Phylogeny of parasitiform mites (Acari) based on rRNA

Molecular Phylogenetics and Evolution 43 (2007) 936–951www.elsevier.com/locate/ympev

Phylogeny of parasitiform mites (Acari) based on rRNA

Hans Klompen a,¤, Mariam Lekveishvili a, William C. Black IV b

a Department of Entomology, Ohio State University, Columbus, OH 43212, USAb Department of Microbiology, Immunology and Pathology, Colorado State University, Ft. Collins, CO 80523, USA

Received 31 May 2006; revised 2 October 2006; accepted 21 October 2006Available online 6 November 2006

Abstract

Acari (mites and ticks) form one the most diverse lineages of arthropods, but basal relationships in the group are still poorly under-stood. The current study addresses this issue for one of its two main lineages, the order Parasitiformes. Relationships are examined at thesubordinal and infraordinal level using complete 18S and partial 28S nuclear rRNA sequence data. Most currently recognized lineagesare recovered with good support, suggesting that nuclear rRNA, and speciWcally 18S rRNA, is very well suited for analyzing relationshipsat this level in this lineage. These results were found despite quite variable rates of sequence evolution, with rates “ratcheting up” from rel-atively low in most non-mite arachnid lineages, to intermediate in Pseudoscorpiones, the mite order Acariformes, and the parasitiformsuborders Opilioacarida, Holothyrida, and Ixodida, to high in the parasitiform suborder Mesostigmata. The most species rich mesostig-matid infraorder, Dermanyssina, shows huge distances to the outgroups, but remarkably low within-group divergence in nuclear rRNA.This suggests the possibility of a relatively recent origin of this lineage.© 2006 Elsevier Inc. All rights reserved.

Keywords: 18S rRNA; 28S rRNA; Acari; Parasitiformes; Evolutionary rates

1. Introduction

As currently conceived, mites (Acari) are divided intotwo orders, the Acariformes and Parasitiformes. Parasiti-formes (DAnactinotrichida) is the smaller of the two, butstill forms one of the largest lineages in the Arachnida withabout 80 described families and more than 12,500 describedspecies (Walter and Proctor, 1999). It is also ecologicallydiverse, including obligate parasites of vertebrates (e.g.,ticks [Ixodida], many Dermanyssoidea), and invertebrates(e.g., Varroa, Diarthrophallidae), soil and arboreal preda-tors, fungivores, and scavengers. There is considerableagreement on several aspects of classiWcation of the group,including assumed monophyly of the four suborders (Opi-lioacarida, Holothyrida, Ixodida, and Mesostigmata) andof many mesostigmatid infraorders or cohorts. However,the monophyly of these groups has not been rigorouslytested, and agreement on relationships among the subor-

* Corresponding author. Fax: +1 614 292 7774.E-mail address: [email protected] (H. Klompen).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2006.10.024

ders and mesostigmatid infraorders is certainly not univer-sal (Camin and Gorirossi, 1955; Hirschmann, 1975;Johnston, 1982; Lehtinen, 1991; Lindquist, 1984; Murrellet al., 2005; Norton et al., 1993). One reason for the lack ofstudies at this taxonomic level is the perception that thereare relatively few morphological characters that can bescored for all groups.

Molecular systematics clearly can make a contributionin this area, but such analyses so far have been appliedalmost exclusively to relationships in ticks, that is the sub-order Ixodida (Black et al., 1997; Dobson and Barker,1999; Klompen et al., 2000; Murrell et al., 1999, 2000,2001, 2003). Studies involving the very diverse suborderMesostigmata have been few and relatively small in scale(Cruickshank and Thomas, 1999; Klompen, 2000; Lekve-ishvili and Klompen, 2004), and only one analysis (Mur-rell et al., 2005) has focused speciWcally on subordinalrelationships in Parasitiformes. The sampling of mites ingeneral, and Parasitiformes in particular, in broader stud-ies of arthropod or chelicerate evolution has also beenweak. The latest comprehensive analysis of Chelicerate

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H. Klompen et al. / Molecular Phylogenetics and Evolution 43 (2007) 936–951 937

evolution (Giribet et al., 2002) included Wve mites, onlythree of which were Parasitiformes, and, recent analysesof arthropod relationships (Mallatt and Giribet, 2006;Regier et al., 2005) featured, respectively, one and none.

The utility of various genes for reconstructing phyloge-netic relationships in Parasitiform mites is therefore notwell established. This is true even for the the large (28S) andsmall (18S) ribosomal RNA (rRNA) genes that are mostcommonly used in studies of arthropod and cheliceraterelationships (Cruickshank, 2002). The 18S rRNA gene isgenerally considered more appropriate for resolving rela-tionships among phyla and superphyla, with the 28S rRNAgene providing more signal at slightly lower taxonomic lev-els (Mallatt et al., 2004; Mallatt and Giribet, 2006). NuclearrRNA genes have great advantages: they are generally easyto amplify, and they appear to contain more signal thanother genes used for higher-order questions in animal phy-logeny (Giribet, 2002). However, nuclear rRNA genes oftenexhibit strikingly diVerent rates of evolution, with atten-dant problems in alignment, and possibly, analysis. Oftencited examples of unusually high rates of sequence evolu-tion of these genes in arthropods s.l. include Onychophora,some Symphyla, Diplura, Zoraptera, Strepsiptera, and Dip-tera (Mallatt et al., 2004; Yoshizawa and Johnson, 2005).Mesostigmata may have to be added to that list. Murrellet al. (2005) demonstrated considerable rate diVerentiationamong Parasitiformes, with rates in Mesostigmata signiW-cantly higher than in other Parasitiformes (or in Acarifor-mes). Unfortunately, the limited taxon sampling in thatstudy did not allow an assessment of possible rate variabil-ity within Mesostigmata, or a comparison with rates innon-mite Arachnida. The latter is important, because 18SrRNA has been used with considerable success at genericand familial levels in Ixodida (Klompen et al., 2000), sug-gesting elevated rates (relative to most Arthropods) even inparasitiform lineages that evolved much slower than Meso-stigmata. At this point there is simply no solid informationon rates in Parasitiformes relative to other Arachnida, oron the general utility of these two rRNA genes in thislineage.

The goal of the current study is thus twofold: (1) toassess the ability of nuclear rRNA genes to recover wellresolved relationships in Parasitiformes at the subordinaland infraordinal level and (2) to document any rate vari-ability that might occur in this lineage.

