmbauer & Günther Krisper

submitted 5. Character evolution within Circumdehiscentiae

Abstract

In the literature, there are several proposals to classify the oribatid mite group

Circumdehiscentiae (”Higher Oribatida”), all being based upon the validity of several diagnostic characters, as for example the development of scalps and centrodorsal setae in nymphs or the presence or absence of the secretory notogastral octotaxic system. However, it is still unclear which of the currently used morphological characters are the most appropriate ones for categorizing the “Higher Oribatida”. The aims of this study were to test the appropriateness of the current classification schemes for the Circumdehiscentiae and to reconstruct the character evolution of these diagnostic traits. Therefore, we traced the evolution of six morphological traits of interest (the four nymphal traits scalps, centrodorsal setae, sclerits and wrinkled cuticle plus octotaxic system and pteromorphs both in adults) on the basis of a molecular phylogenetic hypothesis by means of parsimony, likelihood, Bayesian and stochastic approaches.

The molecular phylogeny based on three nuclear markers (28S rDNA, ef-1α, hsp82) revealed discrepancies to the traditional classification of the five “circumdehiscent” subdivisions, suggesting paraphyly of the four families Scutoverticidae, Ameronothridae,

Cymbaeremaeidae and Achipteriidae. Ancestral state reconstructions of six common diagnostic characters also partially reject the current morphology-based classification due to homoplastic character evolution. The reconstruction of character evolution on the molecular phylogeny suggests multiple convergent evolution of two main traits (octotaxic system, wrinkled cuticle) after a phase of rapid cladogenesis rendering the “Poronota” paraphyletic.

Ancestral character state reconstructions of the four remaining morphological traits suggest single origins and thus their usefulness for categorization within the Circumdehiscentiae.

However, this is only true when looking at a particular trait but not for a combination of traits.

- 105 - 5. Character evolution within Circumdehiscentiae

To conclude, character histories of six morphological traits largely conflict with current morphological classifications of the Circumdehiscentiae, suggesting that the current taxonomic schemes are not appropriate. Only the criterion presence or absence of nymphal scalps (eupheredermous/apheredermous) seems to be applicable to classify the

Circumdehiscentiae in higher groups apart from a recently proposed subdivision into 24 superfamilies.

Key words: Oribatid mites, Circumdehiscentiae, systematic, 28S rDNA, ef-1α, hsp82, character history, parallel evolution.

- 106 - 5. Character evolution within Circumdehiscentiae

Introduction

Morphological characters are traditionally the basis for taxonomy in the animal kingdom. In the last years, morphological analyses were often combined with molecular data to solve a variety of evolutionary and taxonomic problems (Wahlberg et al., 2005; Schaap et al., 2006; Petrusek et al., 2008; Moussalli et al., 2009; Schäffer et al., in press a). The rise of molecular phylogenetics has often led to conflicts in systematic classification. Across many groups of animals, current morphology-based classifications are frequently reflected by new molecular phylogenies as a result of previously undetected homoplastic character evolution

(e.g. Mott and Vieites, 2009; Perkins et al., 2009; Schmitt et al., 2009; Urban and Cryan,

2009; Virgilio et al., 2009).

In the field of acarine research, the classification of higher taxa in mites is still largely unresolved, due to discrepancies of different analytical approaches and the use of different characters and/or data sets (e.g. Norton, 1998; Domes et al., 2007b; Dabert et al., in press).

Comprehensive studies in the form of combined analyses of molecular genetic and morphological data are rare and the few existing investigations in the recent literature are only addressing the family or genus level (e.g. Navajas et al., 1996; Laumann et al., 2007; Schäffer et al., in press a). Within the Acari, there are two superorders, the Parasitiformes and the

Acariformes, of which he latter comprises about seventy percent of the described mite species. One group within the Acariformes, the Oribatida, is the most abundant group of arthropods in the organic soil layer where some species can reach collective densities exceeding 100,000 individuals per m2. Only a few DNA studies dealt with the relationships of some taxa within and between the Oribatida and their related groups (Maraun et al., 2004;

Murrell et al., 2005; Domes et al., 2007a, b) but with only little emphasis on morphological traits and character evolution. Just in one recent study, Maraun et al. (2009) tested whether three particular traits, including two morphological characters, correlate with arboreal life- style in oribatid mites.

- 107 - 5. Character evolution within Circumdehiscentiae

Following Grandjean (1969) the Oribatida consist of six major groups, namely the

Palaeosomata, Enarthronota, Parhyposomata, Mixonomata, Desmonomata and

Circumdehiscentiae. The Circumdehiscentiae, the target of this study, are also named

Brachypylina or “Higher Oribatida”. They are the largest and taxonomically richest group of the Oribatida, and although several studies aimed for resolving the taxonomy of this group, current taxonomic classifications within the Circumdehiscentiae are considered to be questionable. The first and most cited proposal was set by Grandjean (1954) who divided the group into five subdivisions based on three main characters. The first one is the development of scalps in nymphs. A scalp is a part of the exuvia of the gastronotic region that is retained on an emerging nymph (or adult in some genera) after the moult. Grandjean called nymphs that retain scalps as eupheredermous; nymphs that do not as apheredermous. In one family

(Hermanniellidae) nymphs do not retain scalps but the adults possess the tritonymphal scalp; this characteristic is called opsiopheredermous. One further exception concerns the

Oribatellidae: here the species are apopheredermous, meaning nymphs retain scalps which are held away from the body by setae. The second character concerns the three pairs of centrodorsal setae da, dm and dp. If they are lost in nymphs this trait is called dorsodeficient, if setae are present integridorsal. The last trait of Grandjean´s classification is the octotaxic system in adult mites. This system is a special series of originally four pairs of secretory

(Alberti et al., 1997) notogastral porose organs (developed as porose areas or saccules) which can vary in size, shape and number. Species featuring the octotaxic system are called poronotic, species without it pycnonotic. Based on these three characters Grandjean defined the five subdivisions Opsiopheredermata, Eupheredermata, dorsodeficient Apheredermata, normal pycnonotic (=integridorsal) Apheredermata and Poronota. He further divided the

Poronota into three groups according to the appearance of nymphs and larvae (either wrinkled or smooth with micro- or macrosclerits). This classification was also adopted by Wauthy

(2006; available from www.naturalsciences.be/science/collections/mites/elements/) who

- 108 - 5. Character evolution within Circumdehiscentiae included a sixth group, the “Circumdehiscentiae (= Pycno- and Poronota) with wrinkled nymphs” to the Circumdehiscentiae, based on ideas of Grandjean (1954, 1958). In these works, Grandjean assumes that eleven families, including taxa of Eu- and Apheredermata which have nymphs with a wrinkled gastronotic cuticle structure form a monophyletic entity

(“un groupe naturel”). The phylogenetic relationship of the six subdivisions based on a parsimony analysis of 14 morphological characters is shown in Fig. 1 (modified from

Wauthy, 2006; available from www.naturalsciences.be/science/collections/mites/elements/).

Recent hypotheses mostly avoid Grandjean´s old classification scheme. For example, Subías

(2004) simply classified the Circumdehiscentiae into two groups, the “Pycno- and

Poronoticae” and Norton and Behan-Pelletier (2009) presented a system without any higher grouping of the taxa, but assigning the circumdehiscent taxa to 24 superfamilies.

To evaluate which of these classifications is the most appropriate one for the

Circumdehiscentiae, we inferred a molecular phylogeny and traced several diagnostic morphological characters on the phylogeny. Following Grandjean’s (1954) classification, we investigated the three main traits (scalps, centrodorsal setae, octotaxic system) plus three additional characters which are also used for categorization: the presence of micro- and macrosclerits in nymphs, the wrinkled cuticle structure of nymphs, and furthermore the development of so-called pteromorphs in adults. Pteromorphs are humeral projections on the lateral border of the notogaster concealing all or parts of the adducted legs. In some taxa these are immobile fixed structures, whereas in other taxa the base of pteromorphs is de-sclerotized, forming a linear hinge, and inserted with a highly differentiated musculature, allowing for motility of this structure. This trait is only known from many poronotic mites and might potentially serve as diagnostic character within this group.

The aims of the present study are i) testing the evidence of Grandjean´s (1954) classification in contrast to those of Wauthy (2006) for the taxonomy of the

Circumdehiscentiae, ii) examining the character evolution of five hitherto existing diagnostic

- 109 - 5. Character evolution within Circumdehiscentiae traits (four nymphal scalps, centrodorsal setae, sclerits and wrinkled cuticle plus octotaxic system in adults), iii) investigating the potential of pteromorphs for categorization within the

Poronota, and iv) uncovering a potential multiple parallel evolution of the investigated traits within the Circumdehiscentiae. To achieve our goals, we established a molecular phylogeny based on three nuclear markers (28S rDNA, ef-1α, hsp82) that are commonly used for phylogenetic studies in general (Danforth et al., 1999; Chen et al., 2003; Schön and Martens,

2003; Regier et al., 2008; Ruiz et al., 2009) and in particular for phylogenetic inference in mites (Klompen, 2000; Maraun et al., 2004; Schaefer et al., 2006; Laumann et al., 2007), and traced the evolution of our six morphological traits of interest over the molecular phylogeny using parsimony, likelihood, Bayesian and stochastic approaches.

Fig. 1. Phylogenetic reconstruction of Circumdehiscentiae modified after Wauthy (2006; available from www.naturalsciences.be/science/collections/mites/elements/14).

§ = SEM micrographs modified from Hunt et al. (1998)

- 110 - 5. Character evolution within Circumdehiscentiae

Material and Methods

Sample collection

This study includes 40 representatives of all five subdivisions of Circumdehiscentiae

(after Grandjean, 1954): Opsiopheredermata, Eupheredermata, dorsodeficient and pycnonotic

Apheredermata and Poronota (Table 1). Families categorized as the sixth subdivision

“Circumdehiscentiae with wrinkled nymphs” (after Wauthy, 2006) are underlined in the table.

Based on Weigmann (1996) we chose Hermannia gibba (Hermanniidae), which is a member of the Desmonomata, a sister group of the Circumdehiscentiae, as outgroup. Sequences of eight species were obtained from GenBank (see Table 1).

Specimens were extracted from mosses, lichens or soil samples with Berlese-Tullgren funnels and preserved in absolute ethanol. Total genomic DNA was extracted from single individuals applying the CTAB (hexadecyltriethylammonium bromide) method described in

Schäffer et al. (in press b). After DNA extraction, the sclerotized body remnants were mounted on permanent slides and used for species identification using the criteria defined in

Weigmann (2006).

PCR and DNA sequencing

Fragments of 28S rDNA, ef-1α and heat shock protein 82 (hsp82) genes were amplified by polymerase chain reaction (PCR) using the following primers: D3A and D3B

(Litvaitis et al., 1994) for the D3 fragment of the 28S rDNA, 40.71F and 52.RC (Regier and

Shultz, 1997) and EF-SyFwd and EF-SyRev (Schäffer et al., in press a) for ef-1α, hsp1.2 and hsp8.x (Schön and Martens, 2003) for hsp82. Polymerase Chain Reaction (PCR), purification of PCR products and DNA sequencing followed the protocol described in Schäffer et al.

(2008). DNA fragments were purified with SephadexTM G-50 (Amersham Biosciences) following the manufacturer’s instruction and visualized on a 3130xl capillary sequencer

(Applied Biosystems).

- 111 - 5. Character evolution within Circumdehiscentiae

Table 1. Specimens and GenBank accession numbers investigated in this study. Underlined families categorized to the subdivision “Circumdehiscentiae with wrinkled nymphs” (after Wauthy, 2006; available from www.naturalsciences.be/science/collections/mites/elements/).

GenBank Accession No. Classification Species 28S ef-1α hsp82 Outgroup Hermanniidae Hermannia gibba Koch, 1839 AY273530 b EF081327 b DQ090800b gibba Koch, 1839 * * * Opsiopheredermata Hermanniellidae Hermanniella punctulata Berlese, 1908 * * * Eupheredermata Neoliodidae Platyliodes scaliger Koch, 1840 * * * sp. * * * Gymnodamaeidae Arthrodamaeus sp. * * --- Eutegaeidae Eutegaeus curviseta Hammer, 1966 DQ090816b EF081326 b DQ090789b Zetorchestidae Zetorchestes falzonii Coggi, 1898 * * * Niphocepheidae Niphocepheus nivalis Schweizer, 1922 * * * dorsodeficient Apheredermata Liacaridae Liacarus cf. subterraneus Koch, 1841 * * --- Peloppiidae Ceratoppia quadridentata Haller, 1882 * * * normal pycnonotic Apheredermata Carabodidae Carabodes femoralis Nicolet, 1855 AY273508 b EF081325 b DQ090786 b labyrinthicus Michael, 1879 AY273506 b EF093762 b EF093765 b marginatus Michael, 1884 * * * Hydrozetidae Hydrozetes lacustris (Michael, 1882) * * * lemnae Coggi, 1899 * * *

Tectocepheidae Tectocepheus velatus Michael, 1880 EF093757 b EF093763 b EF093770 b minor Berlese, 1903 EF093756 b EF093764 b EF093772 b sarekensis Trägardh, 1910 EF093759 b EF093760 b EF093774 b

- 112 - 5. Character evolution within Circumdehiscentiae

cf. alatus Berlese, 1913

Ameronothridae Ameronothrus maculatus Michael, 1882 * * * Podacarus auberti cf. occidentalis * * * Wallwork, 1966

Cymbaeremaeidae Cymbaeremaeus cymba Nicolet, 1855 GU208575a GU208670a * Scapheremaeus cf. palustris Sellnick, 1924 * * *

Ametroproctidae Ametroproctus lamellatus (Schweizer, 1956) * * * Poronota Galumnidae Galumna cf. obvia Berlese, 1915 * * * Ceratozetidae Trichoribates trimaculatus Koch, 1835 * * * Euzetidae Euzetes globulus Nicolet, 1855 * * * Oribatulidae Phauloppia cf. lucorum Koch, 1841 * * *

Scutoverticidae Scutovertex minutus Koch, 1836 GU208538a GU208633a * sculptus Michael, 1879 GU208550a GU208645a * pannonicus Schuster, 1958 GU208540a GU208635a * pileatus Schäffer & Krisper, GU208544a GU208639a * 2008 “Scutovertex” “pictus” (Kunst, 1959) GU208541a GU208636a * Provertex kuehnelti Mihelcic, 1959 GU208567a GU208662a * Lamellovertex caelatus Berlese, 1894 GU208565a GU208660a * Exochocepheus hungaricus (Mahunka, 1987) GU208570a GU208665a *

Phenopelopidae Eupelops cf. curtipilus Berlese, 1916 * * *

Unduloribatidae Unduloribates undulatus Berlese, 1914 * * *

Achipteriidae Parachipteria punctata Nicolet, 1855 * * * Achipteria coleoptrata Linné, 1758 AY273500 b AY632776 b EF081335 b quadridentata Willmann, 1951 * * * a = Sequences not generated in the framework of this study [obtained from (Schäffer et al., in press a)] b = Sequences obtained from GenBank * = GenBank numbers as recently available as after submission for publication -- = Sequences could not be amplified

- 113 - 5. Character evolution within Circumdehiscentiae

Table 2. Morphological characters and character coding used in this study.