2. Taxonomic background

2.1. Subordinal relationships

We follow Norton et al. (1993) in recognizing four sub-orders within the order Parasitiformes: two small and gen-erally obscure ones, Opilioacarida and Holothyrida, the notvery diverse, but important ticks (Ixodida), and the hugelydiverse Mesostigmata (including groups such as Phytoseii-dae, fowl mites, and Varroa). Some early studies groupedOpilioacarida with Holothyrida in the Onychopalpida(Baker and Wharton, 1952; Camin and Gorirossi, 1955),but most recent studies suggest a sistergroup relationshipbetween Opilioacarida and the Parasitiformes s.s. (e.g.,Lindquist, 1984). Opilioacarida is occasionally evenaccorded its own ordinal status (Opilioacariformes)(Grandjean, 1969; Walter and Proctor, 1999). Relationshipsamong the remaining suborders are far less settled. Forexample, the Holothyrida has been grouped with Mesostig-mata by Johnston (1982) and with Ixodida by Lehtinen(1991) (Fig. 1A). The former study did not provide explicitcharacter evidence, but the latter did. Derived characterstates for the Holothyrida + Ixodida grouping include thepresence of a distinct Haller’s organ, a set of shared muscleinsertions on the subcapitular apodeme allowing ventralbending of the gnathosoma, and, possibly, unusually largesize (for mites) (Lehtinen, 1991). The latter character isshared with Opilioacarida and may well be primitive forParasitiformes (most non-mite Chelicerata are relativelylarge). Lehtinen’s hypothesis is also favored by preliminarymolecular data which place holothyrids very close to Ixo-dida (Dobson and Barker, 1999; Klompen et al., 2000;Murrell et al., 2005). This general understanding of subor-dinal relationships was challenged recently by a 18S rRNA

Fig. 1. Current views of relationships among parasitiform suborders (A) and mesostigmatid infraorders (B and C). (A) Adapted from Lindquist (1984) andLehtinen (1991); (B) Camin and Gorirossi (1955); (C) Johnston (in Norton et al., 1993).

938 H. Klompen et al. / Molecular Phylogenetics and Evolution 43 (2007) 936–951

based analysis suggesting that Mesostigmata is the sister-group of a lineage including not only Holothyrida and Ixo-dida, but also Opilioacarida (Murrell et al., 2005).

2.2. Infraorders of Mesostigmata

Most of the Parasitiform diversity (73 of 80 families inWalter and Proctor (1999) ) is within the suborder Meso-stigmata. Trägårdh (1946) proposed a basic divisionbetween “Agynaspida” (infraorders Sejina and Microgyni-ina, plus some Trigynaspida) and “Eugynaspida” (all otherMesostigmata) based on the absence or presence of distinctgenital shields in the female. However, Trägårdh’s interpre-tation appears to be incorrect, as both Sejina and Microg-yniina have a (single) genital shield (Camin and Gorirossi,1955; Krantz, 1961). As an alternative, Camin andGorirossi (1955) proposed a split between Trigynaspida(infraorders Cercomegistina and Antennophorina) vs.Monogynaspida, based once again on the conWguration ofthe female genital shield(s) (Fig. 1B). The Trigynaspida arecharacterized by three genital shields, the Monogynaspidashare the presence of a single genital shield. Not all hypoth-eses of relationships in the Mesostigmata are based on gen-ital shield characteristics. Hirschmann (1975) proposed aprimary division into Trichopygidiina, with setae on thepygidial shield of the larva, and Atrichopygidiina, withoutsuch setae. The Atrichopygidiina includes most members ofthe Uropodina, the Trichopygidiina includes all other infra-orders. Athias-Henriot (1975) proposed a grouping of Het-erozerconina and Dermanyssina, based on the sharedpresence of highly modiWed structures on the male chelic-erae that function in sperm transfer (podospermous taxa).The remaining Mesostigmata (tocospermous taxa) lacksuch modiWcations. However, this grouping is inconsistentwith sperm morphology, which groups Dermanyssina andParasitina (with “ribbon sperm”) vs. most others, whichhave “vacuolated sperm” (Alberti, 1991). Sperm in Hetero-zerconina is close to the vacuolated type (Alberti et al.,2002). In general, the position of Heterozerconina has beenone of the least understood questions in mesostigmatid sys-tematics. Johnston (in Norton et al., 1993) suggested agrouping of Heterozerconina with Trigynaspida, probablybased on association type (Fig. 1C). He thus rejectedMonogynaspida. In contrast, Klompen (2000) and Lekve-ishvili and Klompen (2004) proposed a grouping of Hetero-zerconina with or within Sejina, based primarily on DNAsequence data. A second issue concerns the relationships ofthe Sejina, Microgyniina, and Uropodina. All three taxa usethe second nymphal instar, the deutonymph, for phoreticdispersal. These deutonymphs share the added characteris-tic of attaching themselves to their host by secretions fromtheir anus. While many Parasitina and some Dermanyssinause deutonymphs for phoresy, these hold on by diVerentmeans. The complex of highly similar modiWcations of theanus of the deutonymphs in Uropodina, some Sejina, and,presumably, some Microgyniina, has been advanced as evi-dence of relationships between Sejina and Uropodina

(Kethley, 1983). A Wnal point of discussion is whether Zer-conina are part of the Dermanyssina (Krantz, 1978), form aseparate infraorder that is the sistergroup to theArctacarina + Parasitina + Dermanyssina lineage (Johnstonin Norton et al., 1993), or form a monophyletic lineage withEpicriina (quite distant from Dermanyssina) (Moraza andLindquist, 1998).

3. Materials and methods

3.1. Extraction and ampliWcation

Most of the template DNA was extracted using either amodiWed CTAB protocol (Black et al., 1997) or the DNeasyTissue Kit (Qiagen Inc.). Given the size of most mite spe-cies, many extractions used more than one whole organismper extraction (see Section 3.4). Primers are drawn fromWhite et al. (1990) and Giribet et al. (1996) for 18S rDNA,and from Hillis and Dixon (1991) and Whiting et al. (1997)for the 28S rDNA fragments.

3.2. PuriWcation and sequencing

The fragments sequenced are the entire 18S (»1800 bp)and two segments of 28S corresponding to the D3–D5 andD9–D10 variable regions (total» 1200 bp). PCR productswere puriWed using Wizard PCR Preps (Promega Corp.).Most sequencing was done at the Plant Microbe GenomicsFacility, Ohio State University, the remaining sequenceswere obtained at sequencing centers of Brigham YoungUniversity, the University of Georgia, Colorado State Uni-versity, and Macrogen Inc., Korea. Products weresequenced in both directions, and assembled using Sequen-cher 4.0 (Genentech Corp.).

3.3. Alignment

Sequences were provisionally aligned using Clustal-X(Thompson et al., 1997). This alignment was adjusted byhand, based on the current understanding of RNA sec-ondary structure (Kjer, 1995). The secondary structuremodels and nomenclature follow Cannone et al. (2002), asimplemented by Gillespie et al. (2005). Some regions weretoo variable for this approach, and were excluded.Regions excluded for 18S included the core of the termi-nal loops of stems H184a and H184b (variable region V2),e23-1, and H1399 (variable region V9), and part of theinterloop between stems H1241b and H1241c. Excludedregions for 28S include two sections of variable region D3(entire stem D3-1 plus interloop between stems D3-1 andD3-2, and terminal loop of stem D3-2), part of the inter-loop beteeen stems H837a and H837b (variable regionD4), and two sections of variable region D10. The totalnumber of excluded positions for both loci is 543 (posi-tions 249–289 326–431 920–933 2011–2022 2235–22572456–2512 2528–2596 2856–2871 3539–3556 3592–3778 incombined matrix). All of these regions were previously


identiWed as Regions of Alignment Ambiguity (RAA) orRegions of Expansion and Contraction (REC) (Gillespie,2004; Gillespie et al., 2005). Exclusion of such regionsclearly discards potential evidence, but this negative isconsidered acceptable given the alternative of includingregions for which primary homology statements areunsupported. The aligned matrix has been deposited atTreebase (study accession number S1671, matrix acces-sion numbers M3024; M3025), annotated alignments,including indicators of secondary structure elements, areavailable from the authors by request.