Taxa sca cd os scl gc pt Hermannia gibba 0 0 0 0 1 0 Hermanniella punctulata 1 0 0 0 0 0 Platyliodes scaliger 2 2 0 0 0 0 Platyliodes sp. 2 2 0 0 0 0 Arthrodamaeus sp. 2 2 0 0 0 0 Eutegaeus curviseta 2 2 0 0 0 0 Zetorchestes falzonii 2 2 0 0 0 0 Niphocepheus nivalis 2 2 0 0 0 0 Liacarus cf. subterraneus 2 2 0 0 0 0 Ceratoppia quadridentata 2 2 0 0 0 0 Carabodes femoralis 3 1 0 0 0 0 Carabodes labyrinthicus 3 1 0 0 0 0 Carabodes marginatus 3 1 0 0 0 0 Hydrozetes lacustris 3 1 0 0 0 0 Hydrozetes lemnae 3 1 0 0 0 0 Tectocepheus velatus* 3 1 0 0 1 0 Tectocepheus minor* 3 1 0 0 1 0 Tectocepheus sarekensis* 3 1 0 0 1 0 Tectocepheus cf. alatus 3 1 0 0 1 0 Ameronothrus maculatus 3 1 0 0 1 0 Podacarus auberti cf. occidentalis 3 1 0 0 1 0 Cymbaeremaeus cymba 3 1 0 0 1 0 Scapheremaeus cf. palustris 3 1 0 0 1 0 Ametroproctus lamellatus 3 1 0 0 1 0 Galumna cf. obvia 3 1 1 2 0 2 Trichoribates trimaculatus 3 1 1 2 0 1 Euzetes globulus 3 1 1 2 0 1 Phauloppia cf. lucorum 3 1 1 1 0 0 Scutovertex minutus 3 1 3 0 1 0 Scutovertex sculptus 3 1 3 0 1 0 Scutovertex pannonicus 3 1 3 0 1 0 Scutovertex pileatus 3 1 3 0 1 0 “Scutovertex pictus” 3 1 0 0 1 0 Provertex kuehnelti 3 1 0 0 1 0 Lamellovertex caelatus 3 1 0 0 1 0 Exochocepheus hungaricus 3 1 3 0 1 0 Eupelops cf. curtipilus 3 1 1 0 1 2 Unduloribates undulatus 3 1 0 0 1 1 Parachipteria punctata 3 1 1 0 1 1 Achipteria coleoptrata 3 1 2 0 1 1 Achipteria quadridentata 3 1 2 0 1 1 sca, scalps (0 = outgroup, 1 = opsiopheredermous, 2 = eupheredermous, 3 = apheredermous); cd, centrodorsal setae (0 = holotrich, 1 = integridorsal, 2 = dorsodeficient); os, octotaxic system (0 = no porose organs, 1 = porose areas, 2 = saccules type1, 3 = saccules type2); scl, sclerits (0 = nymphs nude, 1 = nymphs with microsclerits, 2 = nymphs with macrosclerits); gc, gastronotic cuticle of nymphs (0 = unwrinkled, 1 = wrinkled); pt, pteromorphs (0 = no pt, 1 = pt unmovable, 2 = pt movable).

- 114 - 5. Character evolution within Circumdehiscentiae

Alignment and phylogenetic analyses We sequenced 311-329 bp of the D3 region of the nuclear 28S rDNA, 475 bp of the nuclear ef-1α, and 467-503 bp of the hsp82 gene in 34 specimens (from Liacarus cf. subterraneus and Arthrodamaeus sp. the fragment of hsp82 could not be amplified).

Sequences were verified by comparisons with known oribatid sequences from GenBank and aligned by eye in MEGA 3.1 (Kumar et al., 2004). We removed poorly aligned regions of the alignments of 28S rDNA and hsp82 using the program trimAl (Capella-Gutiérrez et al., 2009) which is a tool for automated alignment trimming. Gap threshold was set to 0.8 and similarity threshold to 0.001. All sequences were combined in a data set with a resulting length of 1298 bp for further analyses.

Phylogenetic reconstructions by neighbor joining (NJ) and maximum parsimony (MP) were conducted in PAUP* (Swofford, 2002), maximum likelihood (ML) in RAxML-7.0.3-

WIN (Stamatakis, 2006) and Bayesian inference (BI) in MrBayes 3.1.2 (Ronquist and

Huelsenbeck, 2003). For NJ analysis, the model of evolution selected by the hierarchical likelihood ratio test (hLRT) implemented in Modeltest 3.06 (Posada and Crandall, 1998) was

GTR+I+G (base frequencies: A = 0.3000, C = 0.2258, G = 0.2846, T = 0.1895; R-matrix:

A↔C = 1.5870; A↔G = 4.1651; A↔T = 1.4535; C↔G = 1.9490; C↔T = 7.5163; G↔T =

1.0000; proportion of invariable sites: I = 0.5227; gamma shape parameter: α = 0.9461). MP analysis was performed using heuristic tree searches applied random addition of taxa and tree bisection-reconnection (TBR) branch-swapping algorithm (1,000 replicates). Statistical support for the resulting NJ and MP phylogenies was assessed by bootstrapping (1,000 replicates). To find the best-scoring ML tree we applied the GTR+I+G substitution model with 40 distinct rate categories, starting from a random tree. Data were partitioned by gene and the ef-1α gene was further partitioned by codon position. Nodes were supported by bootstrapping (500 replicates). For Bayesian Inference we employed the same partitioning scheme as in the ML analysis. Rate heterogeneity was set according to a gamma distribution

- 115 - 5. Character evolution within Circumdehiscentiae with six rate categories (GTR model) for each data partition. Posterior probabilities were obtained from a Metropolis-coupled Markov chain Monte Carlo simulation (2 independent runs; 4 chains with two million generations each; trees sampled every 100 generations), with parameters estimated from the data set. Mixing and convergence to stationary distributions were evaluated in Tracer v.1.4 (Rambaut and Drummond, 2007; available at http://beast.bio.ed.ac.uk/Tracer/). The first 4000 (20%) trees were discarded as burn-ins before a 50% majority-rule consensus tree was constructed from the remaining 16,001 trees.

To assess whether the topologies obtained by the different tree building algorithms differed significantly we performed parsimony based Kishino-Hasegawa (KH; Kishino and

Hasegawa, 1989) tests and likelihood based Shimodaira-Hasegawa (SH; Shimodaira and

Hasegawa, 1999) tests in PAUP*. Although both tests revealed no significant differences between the alternative topologies (except for the MP tree in the SH test), they agreed on the

BI tree as the best tree (Table 3). Thus, all further analyses are based on the BI tree.

Analyses of character evolution

We traced the evolution of six morphological characters (scalps, centrodorsal setae, sclerits and wrinkled cuticle which are all developed in nymphs plus octotaxic system and pteromorphs both in adults) over the molecular phylogeny using parsimony, likelihood,

Bayesian and stochastic approaches. The defined characters with their character codings are shown in Table 2. Information on the studied characters were retrieved from the literature

(Grandjean, 1954; Kunst, 1959; Hammer, 1966; Wallwork, 1966; Behan-Pelletier, 1987;

Ermilov, 2006; Weigmann, 2006; Seniczak et al., 2007; Schäffer and Krisper, 2007; Schäffer et al., 2008; Pfingstl et al., 2008 & in press) and from the body remnants of our specimens.