Preliminary analyses revealed a discrepancy betweenresults for the two gap treatments (gapDmissing andgapDWfth state; see below). To further explore the eVect ofshared indels, additional analyses were conducted exclud-ing nearly all gapped sites (D“no gap” data matrix). Rela-tive to the above set, the following regions were excluded(in some cases expansions of previously excluded frag-ments): 18S: entire terminal loop of stems H61 (variableregion V1), H184b, and H198b; the terminal loop of vari-able region V7; an expanded section of the terminal loop ofstem H1399; the entire interloop between stems H122 andH144, and V7’ and H1118’, and the variable part (RAAregion) of interloop H184b’ to H198b; 28S: the core of theterminal loop of stem H604, an additional section of vari-able region D3 (terminal loop of stem D3-3); the terminalloop (RAA) of variable region D5; the variable part of theinterloop between stems D5’ and H837a’; and an extendedpart of the terminal loop of stem H2127 (variable regionD9). Total number of excluded positions for both loci is851 (positions 83–102 152–192 242–295 311–446 452–459478–497 901–959 1857–1875 1885–1892 2011–2022 2228–2268 2406–2417 2454–2512 2528–2596 2634–26442856–2873 3001–3016 3033–3039 3435–3470 3539–35563592–3778 in combined matrix). The regions excluded inthe “no gap” matrix are similar, though not identical, tothose excluded by Mallatt et al. (2004); Mallatt and Giribet(2006) in their studies of ecdysozoan relationships.

3.4. Vouchering

When dealing with very large mites (e.g., Holothyrida,Ixodida), it was possible to do extractions based on part ofa single specimen, using the remainder as a voucher. How-ever, this procedure was less successful with many othermite species. The use of whole or multiple specimens for asingle extraction ampliWes the need for proper vouchering(Ruedas et al., 2000). A double vouchering system was usedfor most extractions. The Wrst set of vouchers (“secondary”vouchers) was drawn from the same series of specimensfrom which to be extracted material was selected (all speci-men series were drawn from single collections). This mate-rial was used for identiWcation. In addition, the remains ofspecimens used in the actual extraction were recovered andslide mounted as a second set of vouchers (Johnson et al.,2001). The quality of these “primary” voucher specimensvaried from excellent to “nothing but broken parts”, but

was generally suYcient for comparisons with the secondaryvoucher set. Extractions based on mixed species, as estab-lished by comparison of all voucher specimens, were dis-carded. Voucher numbers for both series are listed with thecollection data in Appendix A.

3.5. Taxon selection

The taxon set listed in Table 1 is based on the followingcriteria. (1) Inclusion of at least two representatives of allsuborders of Parasitiformes. This goal was achieved by theinclusion of 18S rRNA GenBank data (no 28S data avail-able) for an undescribed opilioacarid from Australia. Sam-pling for Holothyrida consisted of two exemplarsbelonging to the same family, Allothyridae. Representativesfrom the other two families in that suborder (Holothyridaeand Neothyridae) were not available. (2) Inclusion of atleast two representatives of all infraorders of Mesostig-mata. This goal was not achieved for the small infraordersArctacarina, Epicriina, Microgyniina, and Zerconina, forwhich only a single representative was included. Given therelatively low species diversity in these lineages (Walter andProctor, 1999) this is not considered a major problem. (3)Additional representatives of the lineages that are morediverse (Trigynaspida, Uropodina, Dermanyssina) or thatshowed unusual levels of variability (Sejina). This taxonsampling design allowed preliminary tests of monophylyfor most suborders and infraorders. The total number ofingroup taxa is 54.

We selected 33 outgroup taxa, which allowed (1) a testof monophyly of Parasitiformes, (2) a preliminary estimateof relationships of Parasitiformes with the second miteorder, Acariformes, and with non-mite arachnid orders,and (3) a comparison of relative rates of evolution for thevarious mite groups and non-mite Arachnida. Acariformesis represented by one member of the paraphyletic basalgroup Endeostigmata, and three representatives each ofthe two main acariform lineages, Prostigmata (Trombidi-formes) and oribatid mites (Sarcoptiformes). Two or threerepresentatives (only one available for Palpigradi andThelyphonida) of all other non-mite arachnid order werealso included. Pycnogonida (two representatives) is used asprimary outgroup (following Giribet et al. (2002)). Alldata for non-mite Chelicerata are drawn from GenBank(Table 1).

3.6. Analysis

Homogeneity of base frequencies was tested using astandard �2 test as implemented in PAUP4.0¤ (SwoVord,2002).

Data were analyzed using both parsimony and model-based analysis approaches. Bayesian inference, ratherthan maximum likelihood, was selected as a model-basedapproach because of the size of the matrix and the associ-ated computer time demands. Each approach has itsdrawbacks, parsimony analyses is sensitive to long branch


Table 1Taxa examined and GenBank Accession Numbers

Taxon name Order/Infraorder 18S 28S D3–D5 28S D9–D10

Achelia echinata Pycnegonida AF005438* AF005459* —Callipallene sp. Pycnegonida AF005439* AF005460* —Carcinoscorpius rotundicaudatus Xiphosura U91491* U91493* —Limulus polyphemus Xiphosura U91490* U91492* —Amblypygi sp. Amblypygi AF124933* AF062965* —Paraphrynus sp. Amblypygi AF005445* AF124959* —Aphonopelma sp../hentzi Araneae X13457* AY210803* AY210803*Coelotes terrestris Araneae AJ007986* — —Liphistius bicoloripes Araneae AF007104* AF124960* —Parasiro coiVaiti Opiliones U36999* U91495* —Odiellus troguloides Opiliones X81441* U91500* —Pachyloides thorelli Opiliones U37007* U91508* —Eukoenenia sp. Palpigradi AF207648* AF207653* —Americhernes sp. Pseudoscorpiones AF124934* AF062982* —Roncus cf. pugnax Pseudoscorpiones AF005443* AF124962* —Pseudocellus pearsei Ricinulei U91489* AF124956* —Ricinoididae sp. Ricinulei AF124930* AF062988* —Androctonus australis Scorpiones X77908* AF124955* —Belisarius xambeui Scorpiones AF005442* AF124954* —Pandinus imperator Scorpiones AY210831* AY210830* AY210830*Chanbria regalis Solifugae AF124931* AF062983* —Eusimonia wunderlichi Solifugae U29492* AF124958* —Gluvia dorsalis Solifugae AF007103* AF124957* —Stenochrus portoricensis Schizomida AF005444* — —Trithyreus pentapeltis Schizomida AF124932* AF062990* —Mastigoproctus giganteus Thelyphonida AF005446* AF062989* —Terpnacarus nr. gibbosus Ac “Endeostigmata” AY620904 AY626622 AY626585Horreolanus orphanus Ac Prostigmata AY620907 — AY626588Neochelacheles messersmithi Ac Prostigmata AY620908 AY626626 —Penthaleus minor Ac Prostigmata AY620909 — AY626587Hypochthonius rufulus Ac Oribatida AF022033* AY273495* —Meristolohmannia meristacaroides Ac Oribatida AY620905 AY626623 AY626586Nothrus silvestris Ac Oribatida AF022039* AF515420* —Opilioacarus texanus Op Opilioacaridae AF124935* AF124963 AF120302Opilioacaridae n. sp. Australia Op Opilioacaridae AF287235* — —Allothyrus australasiae gr. Ho Allothyridae AY620910 AY626628 AY626589Allothyrus constrictus gr. Ho Allothyridae AY620911 AY626629 AY626590Ixodes aYnis Ix Ixodidae L76350* — AF120288*Ixodes uriae Ix Ixodidae AF115369* — AF120296*Amblyomma americanum Ix Ixodidae AF291874* AF291874* AF291874*Dermacentor andersoni Ix Ixodidae L76340* AF120311*Rhipicephalus sanguineus (1) Ix Ixodidae AF062986*Haemaphysalis inermis Ix Ixodidae L76338* — AF120309*Argas persicus Ix Argasidae L76353* — AF289194*Argas lahorensis Ix Argasidae L76354* — —Carios puertoricensis Ix Argasidae L76357* — —Ornithodoros turicata Ix Argasidae AF213370* — AF120298*Otobius megnini Ix Argasidae L76356* — AF120297*Acanthodavacarus klompeni Me Cercomegistina AY620942 — AY626592Davacarus gressetti (2) Me Cercomegistina AY620944Asternoseius sp. Me Cercomegistina AY620913* — AY626593Cercoleipus coelonotus Me Cercomegistina AY620914 AY626632 AY626594Antennophorus wasmanni Me Antennophorina AY620915 AY626633 AY626595Euzercon latus Me Antennophorina AY620915* — AY626596Klinckowstroemiella helleri Me Antennophorina AY620943 AY626634 AY626597Megisthanus Xoridanus Me Antennophorina L76341* AY626635 AF120300Micromegistus bakeri Me Antennophorina AY620917 AY626636 AY626598Neomegistus sp. Me Antennophorina AY620918 AY626637 AY626599Passalacarus sylvestris Me Antennophorina DQ144646 — DQ144650Similantennurella spinata Me Antennophorina AY620945 DQ144654 —Microgynium incisum Me Microgyniina AY620919* AY626638 AY626600Diarthrophallus quercus Me Uropodina DQ144647 DQ144655 —Chiropturopoda sp. Me Uropodina AY620920 AY626639 AY626601


attraction, and underestimation of support valuesthrough Jackknife analyses (Alfaro et al., 2003; Simmonset al., 2004), while Bayesian methods may produce bias inclade support due to the standard use of Xat topologicalpriors (Pickett and Randle, 2005), often overestimate con-Wdence levels for especially short nodes (Alfaro et al.,2003; Suzuki et al., 2002), and show greater sensitivity toheterotachy (Kolaczkowski and Thornton, 2004). Asthese sensitivities are not (fully) overlapping, we considercongruent results for the two methods as increasing conW-dence in our results.

Initial parsimony analyses were conducted using theratchet as implemented in PRAP (Müller, 2004). Subse-quent analyses aimed at Wnding all equally most parsimo-nious trees were conducted in PAUP¤4.0. This programwas also used for analyses requiring settings not availablein PRAP (e.g., gapsD Wfth state), using heuristic searcheswith 100 random taxon addition sequences to avoid localoptima. Additional replications were used when less than5 of the replications generated the shortest trees. For allanalyses gaps were treated either as missing data or as aWfth state. The Wrst approach underestimates the impor-tance of indels, the second overestimates it. Identicalresults for both approaches suggest robustness relative toparameter variability. Elimination of all gapped sites, as isstandard in Maximum Likelihood based analyses, doesnot allow exploration of the eVect of indels. This discard-ing of potential evidence seems particularly unfortunatewhen dealing with lineages where length variability is

common. Incongruence Length DiVerence (ILD) (Micke-vich and Farris, 1981) was calculated to examine possibleconXicting signals among the various components of thedata set. Two sets of partitions were explored: 18S vs. 28S,and all segments as delimited by sequencing primers (3 for18S, and 2 for 28S). Lineage support was measured by cal-culating Bremer Support (BS) (Bremer, 1988) and by jack-knife (JS) analysis (Lanyon, 1985). Jackknife analyseswere executed using the settings: 37% deletion, emulate“JAC” resampling, 1,000 replications, “random additionsequences”D 1, and “hold trees” D 2 (Freudenstein et al.,2004).

For Bayesian analyses we used the “no gap” matrix only(following standard approaches in ML/Bayesian analyses).The model selected was a general time reversible (GTR)model of nucleotide substitution with a proportion ofinvariant sites (I) and a gamma distribution (�) of among-site rate heterogeneity. This model provided the best Wt asjudged by both the Akaike (AIC) and Bayesian (BIC)Information Criterion approach as implemented in Model-test 3.7 (Posada and Crandall, 1998). Proportion of invari-able sites is 26.6% with a gamma shape parameter of 0.6092.The GTR+I+� model was implemented in MrBayes 3.1(Ronquist and Huelsenbeck, 2003) with the commandnstD6 ratesD invgamma. The number of categories used toapproximate the gamma distribution was set at 4, and fourMarkov chains were run for 1,000,000 generations. Stabil-ization of model parameters (burn-in) occurred around250.000 generations. Every 100th tree after stabilization

Table 1 (continued)

Ac: order Acariformes; Op: Opilioacarida; Ho: Holothyrida; Ix: Ixodida; Me: Mesostigmata; —: no sequence available. * sequence drawn from GenBank.(1) Dermacentor: 18S, 28S (D9–D10) D. andersoni, 28S (D3–D5) Rhipicephalus sanguineus (2) Davacaridae: 18S NS14 and 28S (D9–D10) Acanthodavaca-rus klompeni; 18S NS58 Davacarus gressetti.

Taxon name Order/Infraorder 18S 28S D3–D5 28S D9–D10

Macrodinychus sellnicki Me Uropodina AY620921 AY626640 AY626602Oplitis sp. Me Uropodina AY620922 AY626641 —Polyaspis lamellipes Me Uropodina AY620923 AY626642 AY626604Prodinychus sp. Me Uropodina AY620924* AY626643 AY626605Trachytes baloghi Me Uropodina DQ279720 — —Trigonuropoda sp. Me Uropodina DQ144648 DQ144656 —Uroobovella sp. Me Uropodina DQ144649 DQ144657 DQ144651Uropoda orbicularis Me Uropodina AY620926* AY626645 AY626607Discozercon sp. Me Heterozerconina AY620927* — AY626608Narceoheterozercon ohioensis Me Heterozerconina AY620928* — AY626609Asternolaelaps sp. Me Sejina AY620929* AY626646 AY626610Epicrosejus sp. Me Sejina AF287237* AY626647 AY626611Sejus carolinensis Me Sejina AY665724* — DQ144652Uropodella laciniata Me Sejina AY620930* AY626648 AY626612Epicrius longisetosus. Me Epicriina AY620931 AY626649 —Zercon sp. Me Zerconina AY665725* DQ144658 DQ144653Proarctacarus oregonensis Me Arctacarina AY620933* AY626651 AY626614Pergamasus cf. canestrinii Me Parasitina AY620934 AY626652 AY626615Phorytocarpais Wmetorum Me Parasitina AY620935* AY626653 AY626616Poecilochirus sp. Me Parasitina AY620936 AY626654 AY626617Cosmolaelaps triWdus Me Dermanyssina AY620937 AY626655 —Gamasiphis pulchellus Me Dermanyssina AF115374 AY626656 AL120301Lasioseius sp. Me Dermanyssina AY620938 AY626657 AY626618Macrocheles sp. Me Dermanyssina AY620939 AY626658 AY626619Varroa destructor Me Dermanyssina AY620940 AY626659 AY626620Veigaia sp. Me Dermanyssina AY620941 AY626660 AY626621


(burn-in) was sampled to calculate a majority consensustree. Values on this tree are interpreted as posterior proba-bility values of the nodes.