Regarding the porose organs, we designed two different data sets to analyze the ancestral states, one with porose organs (regardless which type) as either absent (0) or present (1) and one coded with the different types found in oribatid mites: porose organs absent (0), areae

- 116 - 5. Character evolution within Circumdehiscentiae porosae (1), saccules type1 (2), saccules type2 (3) [saccules were sub-divided with reference to Alberti et al., 1997]. The program Mesquite v.2.71 (Maddison and Maddison, 2009) was used to reconstruct the character states at ancestral nodes of the phylogeny. We performed parsimony and likelihood analyses using the “trace character over trees” method which summarizes ancestral state reconstructions over a series of trees. All reconstructions were integrated over the last 6001 post burn-in trees of the Bayesian analysis. The BI tree (as the best one according to KH and SH tests) was taken for summarizing the ancestral states on a consensus tree. Model of evolution for these reconstructions was the Markov k-state 1 (Mk1) parameter model, with equal probability for any particular character change. For the ancestral state reconstruction we also applied a fully Bayesian approach as implemented in the software

SIMMAP v. 1.5 (Bollback, 2006; available at http://www.simmap.com/) which was also used for stochastic character mapping. To accommodate uncertainties in the phylogeny we used

1000 randomly sampled trees of the MCMC analysis. SIMMAP uses the Mk class of models for morphological characters. Twenty-seven nodes (Fig. 2) were selected based on their posterior probability support values (only those nodes with PP ≥ 90 were included). The settings were a bias parameter for two-state characters following a discrete beta distribution with k=19 categories and α=1.0. Overall rate parameters were α=3.0, β=2.0 and k=60.

Number of samples and number of prior draws were set to 10. Tree lengths were rescaled to

1.0 before the rate is applied. All trees were rooted with Hermannia gibba as outgroup which was excluded from the analyses but was used to establish the root position.

Testing alternative hypothesis of character evolution

Alternative hypotheses regarding character evolution were compared to the BI tree by means of SH tests. Following alternative phylogenetic trees [created in MacClade v. 3.04

(Maddison and Maddison, 1992)] were evaluated: Monophyly of a) Apheredermata, b) the dorsodeficient nymphs, c) nymphs with macrosclerits, d) “Circumdehiscentiae with wrinkled

- 117 - 5. Character evolution within Circumdehiscentiae nymphs”. Furthermore, we tested i) a single origin of pteromorphs and ii) the monophyly of the family Scutoverticidae.

Table 3. Comparison of alternative phylogenetic hypotheses. Kishino-Hasegawa (KH; Kishino and Hasegawa, 1989) and Shimodaira-Hasegawa tests (SH; Shimodaira and Hasegawa, 1998) were used to assess whether the topologies of NJ, MP, BI and ML differed significantly. * P<0.05.

KH test SH test

Tree tree length diff. s.d. (diff) t P -lnL Δ-lnL P

NJ 22 15.99451 1.3755 0.1692 15062.54325 33.93225 0.164

MP 24 17.08160 1.4050 0.1603 15082.57937 53.96837 0.032*

ML 6 10.20061 0.5882 0.5565 15032.95428 4.34328 0.703

BI best 15028.61100 best

Results Phylogenetic analysis

Pairwise sequence divergence (uncorrected p-distance) between the investigated species ranged from 0 to 11 % in the D3 fragment of 28S rDNA, from 1 to 22 % in the ef-1α gene and from 2 to 26 % in the hsp82 gene. In the combined data set, pairwise differences ranged from 8 to 19 %.

Tree topologies of the four phylogenetic algorithms were almost identical. Only slight differences were observed with respect to the tree building algorithm used. All four analyses showed high statistical support at the base of the phylogeny but gave no resolution at the inner nodes. MP yielded 9 most parsimonious trees with a length of 3,105 steps (CI excluding uninformative characters = 0.2700; RI = 0.5039; RC = 0.1467). An evaluation of the phylogenetic hypotheses obtained from NJ, MP, ML and BI by means of KH and SH tests revealed no significant differences between the alternative topologies except for the MP tree in the SH test (Table 3). Compared to the other three tree topologies the MP tree showed a

- 118 - 5. Character evolution within Circumdehiscentiae slightly different branching order albeit with low bootstrap support. Only the best tree of both tests - the BI tree - is shown (Fig. 1). The analyses resulted in a tree that is highly congruent with previous molecular studies (Maraun et al., 2004; Domes et al., 2007b; Maraun et al.,

2009).Compared to the traditional classification, our results show some discrepancies. None of the five or six, respectively, major subdivisions seem to be monophyletic. In particular, the

“Eupheredermata”, “dorsodeficient Apheredermata” and “Poronota” appeared as para- or polyphyletic as has been already suggested by Maraun et al. (2004). Wauthy´s (2006) subdivision “Circumdehiscentiae with wrinkled nymphs” clusters together in a major clade also including three species of Poronota (Trichoribates trimaculatus, Euzetes globulus and

Galumna cf. obvia), albeit with low statistical support. The two “circumdehiscent” species

Cymbaeremaeus cymba and Ametroproctus lamellatus form a well supported more basal clade rendering the “Circumdehiscentiae with wrinkled nymphs” unambiguously paraphyletic. The placement of Cymbaeremaeus cymba in our phylogeny is almost identical to that of Maraun et al. (2009).

In case of the family Scutoverticidae, it became evident that the investigated species do not form a monophylum, but rather constitute two distinct clusters: one includes exclusively species of the genus Scutovertex, whereas the other one comprises members of the three other genera - Provertex kuehnelti, Lamellovertex caelatus, Exochocepheus hungaricus - plus “Scutovertex pictus” and furthermore Ameronothrus maculatus and Scapheremaeus cf. palustris, thus rendering the family paraphyletic. The two alternative hypotheses (Fig. 3, hypotheses 6a-b) enforcing a monophyletic family Scutoverticidae in the molecular phylogeny, tested with a SH test, resulted in significantly worse fits to the data (Table 4). The aforementioned results also imply paraphyly of the families Ameronothridae and

Cymbaeremaeidae. Moreover, Achipteria coleoptrata clusters with Parachipteria punctata and Achipteria quadridentata as sister taxon, also rendering the Achipteriidae paraphyletic.

- 119 - 5. Character evolution within Circumdehiscentiae

Fig. 2. Bayesian 50% majority rule consensus tree of 40 representatives of all five subdivisions of Circumdehiscentiae (after Grandjean, 1954): Opsiopheredermata (violet), Eupheredermata (green), dorsodeficient Apheredermata (orange), pycnonotic Apheredermata (blue) and Poronota (red). Tree based on a combined data set of all available fragments of the 28S rDNA, ef-1α and hsp82 genes. Posterior probabilities for BI are shown when higher than 50. Numbers at nodes indicate nodes that have been used to access ancestral states.

- 120 - 5. Character evolution within Circumdehiscentiae

Fig. 3. Alternative phylogenetic hypotheses (highlighted in red) were compared to the Bayesian inference tree (grey). We tested the monophyly of Apheredermata (hypothesis 1), of dorsodeficient nymphs (hypothesis 2), of nymphs with macrosclerits (hypotheses 3a-c), of “Circumdehiscentiae with wrinkled nymphs” (hypothesis 4), of pteromorphs (hypotheses 5a-b) and of the family Scutoverticidae (hypotheses 6a-b).

- 121 - 5. Character evolution within Circumdehiscentiae

Table 4. Comparison of alternative phylogenetic hypotheses. A Shimodaira-Hasegawa test (SH; Shimodaira and Hasegawa, 1998) was used to assess whether the topologies of six phylogenetic hypotheses (see Fig. 3) differed significantly from the phylogeny resulted of a Bayesian inference (BI) analysis. * P<0.05.