Following Mallatt et al. (2004) we interpret posteriorprobabilities equal or over 95% in Bayesian trees, andjackknife values equal to or over 60% as signiWcantsupport.

Sequence divergence was initially assessed by calculatingaverage uncorrected “p” distances of all taxa relative to theoutgroup, followed by relative rate analyses (always usingPycnogonida as outgroup). Pair-wise relative rate analyseswere executed in HyPhy v0.98 (Kosakovsky Pond et al.,2005), using the HKY85 model, and local parameters. Aspair-wise analyses may hide taxonomic eVects, we checkedfor such artifacts by analyzing a subset of data usingRRTree vs. 1.1 (Robinson-Rechavi and Huchon, 2000),which allows the use of a guide tree to incorporate eVects oftopology. The tree in Fig. 3 was used, but with all weaklysupported clades collapsed. Lineages compared includedamong non-mites Xiphosura, Tetrapulmonata, Opiliones,Pseudoscorpiones, Ricinulei, Scorpiones, and Solifugae;among acarine lineages, Acariformes, Opilioacarida,Holothyrida + Ixodida, and Mesostigmata. RRTree wasexecuted using only 18S rRNA data and a subset of taxa(see Table 3; Appendix B).

4. Results

4.1. Systematics

4.1.1. Data structureAverage nucleotide frequencies (A 26.0%; C 21.7%; G

27.1%; T 25.2%) suggest no substantial overall bias(PD0.00 in �2 test of homogeneity across all taxa(dfD258); same result for analyses across Parasitiformesonly (dfD159)), but there is moderate variation amongestablished lineages. The most striking deviation fromequal distributions occurs in the lineage of Arctacarina +Parasitina + Dermanyssina, with relatively low frequenciesof C (17.8%), and higher levels of A and T, respectively,28.1% and 29.0% (PD 0.99 in �2 test of homogeneity(dfD27)).

4.1.2. ConXict among partitionsThere is no major conXict among any of the partitions.

ILD values for, respectively, the 18S vs. 28S partition andthe partition based on the Wve fragments as deWned bysequencing primers, are 0.86 (0.70 for gapsDWfth) and 2.64(2.53 for gapsDWfth).

4.1.3. Partial analyses (parsimony only)After exclusion of uninformative characters and unalig-

nable regions, the 18S and 28S matrices retained, respec-tively, 828 and 508 informative characters (881 and 537 ifgapsDWfth state), total for combined 1336 (1418).

The analysis based on 18S data generated 12 equally mostparsimonious trees of length 6716, CID0.26, RID0.63 (for

gapsDWfth state: 21 trees, length 7140, CID0.27, RID0.64).The strict consensus tree for either gap treatment (Fig. 2)shows monophyly of the Acari, although with only modestsupport (Jackknife Support (JS)D68/62%). In contrast, sup-port for the two constituent orders of Acari, Acariformes andParasitiformes, and the four suborders of Parasitiformes isgenerally strong (JSD>95%; 88/86% for Ixodida). WithinMesostigmata, Trigynaspida (DCercomegistina + Antenn-ophorina), Uropodina + Microgyniina, and Gamasina(DEpicriina+ Zerconina +Arctacarina + Parasitina + Dermanyssina) are all monophyletic under both gap treatments,although support for monophyly of these lineages, and forrelationships among them, varies. Parasitina is alwaysincluded in Dermanyssina. The grouping of Sejina and Het-erozerconina (DSejina s.l. (Lekveishvili and Klompen, 2004))is monophyletic in 8 of 12 equally most parsimonious trees.Overall, resolution and support are quite good. In terms ofsupport, 20 of a possible 53 groupings in Parasitiformesare very strongly supported (JS >95% under both gaptreanstments).

The analyses based on 28S data only, generated 126trees of length 3278 (CID 0.31, RID 0.59) in analyses withgaps treated as missing data (gapsD Wfth state: 113 treesof length 3590, CID 0.33, RI D 0.59). Overall resolutionand support is much weaker than for 18S. The fourparasitiform suborders are monophyletic, but someestablished groupings, including Parasitiformes, are notrecovered in any of the equally most parsimonioustrees. Of the four main lineages of Mesostigmata noted forthe 18S partial analysis, Trigynaspida, Uropodina +Microgyniina, Gamasina, and Sejina + Heterozerconina,only Trigynaspida is recovered, and that with weak support(JS < 50%). Dermanyssina is monophyletic undergapDmissing, but is paraphyletic relative to Parasitina(as in 18S) for gapD Wfth state. Some of this lack of reso-lution and support is undoubtedly due to missing data, asthe 28S data set includes relatively more missing datathan the 18S set. On the other hand, it also provides anindication that these segments of 28S by themselves donot have suYcient resolving power for this question. Useof complete 28S sequences is likely to provide betterresults (Mallatt et al., 2004).

4.1.4. Total evidenceResults of the parsimony and Bayesian analyses are

shown in Figs. 3 and 4. The parsimony analyses resulted in98 equally most parsimonious trees of length 10081,CID 0.27, RID 0.63 (13 trees, length 10806, CID0.29, andRID0.62 for gapsDWfth state). The topologies for the strictconsensus trees for the two gap treatments (Fig. 3) arelargely compatible, although results for the two treatmentsdiVer in arrangement of the taxa in Sejina and Heterozer-conina.

The combined analyses (parsimony and Bayesian)weakly support monophyly of Acari (Bremer Support(BS)D 3/2; JSD 69/57%; posterior probability (PP)D 82%),with Pseudoscorpiones, Palpigradi, Solifugae, and a


Fig. 2. Strict consensus of eight equally most parsimonious trees based on 18S rRNA data only (gaps D missing). Values below branches indicate Jackknifesupport (gaps D missing/gaps D Wfth state). Order abbreviations for outgroups: Ac: Acariformes; Am: Amblypygi; Ar: Araneae; Op: Opiliones; Pa: Palpi-gradi; Ps: Pseudoscorpiones; Py: Pycnogonida; Ri: Ricinulei; Sc: Scorpiones; So: Solifugae; Sz: Schizomida; Th: Thelyphonida; Xi: Xiphosura.


Fig. 3. Strict consensus of 98 equally most parsimonious trees based on combined 18S and partial 28S rRNA data (gaps D missing). Values above branchesindicate Bremer support (gaps D missing/gaps D Wfth state; calculated only for basal branches), values below branches indicate Jackknife support(gaps D missing/gaps D Wfth state). Inset: arrangement of Sejina s.l. with gaps D Wfth state. xx: incompatible topologies under diVerent gap treatments.Order abbreviations for outgroups as in Fig. 2.


grouping of all other Arachnida as possible outgroups toAcari. Support for any speciWc hypothesis of sistergrouprelationships of mites is weak in all analyses. However, the

two main lineages of Acari, Acariformes and the targetgroup, Parasitiformes, are very strongly supported (BS > 8,JS > 95%; PPD100).

Fig. 4. Bayesian analysis tree (GTR + I + �� ) calculated for “no gap” data set with combined 18S and partial 28S data. Values below branches indicateposterior probabilities. Order abbreviations for outgroups as in Fig. 2.


Similarly, support for monophyly of three (out of four)parasitiform suborders is very strong. Ixodida has moder-ate support (BSD 3/4, JSD 87/78%; PPD 83%), but thegrouping of Ixodida and Holothyrida is very strongly sup-ported (BSD18/18, JSD 100/100%; PPD 100%). Otherhypotheses of relationships among the suborders are lessclear. The traditional grouping of Ixodida, Holothyrida,and Mesostigmata (DParasitiformes s.s., Anactinotrichidasensu Van der Hammen (1961); Lindquist, 1984; Walterand Proctor, 1999) is moderately supported in Bayesiananalyses (PPD92%), but monophyly of Parasitiformes s.s.in parsimony analyses requires 4–6 extra steps, dependingon gap treatment. The alternatives, Opilioacarida withMesostigmata (weakly supported in all “most parsimoni-ous” trees, BSD1/3; JSD58/62%) or Opilioacarida withHolothyrida + Ixodida (requires an additional 1 or 3 steps),are also poorly supported.

Given the limited taxon sampling for those groups, theanalysis does not provide new data on relationships withinOpilioacarida or Holothyrida. Relationships within Ixo-dida follow results of previous analyses (e.g., Klompenet al., 2000), including monophyly of the families Ixodidaeand Argasidae. Relationships within Mesostigmata showsigniWcant support for Trigynaspida (BSD 35/39; JSD 100/100%; PPD100%), Uropodina + Microgyniina (BSD13/12;JSD99/99%; PPD100%), Sejina + Heterozerconina(BSD3/10; JSD72/96; PPD100%) and Gamasina(BSD10/20; JSD 88/99; PPD 100%). Relative to the resultsfor 18S alone, the addition of partial 28S data adds weak,but consistent, support for Monogynaspida, and modestsupport for the grouping of Sejina + Heterozerconina. Add-ing 28S also increases support for some relationships withinDermanyssina. The number of lineages within Parasitifor-mes with strong support increases from 20 in 18S only to 21in the combined parsimony analyses.

While parsimony and Bayesian analyses agree on mono-phyly of these four major lineages in Mesostigmata, they dis-agree (in part) on relationships among them. Both analysismethods provide signiWcant support for a grouping of Sejinas.l. and Gamasina BSD10/13; JSD93/99; PPD100%), butwhile parsimony methods weakly favors a sistergroup rela-tionship of Trigynaspida and Monogynaspida (Fig. 3), Bayes-ian analyses weakly favor a sistergroup relationship betweenUropodina s.l. and “all other Mesostigmata” (Fig. 4).

Relative to traditional classiWcations, monophyly ofmost infraorders is upheld in all analyses. Exceptionsinclude Cercomegistina and Antennophorina (both require2/1 added steps), Dermanyssina s.s. (without Parasitina)(10/12 added steps), and Sejina. Monophyly of the latterinfraorder requires 1 added step in parsimony analyses ifgapDmissing (Fig. 3), but is strongly supported (BSD 8,JSD94%) if gapDWfth state (Fig. 3 inset). This very strongand unexpected diVerence in results of parsimony analysesrelative to gap treatment can be traced to a set of shareddeletions in Gamasina in variable region D9 (loop H2127)of 28S. The coding approach of gapDWfth state results instrong added support for Gamasina, indirectly providing

support for Sejina s.s. Analyses using the “no gap” matrix,which excluded the aVected region (not shown), are consis-tent with this hypothesis. Using that matrix, Sejina s.l.(including Heterozerconina) is the sistergroup to Gamasina(gapDmissing: 3 trees, length 8302, CID0.29, RID0.63;gapDWfth state: 2 trees, length 8687, CID0.30, RID0.64).The remaining parts of the resulting consensus trees arefully compatible with the results for the more expandedcharacter set. Not surprisingly, given use of the same “nogap” matrix, results of the Bayesian analyses agree oninclusion of Heterozerconina within Sejina, and sistergrouprelationships of Sejina s.l. with Gamasina, but relationshipsin Sejina s.l. show a third permutation, with Heterozercon-ina as sistergroup to Sejidae (Fig. 4). While the close associ-ation of Sejina s.s. and Heterozerconina [DSejina s.l. ofLekveishvili and Klompen (2004)] is conWrmed in all analy-ses, the current data set does not resolve within-group rela-tionships in that lineage with any conWdence.

5. Rates

Average uncorrected “p” distances (relative to the two pyc-nogonid outgroup taxa) vary from relatively low in most non-mite Chelicerata (0.078–0.097), to quite high in Mesostigmata(0.180–0.213) (Table 2). Values for Pseudoscorpiones, Acari-formes, and the clade of Ixodida+Holothyrida are intermedi-ate. Uncorrected “p” distances most likely underestimate theactual divergence level, especially at high rates of evolution,because they do not include any mechanism to account forreversals. Maximum likelihood methods should allow correc-tion for such biases. Still, all relative rate analyses, whetherpair-wise without consideration of topology or comparison oflineages including consideration of topology, suggest the samethree distinct “classes” of taxa, each characterized by signiW-cantly diVerent rates (Table 3; Appendix B). Rate diVerencesamong most non-mite chelicerate taxa are insigniWcant, butall comparisons between these taxa and either Pseudoscorpi-ones or any of the four mite lineages tested (Acariformes, Opi-lioacarida, Holothyrida+Ixodida, Mesostigmata) were highly

Table 2Average uncorrected “p” distances relative to Pycnogonida

N Average “p” distance SD

Xiphosura 2 0.081 0.002Amblypygi 2 0.078 0.002Araneae 3 0.087 0.002Opiliones 3 0.090 0.003Scorpiones 3 0.092 0.004Solifugae 3 0.097 0.004Schizomida 2 0.078 0.007Pseudoscorpiones 2 0.117 0.004Acariformes 7 0.121 0.019Parasitiformes 54 0.176 0.044Holothyrida/Ixodida 12 0.112 0.010Mesostigmata 41 0.198 0.024Trigynaspida 11 0.199 0.021Uropodina s.l. 11 0.192 0.023Sejina s.l. 6 0.180 0.017Gamasina 13 0.213 0.023


signiWcant (P<0.001). Second, rates among Pseudoscorpi-ones, most Acariformes, and the large-bodied suborders ofParasitiformes (Opilioacarida, Holothyrida, Ixodida) are gen-erally not signiWcantly diVerent. Some prostigmatid Acarifor-mes (Neochelacheles, Horreolanus) appear to have moreelevated rates as shown by pair-wise relative rate tests. Addi-tional sampling should allow a better assessment of the distri-bution of high rates in Prostigmata. Rates in Mesostigmataare in yet another “class”: all lineage comparisons betweenMesostigmata and any other lineage tested were highly sig-niWcant (Table 3). A few pair-wise tests involving comparisonsbetween some Mesostigmata (Polyaspis, Asternolaelaps, Zer-con) and either Neochelacheles or Horreolanus (Acariformes)showed less striking diVerences (P»0.01). These results sug-gest a gradual ratcheting up of evolutionary rates in 18SrRNA. From relatively low in most Chelicerata, to moder-ately high in Pseudoscorpiones, (many) Acariformes, and thelarge bodied Parasitiformes, to quite high in Mesostigmata(and possibly some Acariformes).