SH test

Tree -lnL Δ-lnL P BI 15028.61100 best hypothesis 1 15061.84896 33.23797 0.219 hypothesis 2 15032.78871 4.17771 0.888 hypothesis 3a 15052.54352 23.93253 0.332 hypothesis 3b 15050.58891 21.97791 0.397 hypothesis 3c 15055.59293 26.98194 0.265 hypothesis 4 15132.98291 104.37191 0.000* hypothesis 5a 15042.30004 13.68904 0.644 hypothesis 5b 15064.06749 35.45650 0.189 hypothesis 6a 15105.98213 77.37114 0.004* hypothesis 6b 15123.07471 94.46371 0.001*

Ancestral state reconstruction

The results from the ancestral state reconstruction of scalps, centrodorsal setae, porose organs, sclerits in nymphs, nymphal cuticle structure and pteromorphs are shown in Figs. 4-7. The reconstruction yielded no conflicts between parsimony, likelihood and Bayesian analyses except for some nodes, depending on the studied character, whereas the Mk1 model reconstructed them with greater uncertainty (equivocal) than the stochastic mapping which is not uncommon, as recent studies have shown (Ricklefs, 2007; Eckman, 2008; Hinchliff and

Roalson, 2009). For example, nodes 10 and 13 (Figs. 5A-B, 6A) are ambiguously reconstructed in the MK1 model compared to the stochastic mapping which only considered the two character states present in the tip taxa as probable for the ancestral state reconstruction

(Table 5). This is also exemplarily the case for the nodes 5, 6 and 8 in scalp evolution (Fig.

4A) or nodes 19 and 20 in the pteromorphs (Fig. 7).

- 122 - 5. Character evolution within Circumdehiscentiae

Fig. 4. Cladograms of the Circumdehiscentiae based on parsimony (left cladogram) and likelihood (right cladogram). Ancestral state reconstructions of (A) scalps in nymphs (B) centrodorsal setae in nymphs.

- 123 - 5. Character evolution within Circumdehiscentiae

Fig. 5. Cladograms of the Circumdehiscentiae based on parsimony (left cladogram) and likelihood (right cladogram). Ancestral state reconstructions of (A) porose organs (two-stated character coding) and (B) the different types of porose organs found in oribatid mites. Light micrographs with differential interference contrast showing one porose area of Trichoribates trimaculatus, one saccule type1 of Achipteria coleoptrata and one saccule type2 of Scutovertex pannonicus (from left to right). Scale bars: 10µm.

- 124 - 5. Character evolution within Circumdehiscentiae hs p Pteromor Wrinkles Sclerits an -2 an g Porose (P.) or (P.) Porose an -1 an Probability for ancestral states ancestral for Probability g Centrodorsal setaeCentrodorsal or P. s p Scal 06030110 10 1000 10 1000 00 10 1000 00 10 1000 00 10 100 1000 00 10 100 00 10 100 10 100 100 123 012 00 01 01010 0123 100 00,630,37001 001 12 0000,010,99001 10 01 0100,920,08001 1000 012 010 00,890,11001 00001 10001 00,030,9710 100 0 010 00001 10 00 01001 010 1000 0 00 01 100 0 010 10 00 01 100 001 10 1000 01 100 001 010 1000 00 1 100 0001 010 10 00 10 0,990,011000 001 010 1000 10 100 001 010 10 00 1 100 00 10 010 1000 100 01 1000 00 100 0 00 00,080,910 01 1 0 0001 01 100 0001 010 100 0 0001 010 01 1 1001 010 01 0001 1 0,990,011000 001 010 0001 00 1 0,960,041000 0 0,99 010 00 01 0,970,031000 0 01 100 0,01 0 100 0 0,93 1 1 0,07 1 1 0 0 0 1 0 0,06 0 0,01 0 0,94 0,39 0,99 0 0,61 0 0,02 0 0,98 0,84 0 0 0,01 0 1 0 0 0 0 0 0,14 0 0,33 0 0 0,67 0 0,67 0,21 0 0,33 1 0 0,13 0,5 0 1 0,5 0 0,13 0 0 0,87 0,04 1 0,85 0 0,01 0,11 0,67 1 0,32 0 0 001 010 10 1000 00 01 100 The probability for ancestral states ofcharacters six studied 2. at 27nodes Fig. from ,1,9 001 10 1000 00 10 100 0,010,010,990 0,95 0,04 0,01 0 0,99 1 0 1 0 0 0 0 0 1 0 1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Table 5.Table Node

- 125 - 5. Character evolution within Circumdehiscentiae

Table 6. Analysis of character histories. Number of transformation from (A) opsio- [1] to eu- [2] and apheredermous [3]; [0] here neglected because it served for outgroup definition; (B) nymphs holotrich [0] to integridorsal [1] and dorsodeficient [2]; (C) porose organs absent [0] to present [1]; (D) no porose organs [0] to porose areas [1] to saccules type1 [2] and saccules type2 [3]; (E) nude nymphs [0] to nymphs with micro- [1] and macrosclerits [2]; (F) unwrinkled [0] to wrinkled [1] gastronotic cuticle structure (of juveniles); (G) no pteromorphs [0] to unmovable [1] and movable [2] pteromorphs.

Posterior expectations Posterior expectations (A) State 1 Ö 2 0.24 State 2 Ö 0 0.46 State 1 Ö 3 0.35 State 2 Ö 1 1.01 State 2 Ö 1 0.91 State 2 Ö 3 0.08 State 2 Ö 3 2.14 State 3 Ö 0 0.48 State 3 Ö 1 0.49 State 3 Ö 1 0.20 State 3 Ö 2 2.57 State 3 Ö 2 0.06 (B) (E) State 0 Ö 1 0.40 State 0 Ö 1 1.57 State 0 Ö 2 0.77 State 0 Ö 2 3.49 State 1 Ö 0 0.50 State 1 Ö 0 0.58 State 1 Ö 2 1.46 State 1 Ö 2 0.10 State 2 Ö 0 1.50 State 2 Ö 0 0.66 State 2 Ö 1 1.62 State 2 Ö 1 0.09 (C) (F) State 0 Ö 1 7.91 State 0 Ö 1 6.49 State 1 Ö 0 5.11 State 1 Ö 0 6.28 (D) (G) State 0 Ö 1 3.46 State 0 Ö 1 2.72 State 0 Ö 2 1.39 State 0 Ö 2 1.29 State 0 Ö 3 2.49 State 1 Ö 0 1.26 State 1 Ö 0 1.34 State 1 Ö 2 1.34 State 1 Ö 2 0.24 State 2 Ö 0 0.64 State 1 Ö 3 0.13 State 2 Ö 1 0.32

The reconstruction of character evolution of the nymphal scalps (Fig. 4A) and centrodorsal setae (Fig. 4B) revealed a more or less single origin of the different character states. In the scalps, only the two apheredermous species Ceratoppia quadridentata and

Liacarus cf. subterraneus and the eupheredermous Arthrodamaeus sp. cluster together with - 126 - 5. Character evolution within Circumdehiscentiae specimens of another subdivision. The phylogenetic placement of the latter species also implies that integridorsal nymphs evolved twice. But in both cases, the alternative hypotheses, assuming on the one hand the monophyly of Apheredermata (Fig. 3, hypothesis 1), and on the other hand the monophyly of dorsodeficient nymphs (Fig. 3, hypothesis 2), were not rejected by the SH-test (Table 4). The two-state data set of the porose organs revealed multiple evolution of this character (Fig. 5A). This agrees with the ancestral state reconstruction of the second data set (Fig. 5B; coded with the different types of porose organs) in which porose areas, saccules type1 and 2 do not share one most recent common ancestor (MRCA). The fact that these organs have evolved more than once independently, is also shown by the number of transformations (from state 0 to 1/2/3) in Table 6. More precisely, saccules type2 evolved two times and porose areas also at least twice. Particular types of porose organs are typically restricted to one phylogenetic lineage, with some exceptions. In one family (Achipteriidae), two different types of porose organs are found. Whereas species of the genus Achipteria possess porose areas, the genus Parachipteria has saccules type1, which is expected to be the derived form (Grandjean, 1959). However, our data indicate that the transformations from no porose organs to saccules type1 and saccules type1 to porose areas, respectively, is more likely than the other way round (Table 6). Ancestral state reconstructions of the development of sclerits in poronotic nymphs indicated multiple independent origin of this trait (Fig. 6A).