Pair-wise tests also reveal some striking rate variabilitypatterns within Mesostigmata. Most of the four major lin-eages noted in the phylogenetic analyses, Trigynaspida,Uropodina s.l., Sejina s.l., as well as “Epicriina plus Zercon-ina” include taxa with very diVerent rates. These diVerencescannot all be explained as phylogenetic eVects, because sis-ter taxa may show signiWcantly diVerent rates (e.g., Astern-olaelaps and Uropodella), while distantly related taxa shareapparently similar rates (e.g., Euzercon, Uroobovella). Thestriking exception to this pattern is Dermanyssina s.l. (thatis including Parasitina). Not one pair-wise comparisonwithin that lineage shows highly signiWcant rate diVerences.

6. Discussion

6.1. Systematics

Overall, the majority of basal relationships in Parasiti-formes are well supported in all analyses (parsimony orBayesian). Even so, a few issues generated contradictory

results, and are thus considered unresolved. In terms ofrelationships among the suborders, the traditional, mor-phology based, view is that Opilioacarida is the sistergroupto a lineage including the remaining suborders (Parasitifor-mes s.s.). This view was challenged by Murrell et al. (2005)in their analyses of 18S rRNA sequences. They groupedOpilioacarida within Parasitiformes s.s. as sistergroup toHolothyrida + Ixodida. Our parsimony analyses supportthe inclusion of Opilioacarida in Parasitiformes, but as sist-ergroup to Mesostigmata, rather than to theHolothyrida + Ixodida lineage (and with weak support). Incontrast, the Bayesian analyses are consistent with the moretraditional view of Opilioacarida as sistergroup to Parasiti-formes s.s. Based on these indecisive results we do notbelieve that the traditional view can be rejected based onour analyses. In contrast, support for sistergroup relation-ships of the suborders Holothyrida and Ixodida is quitestrong in all analyses, consistent with previous morphology(Lehtinen, 1991) and molecular based hypotheses (Murrellet al., 2005).

At the infraordinal level within Mesostigmata, the tra-ditional division into Mono- and Trigynaspida (Caminand Gorirossi, 1955) is (weakly) supported in parsimonyanalyses, but rejected in Bayesian analyses. The latterweakly support a solution closer to the Hirschmann(1975) hypothesis of a basal position of Uropodina. Thisoption requires three (7 if gapD Wfth state) added steps inparsimony analyses. A third alternative, with a basalposition of Sejina, is strongly rejected, requiring 11 (16)added steps in parsimony analyses. Given the lack ofclear resolution, we prefer to leave the relationship ofTrigynaspida, Uropodina s.l., and the Sejina + Gamasinalineage as an unresolved trichotomy. The commonly sug-gested relationship of Sejina with Microgyniina isstrongly rejected, requiring 54 (56) added steps (thesenumbers get reduced somewhat using a constraint mak-ing the grouping of Microgyniina, Sejina, and Heterozer-conina monophyletic (43 (47)). Instead a novelhypothesis, featuring a close association of Microgyniina

Table 3Relative rates of nucleotide substitution among the main lineages for 18S rRNA

Numbers listed are P-values for comparisons of diVerent lineages (further details in Appendix B). Note: Tetrapulmonata includes Araneae, Schizomida, Thelyphonida, Amblypygi; Holothyrida/Ixodida includes both Holothyrida, Argas persicus, Cariospuertoricensis, Ornithodoros moubata, Ixodes aYnis, Amblyomma americanum, and Dermacentor variabilis; Mesostigmata includes Acanthodavacarus klom-peni, Megisthanus Xoridanus (Trigynaspida), Polyaspis lamellipes, Uropoda orbicularis (Uropodina s.l.), Epicrosejus sp., Narceoheterozercon ohioensis (Sej-ina s.l.), Zercon sp. Varroa destructor (Gamasina).

N Xi Te Op Ri Sc So Ps Ac Opil HoIx

Xiphosura (Xi) 2Tetrapulmonata (Te) 8 0.689Opiliones (Op) 3 0.344 0.068Ricinulei (Ri) 2 0.262 0.300 0.017Scorpiones (Sc) 3 0.056 0.003 0.232 0.002Solifugae (So) 3 0.108 0.020 0.374 0.004 0.756Pseudoscorpiones (Ps) 2 0.000 0.000 0.000 0.000 0.000 0.000Acariformes (Ac) 7 0.000 0.000 0.000 0.000 0.000 0.000 0.379Opilioacarida (Opil) 2 0.000 0.000 0.000 0.000 0.000 0.000 0.811 0.271Holothyrida + Ixodida (HoIx) 8 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.829 0.239Mesostigmata 8 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000


with Uropodina (Uropodina s.l.), is very well supportedin all analyses, while Sejina + Heterozerconina (Sejinas.l.) always groups as sistergroup to the Gamasina. Thisimplies that the characteristic of having deutonymphsthat attach to their phoretic host by anal secretions may(1) have multiple origins or (2) be primitive for Mesostig-mata with subsequent losses in Heterozerconina andGamasina (and Trigynaspida if Uropodina s.l. is thesistergroup to all other Mesostigmata). The grouping ofHeterozerconina with Sejina is consistent with earlierresults based on EF-1� sequence data (Klompen, 2000),and a combination of 18S, EF-1�, COI and morphology(Lekveishvili and Klompen, 2004). Thus several semi-independent analyses using diVerent molecular charactersets suggest a relationship for which no obvious support-ing morphological evidence is available (Lekveishvili andKlompen, 2004). The grouping of Zerconidae andEpicriidae (D Epicriina s.l.) (Moraza and Lindquist,1998) is well supported in parsimony analyses, butreceives no signiWcant support in Bayesian analyses. Wetentatively accept this grouping, but it needs furthertesting using a broader taxon sampling. Finally, theGamasina (Epicriina+Zerconina+Arctacarina+Parasitina+Dermanyssina) is well supported in all analyses.