With Phauloppia cf. lucorum we only had one representative of the character state “nymphs with microsclerits” why we focused specifically on species showing “nymphs with macrosclerits”. According to the reconstruction of Mesquite (Fig. 6A), this trait could have evolved at least two times but also in this case testing a monophyly of “nymphs with macrosclerits” (Fig. 3, hypotheses 3a-c) was not rejected by SH test (Table 4).

- 127 - 5. Character evolution within Circumdehiscentiae

Fig. 6. Cladograms of the Circumdehiscentiae based on parsimony (left cladogram) and likelihood (right cladogram). Ancestral state reconstructions of (A) sclerits in nymphs (B) wrinkles in nymphs and (C) pteromorphs in adults. * SEM micrograph from Hunt et al. (1998).

- 128 - 5. Character evolution within Circumdehiscentiae

The ancestral state reconstructions of the gastronotic cuticle structure of juveniles suggested that wrinkles in nymphs evolved independently at least two to maximum four times

(Fig. 6B). Testing the hypothesis enforcing a monophyletic subgroup “Circumdehiscentiae with wrinkled nymphs” (Fig. 3, hypothesis 4) resulted in a significantly worse fit to the

Bayesian inference tree (Table 4). The number of transformations from unwrinkled (0) to wrinkled (1) character state and vice versa are nearly the same (6.49 and 6.28, respectively) meaning that both ways are equally likely. The results of the ancestral state reconstruction of pteromorphs (Fig. 7) indicated that this structure evolved two times within the Oribatida, but the alternative hypothesis, assuming a monophyletic clade of species owning pteromorphs

(Fig. 3, hypotheses 5a-b), was not rejected by the SH test (Table 4). Our assumption that motile pteromorphs evolved from immotile ones is equally probable than from no (0) to motile (2) ones, as the number of transformations in the stochastic mapping analysis showed

(Table 5).

Fig. 7. Cladograms of the Circumdehiscentiae based on parsimony (left cladogram) and likelihood (right cladogram). Ancestral state reconstructions of pteromorphs in adults. * SEM micrograph from Hunt et al. (1998).

- 129 - 5. Character evolution within Circumdehiscentiae

Discussion Molecular phylogeny

Phylogenetic analyses of the Circumdehiscentiae based on a combined data set of fragments of three nuclear genes revealed a tree topology similar to a previous study using the

18S rDNA gene (Maraun et al. 2009), albeit with higher statistical support for most important nodes in the recent study (note, that taxon sampling differs slightly between the two studies).

Splits within the Apheredermata especially within the poronotic mites are not well supported statistically. This is a strong indication that this group underwent a phase of rapid cladogenesis at a certain time in the past. Comparing the 18S tree with our results in detail revealed a different placement of Scapheremaeus (cf.) palustris in both phylogenies. While it is grouped with Ameronothrus maculatus and four specimens of the family Scutoverticidae in our phylogeny, it is placed with Eremaeozetes sp. as sister taxon to Tectocepheus velatus in the 18S tree, but we emphasize that Maraun et al. (2009) had no representatives of the

Scutoverticidae in their data set. The placement of S. cf. palustris and A. maculatus within the

Scutoverticidae renders not only this family but also the Cymbaeremaeidae and

Ameronothridae paraphyletic. Our results show that there is no close relationship of A. maculatus and the genus Podacarus auberti cf. occidentalis as already supposed by

Grandjean (1955). However, Weigmann and Schulte (1977) unified the seven genera of originally three families (Ameronothridae, Podacaridae, Aquanothridae) into one single family Ameronothridae, justifying it with the words: “Groups of genera did not differ by decided gaps of morphological characters, and so these groups should not be regarded as monophyletic groups of the rank of families” (p. 166, line 2 ff.). But the placement of A. maculatus within members of the Scutoverticidae is still not so devious considering that taxa of this genus were firstly described and assigned as “Scutovertex” species until Berlese (1896) summarized them in the genus Ameronothrus because, unlike the “real” Scutovertex, they do not possess pedotecta. Furthermore, this genus has brachytracheae (respiratory internalized

- 130 - 5. Character evolution within Circumdehiscentiae porose organs) in its legs, as do Provertex, Exochocepheus (both Scutoverticidae) and

Scapheremaeus. Whether the family Scutoverticidae either should be split up or extended with additional taxa (S. cf. palustris, A. maculatus and maybe others) remains unclear until more species of the remaining genera and closely related families (e.g. Ameronothridae,

Passalozetidae, Licneremaeidae) are included in a comprehensive phylogenetic study. With respect to the brachytracheae it must be mentioned that they are also present in the oribatid genera Cymbaeremaeus, Teleioliodes, Platyliodes and Neoliodes. Whereas brachytracheae are typically found in adults only, they are already present in larvae in the genera Teleioliodes,

Platyliodes and Neoliodes (Grandjean, 1934). But with the backgrounds of our results, convergent evolution of this character as well seems not to be devious.

Ancestral state reconstruction

The aim of this study was to investigate which current classification scheme for circumdehiscent mites is the most appropriate one. Therefore we reconstructed the ancestral states of six partially diagnostic traits that are commonly used for taxonomic assignment of oribatid mites. According to our results, two of the three main traits after Grandjean (1954), namely scalps and centrodorsal setae both in nymphs are good tools to classify the investigated taxa on the one hand into Opsio-, Eu- or Apheredermata and on the other hand into species with nymphs which are holotrich, integridorsal or dorsodeficient (Figs. 4A-B).

On the contrary, reconstruction of the character history of the octotaxic system in adults which is eponymous for the Poronota suggested multiple evolution of this diagnostic character (Figs. 5A-B). The general model of the origin of saccules proposed by Grandjean

(1934, 1959) is that porose areas invaginated and formed a saccule having a lumen encircled by porose walls. Thus, porose areas would represent the plesiomorphic character state.

Concerning the question whether the small porose areas or minute saccules in various

Licneremaeoidea (e.g. Licneremaeus, Scutovertex) do either represent a numerical and size

- 131 - 5. Character evolution within Circumdehiscentiae regression or the plesiomorphic state of the typical octotaxic system, Norton and Alberti

(1997) argued that the early evolution of porose organs started small because they are small, even minute in Licneremaeoidea (in our study called saccules type2), in their opinion the most early-derived group of Poronota. According to the numerical regression, it should be mentioned that the octotaxic system is often reduced to one, two or three pairs of organs.

Among the Scutoverticidae, for example, no species is known to have the full complement of four pairs leading to the hypothesis that four pairs do not represent the ancestral state for this family. Norton and Alberti (1997) noted that the earliest homologue of the octotaxic system might be a single pair of dermal glands that evolved by gene-duplication or by modification of developmental controls to four pairs. Our results of the ancestral state reconstructions now showed, that, whatever character coding [either two-stated (Fig. 5A) or organ-type specific

(Fig. 5B)] is used, the octotaxic system evolved independently many times in parallel within the Poronota. Furthermore, there are no indications that either porose areas or saccules type2 are the plesiomorphic state of the porose organs as proposed by Grandjean (1934; 1959) and

Norton and Alberti (1997), respectively. Concerning the weakly supported nodes within the

Poronota (Fig. 2), one could hypothesize that porose areas still can be traced back to one

MRCA. Unequivocally is the dual origin of saccules type2, present in Scutovertex and

Exochocepheus hungaricus, from an ancestor lacking porose organs (Fig. 5B). These results unambiguously reject the general hypothesis that Poronota represent a natural, monophyletic subdivision based on the existence of an octotaxic system (Grandjean, 1954). In a later study, already Grandjean (1958) himself supposed that Pycno- and Poronota “ne sont pas des groupe phylétiques” (p. 139, line 8 ff.) and that the presence or absence of these organs alone is not sufficient for a grouping to Pycno- or Poronota. Also Woas (1990) suggested that secretory porose organs probably represent functional adaptations potentially leading to a multiple independent evolution of this morphological character complex. Additionally, it must be noted that in many poronotic families and genera species can have various types of porose

- 132 - 5. Character evolution within Circumdehiscentiae organs (see table 1 in Norton and Alberti, 1997). For example, both porose areas and saccules

(type1) are found among species of Achipteriidae (Table 2) or Trichoribates (Behan-Pelletier,

1985). In this regard a potential parallel or convergent evolution of the octotaxic system should already have been a point of discussion in former time. In addition to the three main characters Grandjean (1954) used the appearance of nymphs and larvae for a classification within Circumdehiscentiae. He divided the Poronota in three types: 1) species with wrinkled nymphs; 2) species having nymphs with macrosclerits, and 3) species having nymphs with microsclerits. Furthermore, Grandjean (1954, 1958) postulated that those taxa with wrinkled nymphs should form a monophyletic group regardless if they are pycno- or poronotic (see

Introduction) and that they could represent an intermediate group (he formulated it as “à cheval sur la limite” meaning “at the frontiers”) between the pycnonotic Apheredermata and

Poronota. This concerns taxa of the following 11 families: Podacaridae, Charassobatidae,

Ameronothridae, Scutoverticidae, Tectocepheidae, Passalozetidae, Cymbaeremaeidae,

Licneremaeidae, Achipteriidae, Tegoribatidae and Phenopelopidae (formerly Pelopsidae).

Wauthy (2006; www.naturalsciences.be/science/collections/mites/elements/) followed this proposal and named the group “Circumdehiscentiae with wrinkled nymphs”. Testing the value of these sub-classifications, we defined two different data sets investigating the presence of sclerits on the one hand and on the other hand of wrinkled nymphs alone (Figs.

6A-B). Whereas taxa having nymphs with either micro- or macrosclerits could each form monophyletic groups (Table 4, the wrinkled nymphal cuticle structure evolved in parallel multiple times among Circumdehiscentiae. This clearly rejects the hypotheses of Grandjean

(1954, 1958) and Wauthy (2006) that taxa with wrinkled nymphs are monophyletic. Ancestral state reconstructions also showed that the MRCA of Circumdehiscentiae had unwrinkled nymphs (Fig. 6B), thus rejecting the assumption of Norton and Behan-Pelletier (2009) that wrinkles in nymphs - because of their occurrence in apheredermous and eupheredermous taxa

- seem to be the plesiomorphic or even ancestral state in Circumdehiscentiae. The

- 133 - 5. Character evolution within Circumdehiscentiae pteromorphs occurring only (with exception to the eupheredermous Microzetidae) in adult poronotic mites were the last studied character. Travé (1996) formulated that the presence or absence of this trait could serve as differentiation criterion within the Poronota. This hypothesis is clearly supported by our data which yielded monophyly of taxa having pteromorphs (Fig. 7).

Character evolution conflicting current classification

Our study on ancestral states within the Circumdehiscentiae shows that some previously used diagnostic characters are not useful to classify the taxa. However, specific traits are still of appreciable value such as the presence of nymphal scalps or centrodorsal setae. Scalp retention is often correlated with the nature of dehiscence which goes up after a striking process in immature instars. If the metamorphosis fails, for example due to a genetic defect, the molting individual would stall and die. Furthermore, the absence of centrodorsal setae correlates with the scalps because species retaining scalps on their notogaster are dorsodeficient (have lost setae da, dm, dp). Thus, dorsodeficient nymphs correspond to the

Eupheredermata and integridorsal nymphs to the Apheredermata, respectively. As an exception the two families Ceratoppiidae and Liacaridae group with the Apheredermata with dorsodeficient nymphs, thus suggesting a possible lost of scalps in nymphal stages and adults.

Altogether, two of Grandjean´s main traits could serve for classification of the

Circumdehiscentiae on its own. But his third character, the octotaxic system in “Poronota”, is clearly not suitable as already above mentioned. The parallel evolution of this trait could be argued with a possible correlation of secretory notogastral porose organs and ecology. Norton and Alberti (1997) still noted that poronotic mites with modified porose areas (saccules, multiplications) inhabit non-soil microhabitats (e.g. mosses and lichens on rocks or trees) and therefore internalization may reduce water-loss through porose surface. But they finally stated that there is no biological or ecological correlation that would help to understand frequent

- 134 - 5. Character evolution within Circumdehiscentiae convergent evolution. We hypothesize that maybe there is no correlation of the secretory organs with the present ecology. Considering the old age of Oribatida - indisputable fossil record from Devonian sediment (Shear et al., 1984; Norton et al., 1988) - it might be more adequate to search for congruent environmental conditions in the past, when the explosive radiation within the Apheredermata took place. Applicable in this context are the reasons for multiple evolution of a trait formulated by Wiens et al. (2006). Typically, studies on character evolution focus on either the potential advantages of a trait in a given environment (McPeek et al., 1996; Martins, 2000; Grant, 2003) or mechanisms that create novel phenotypes

(Wilkins, 2002; Carroll et al., 2005). However, Wiens et al. (2006) stated that these investigations may be necessary to elucidate why a trait has evolved in a particular instance but not why it has evolved multiple times. In his mind at least two additional factors are important, whereof one is most appropriate for our study: the biogeographic context of the selective environment. This means that a trait which is adapted to a selective environment may evolve multiple times in geographically isolated regions with identical selective environments.

A monophylum “Circumdehiscentiae with wrinkled nymphs” was clearly rejected by the results of the ancestral state reconstructions, implying that the wrinkled nymphal cuticle structure is of little value for classification. The potential function of these wrinkles was already well studied by Smrž (2007) for some oribatid taxa. Thereby he recovered on the one hand two different types of wrinkling (e.g. Hermannia gibba versus Scutovertex minutus) and on the other hand a possible correlation between environmental conditions and type of wrinkling. Thus, it is not unusual that due to similar environmental conditions and life history strategies, parallel evolution of the wrinkling structure took place.

Our study further revealed that pteromorphs might be useful as a potentially diagnostic character within the Apheredermata but more species having this character must be included in order to confirm this hypothesis.

- 135 - 5. Character evolution within Circumdehiscentiae

Conclusions

Ancestral state reconstructions of six diagnostic characters revealed some conflicts to the current morphological classification within the oribatid mite group Circumdehiscentiae.

Two of these presumed diagnostic characters, the octotaxic system (eponymous for the subdivision “Poronota”)and the wrinkled gastronotic cuticle of nymphs (taxa having these nymphs were hypothesized to be monophyletic within “poronotic” mites) were inferred to have evolved multiple times independently, subsequent to an explosive radiation of the

“Higher Oribatida” into its major lineages. One likely reason for the parallel or convergent evolution of particular traits could be based on the biogeographic context of the selective environment, meaning that evolution could produce similar phenotypes in different geographically isolated habitats (Wiens et al., 2006). In contrast, ancestral character reconstructions of two other traits (scalps and centrodorsal setae, both in nymphs) revealed a monophyletic origin of these characters, thus, indicating their importance for taxonomic assignment in circumdehiscent mites. Both characters correlate with each other whereof the dehiscence accountable for the presence or absence of scalps is an important factor during the genesis of the mites which of course has to be under selective pressure.