The study was not designed to deal with relationships athigher taxonomic levels, but a few results are worth men-tioning. Monophyly of both mite orders, Parasitiformesand Acariformes, is very well supported, although the ques-tion of monophyly of Acariformes should probably berevisited with a more expanded taxon set. Monophyly ofmites as a lineage is also supported in all analyses, butalways with relatively weak support. This result mirrorsuncertainty about this issue in morphology based literature(Dubinin, 1957, 1962; Van der Hammen, 1977, 1979; Lind-quist, 1984; Zakhvatkin, 1952). That being said, monophylycannot be rejected based on available evidence. With regardto the question of relationships of mites to other orders ofArachnida, results are even less clear. The traditional asso-ciation or Acari with Ricinulei (Lindquist, 1984; Shultz,1990) is not well supported. Monophyly of a lineage ofAcari + Ricinulei requires 14 added steps (for both gaptreatments) in parsimony analyses. Hypotheses of closerelationships with Pseudoscorpiones, Palpigradi, or Solifu-gae, or even with a lineage including most of the non-miteChelicerata show a marginally better Wt. Overall, 18SrRNA and the fragments of 28S rRNA analyzed by them-selves do not appear to be well suited for answering thisquestion.

In summary, the analyses support most currently rec-ognized groupings within Parasitiformes, suggestingalternatives only in cases that were inconclusive based onprevious work. These result are achieved despite consid-erable diVerences in rates among lineages, and attendantpotential for artifacts due to long-branch attraction inparsimony analyses. The highly similar results of parsi-mony and Bayesian analyses suggest that long branchattraction does not generate any major artifacts in this

data set. Apparent artifacts (due to long-branch attrac-tion?) led Murrell et al. (2005) to prefer an analysis thateliminated all high variable positions, leaving only 115informative positions. In their analysis this stronglyimproved branch support (relative to analyses includingthe more variable sites). In contrast, we used 828 infor-mative positions, while still achieving good branch sup-port and avoiding obvious artifactual results. ThediVerence is most likely due to the inclusion of more taxa(e.g., 54 vs. 9 Parasitiformes), supporting the generalnotion that dense taxon sampling may be needed for taxawith high and highly variable rates. Overall, the strongsupport for most lineages suggests great utility of 18SrRNA sequence data at the infraordinal, familial andeven generic level in basal Mesostigmata. The high rateof evolution of the 18S rRNA gene in Mesostigmata hasclearly shifted the optimal range for this locus to muchlower taxonomic levels than usual. As for the 28S rRNAgene, the fragments used in this study are not very infor-mative at this taxonomic level. It would be worthwhile torevisit this issue for Parasitiformes when using largerfragments of that gene (Mallatt et al., 2004; Mallatt andGiribet, 2006).

6.2. Rate

It is tempting to speculate on causes for the remark-able diVerence in rate among diVerent arachnid orders.DiVerences between mites and Pseudoscorpiones on theone hand, and all other Chelicerata on the other, or thediVerences between Mesostigmata and all other chelicer-ates, might be attributed to shorter life spans, resultingin more germ-line divisions per absolute unit time( D generation time eVect (Laird et al., 1969)). Generationtimes in Mesostigmata are often measured in weeks ormonths (Murrell et al., 2005; Walter and Proctor, 1999)which makes them are generally shorter than those ofIxodida (one to several years) and many non-mite Chelic-erata (there are no data on longevity for Opilioacarida orHolothyrida). However, this picture is certainly not clear.It does not explain why rates in sister taxa such as Cariospuertoricencis and Ornithodoros moubata, or Asternola-elaps sp. and Uropodella laciniata are so strikingly diVer-ent (Fig. 4). Murrell et al. (2005) noted a range of otherproposed causes, including metabolic rate, body size,cladogenesis, altitude, DNA replication and repaireYciency and type of polymerase (Martin and Palumbi,1993; Mindell and Thacker, 1996). We are somewhatskeptical of the suggestion by Murrell et al. (2005) thatdiVerences in metabolic rate might explain the observedpattern. The number of cell divisions in the germ-line ofarthropods in each generation is relatively constant, irre-spective of metabolic level. It is quite possible that highermetabolic rates will result in more cell division in mito-chondria or in somatic cell lines (thus increasing possibil-ities for mutations), but it is unclear how this reasoningwould aVect germ-line nuclei, when germ cells are largely


in meiotic arrest for much of an organisms life time.Clearly this issue needs to be revisited with additionaldata on generation times, germ-line development, dataon DNA replication and repair eYciency, etc.

The rate comparisons did reveal an odd pattern relativeto Dermanyssina s.l. Dermanyssina s.l. is by far the mostspecies rich extant lineage of Parasitiformes (8,600 speciescurrently described, total estimated between 80,000 and160,000), and may represent 2/3 to 3/4 of all species leveldiversity in Parasitiformes (Walter and Proctor, 1999). Ithas undergone signiWcant evolution in its rRNA genes, as isshown by the high average divergence levels relative to thepycnogonid outgroups (Table 2). The lineage is alsoextremely well supported with numerous apomorphies sup-porting both the lineage of Arctacarina + Dermanyssina s.l.,and Dermanyssina s.l. itself (Figs. 3, 4). Yet Dermanyssinais the only major group of Mesostigmata that shows mini-mal within-group sequence diVerentiation (Fig. 4). Thisoverall pattern suggests either highly variable rates of evo-lution within a single lineage (Wrst very high, than very low)or (perhaps more realistic) extensive extinction along thelineages leading to Arctacarina + Dermanyssina s.l. and toDermanyssina s.l. itself, combined with a relatively recentorigin of Dermanyssina s.l. The notion of a recent origin ofDermanyssina s.l. is not an obvious one given available dis-tribution data. Epicriina, Zerconina, and Arctacarina occurexclusively or largely in the Northern Hemisphere, consis-tent with (though certainly not proof of) a relatively recent(Laurasian) origin, but most superfamilies in Dermanyssina(Rhodacaroidea, Ascoidea, Phytoseioidea, Eviphidoidea,Dermanyssoidea) are cosmopolitan, with Rhodacaroideareaching its peak diversity in the Southern Hemisphere.Only the basal superfamilies in the lineage, Veigaioidea andParasitoidea, might be inferred to have a less than cosmo-politan distribution (skewed towards the Northern hemi-sphere in both cases). Clearly, if this group is relativelyrecent, it has been extraordinary successful in dispersing.

Independent data on the age of the group is diYcult toobtain. The oldest fossils of Parasitiformes are ticks fromthe mid Cretaceous (Klompen and Grimaldi, 2001;Poinar and Brown, 2003), and the oldest fossils of Der-manyssina are from the Eocene (Witalinski, 2000). Whileconsistent with a hypothesis of a relatively recent originof Dermanyssina s.l., this observation may be artefactualgiven that the fossilization rate of parasitiform mites ingeneral appears to be quite low. A recent origin of thelineage would therefore leave us with an(other) exampleof a group where the most recent of the major lineages isby far the most successful in terms of species richness, aswell as ecological and geographic range.

Acknowledgments

We thank Gerald W. Krantz, Beverly S. Gerdeman,David E. Walter, Barry OConnor, Steve Goodman, JohnMoser, and Rodger Mitchell for providing specimens. Theresearch was supported by NSF DEB-0108359 (HK).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ympev.2006.10.024.

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Phylogeny of parasitiform mites (Acari) based on rRNA

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