To conclude, the present study clearly shows that the current classification schemes of the Circumdehiscentiae are inappropriate. In our opinion, the most recent proposal by Norton and Behan-Pelletier (2009) with 24 superfamilies and no higher groupings best reflects the taxonomic situation /uncertainty within the “Higher Oribatida”. Furthermore, the present study showed that two additional traits (sclerits in nymphs and pteromorphs in adults) might serve for categorizations within the Circumdehiscentiae. However, for future prospects to clarify the taxonomy of the circumdehiscent mites it is necessary to increase taxon sampling and to search for additional morphological traits that might serve as diagnostic characters for taxonomic assignment.

- 136 - 5. Character evolution within Circumdehiscentiae

Acknowledgments

Financial support was provided by the Austrian Science Fund (FWF, project number P19544-

B16). We are grateful to Ernst Ebermann, Peter Horak, Julia Knapp, Heinrich Schatz and

Johan Witters for providing moss samples for our study and Günther Raspotnig for the SEM- micrograph of Liacarus subterraneus. Furthermore, the authors thank Ferdinand Hofer and his team at the Research Institute for electron Microscopy (FELMI) for the cooperation in making SEM-micrographs.

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- 144 - Conclusions

Conclusions

The aims of the present work were to investigate, by means of molecular genetic approaches, several evolutionary questions within the family Scutoverticidae and to evaluate the validity of various morphological classification schemes of the Circumdehiscentiae.

Molecular phylogenetic analyses based on a total of four molecular markers revealed remarkable new insights into the evolutionary history and mechanisms of European scutoverticid mites. A molecular phylogeny clearly confirmed the taxonomic discreteness of several formerly and recently described scutoverticid species, representing the four genera Scutovertex, Lamellovertex,

Provertex and Exochocepheus. Moreover, during the investigations three new species have been described according to evidence from foregone genetic studies and at least two further undescribed species have been identified. Furthermore, the data revealed the paraphyly of the genus Scutovertex resulting from the grouping of S. pictus with specimens of the three remaining genera mentioned above. With the help of genetic data it was herein possible to support the morphology-based hypotheses that S. pictus does not belong to the genus Scutovertex. But so far, it was not possible to transfer “S.” pictus into another genus because morphological as well as molecular data rather indicate its representation as a distinct genus. The most remarkable result, however, was the corroboration of the partially expected occurrence of cryptic diversity also within the Scutovertex. The widespread “species” Scutovertex sculptus seems to be a complex of at least two cryptic species showing allopatric distributions in eastern (Russia, Austria, Sweden) and western (France, Ireland) Europe, respectively. Furthermore, phylogenetic analyses based on three nuclear markers revealed the paraphyly of the family itself, with members of two other families (Ameronothrus maculatus, Scapheremaeus cf. palustris) unambiguously placed within the Scutoverticidae. According to these findings, several conclusions on the taxonomy of the

- 145 - Conclusions family in general and in particular of the genus Scutovertex can be formulated. It is clear that species diversity within Scutovertex seems to be much higher than initially expected. However, no intraspecific morphological variation was observed. This also means that even minor morphological differences might be already regarded as evidence for a potentially new species.

Also the fact that authors in former literature pictured different drawings of as they thought “one and the same” species is a further indication for the presence of a yet undetected high diversity within the family Scutoverticidae. The presence of cryptic diversity within scutoverticid mites is not unexpected since cryptic diversity is generally high in little studied, small-size and short time generation organisms. Indeed, as already mentioned, several new species have been identified in the framework of this thesis. Detection of these new species proofed to be only possibly due to the application of molecular and morphological approaches, highlighting the importance of using a combined approach for disentangling the evolutionary history and detecting cryptic diversity in small organisms. For future prospects, also descriptions of new mite species should not only base on morphological characters alone but also include other analytical (molecular genetic or biochemical data) approaches, such as it was done for the new species Scutovertex pileatus described in the present work.

In an enlarged study dealing with intraspecific variability of two Austrian Scutovertex species remarkable differences in mtDNA diversity have been detected. Divergence estimates in Austrian

S. minutus are much lower than in Austrian S. sculptus. A possible difference in the reproductive mode could be one reason therefor. But both species reproduce sexually and albeit the sex-ratio of the studied S. minutus samples was clearly female biased, it is rather unlikely that differences in the reproductive mode are primarily responsible for the different diversity estimates in S. minutus and S. sculptus, because quite a few male individuals were also identified in most populations of S. minutus. The difference is surely founded in the dispersal ability of the species.

- 146 - Conclusions

Scutovertex minutus seems to be a poor disperser as it is considered for oribatid mites. This species has a low overall genetic diversity which is accompanied by a pronounced phylogeographic structure indicating spatially limited dispersal ability. On the contrary, S. sculptus represents a large panmictic population with very divergent haplotypes at single sampling sites suggesting a high colonizing capacity and dispersal ability, maybe attributed to a potential phoresy on birds. During this study it was further confirmed that S. sculptus shows a high tolerance for and adaptation to different environmental conditions. The initial hypothesis, assuming high levels of genetic differentiation among populations from different habitat types, was clearly rejected. This means that S. sculptus is able to inhabit several types of soils (saline soil, mosses on rocky habitats etc.) implying high physiological adaptations. Thus, it is not understated when speaking from S. sculptus as an extremist “par excellence”.

In the last part of my thesis, the main focus was to evaluate the utility of various currently used taxonomic classification schemes of the Circumdehiscentiae. Tracing six diagnostic morphological characters on a molecular phylogeny revealed interesting results regarding the character evolution of these traits. Whereas the utility of four characters for circumdehiscent categorization was supported by single origins of the characters within Circumdehiscentiae, results of two main traits (the octotaxic system and the wrinkled gastronotic cuticle of nymphs) strongly conflict current morphological classification schemes as a result of multiple independent evolution after an explosive radiation. Results of ancestral character state reconstructions imply that all currently applied classification schemes are not appropriate as they are and that, judging from our results, the most recent proposal with 24 superfamilies without any higher groupings is the best one at the current point of our knowledge. Furthermore, two additional traits (sclerits in nymphs and pteromorphs in adults) might serve for categorizations within the

Circumdehiscentiae when looking at a particular trait alone but not for a combination of the traits.

- 147 - Conclusions

However, it is certainly necessary to increase taxon sampling and to find “new” possible classification traits before proposing a “right” classification for Circumdehiscentiae.

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Schäffer Sylvia Universitätsplatz 2 8010 Graz AUSTRIA Phone: 0316-380 5611 E.mail: [email protected]

Curriculum Vitae

Personal data ƒ Nationality: Austria ƒ Date and place of birth: 15.10.1980 Graz

School education 1987 – 1991 Elementary school Elisabeth / Graz

1991 – 1999 BG & BRG Seebacher / Graz

Universitary education 1999 - 2005 Studies in biology/zoology at the Karl- Franzens (KF) University Graz 2004 – 2005 Diploma thesis titled “Taxonomische und ökologische Untersuchungen an heimischen Vertretern der Hornmilbengattung Scutovertex (Oribatida, Scutoverticidae)” in the group of Dr. Günther Krisper

Teaching experience SS 2005 – SS 2007 Teaching Assistent „Tiermorphologisches Proseminar” WS 2007 Teaching Assistent SE Evertebrata SS 2008 & 2009 Tutor SE Spezielle Zoologie Teil: Analysemethoden der phylogenetischen Systematik WS 2008 & 2009 Tutor SE Molekulargenetische Arbeitsmethoden

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