Molecular Phylogenetics and Evolution Vol. 11, No. 2, March, pp. 296–307, 1999 Article ID mpev.1998.0583, available online at http://www.idealibrary.com on Phylogeny of the Arachnid Order Opiliones (Arthropoda) Inferred from a Combined Approach of Complete 18S and Partial 28S Ribosomal DNA Sequences and Morphology Gonzalo Giribet,*,1 Maria Rambla,* Salvador Carranza,† Jaume Bagun˜a`,† Marta Riutort,† and Carles Ribera*,2 *Departament de Biologia Animal and †Departament de Gene` tica, Universitat de Barcelona, Avinguda Diagonal 645, 08071 Barcelona, Spain Received February 18, 1998; revised July 18, 1998 times rather deep in the soil. About 90 species are The phylogenetic relationships among the main evo- strictly cavernicolous (Rambla and Juberthie, 1994). lutionary lines of the arachnid order Opiliones were Three suborders of Opiliones are widely accepted: investigated by means of molecular (complete 18S Cyphophthalmi, Laniatores, and Palpatores. Approxi- rDNA and the D3 region of the 28S rDNA genes) and mately 5000 species of Opiliones have been described. morphological data sets. Equally and differentially Cyphophthalmi (with about 100 species) have very weighted parsimony analyses of independent and com- discontinuous distributions, occurring mainly in tropi- bined data sets provide evidence for the monophyly of cal, subtropical, and temperate regions (Juberthie, the Opiliones. In all the analyses, the internal relation- 1988). Laniatores is the dominant group of Opiliones in ships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpa- southern latitudes, found mainly in America, tropical tores, Dyspnoi Palpatores, and Laniatores. The Cy- Asia, southern Africa, and Australia. Both Cyphoph- phophthalmi are monophyletic and sister to a clade thalmi and Laniatores are poorly represented in Eu- that includes all the remaining opilionid taxa rope. Palpatores have fewer species than Laniatores; Phalangida). Within the Phalangida the most sup- however, they are distributed worldwide and include؍) ported hypothesis suggests that Palpatores are para- the most commonly known Opiliones in the northern phyletic, as follows: (Eupnoi (Dyspnoi ؉ Laniatores)), hemisphere. Fossil species from the Carboniferous are but the alternative hypothesis (Laniatores (Eup- known and several genera are present in the Baltic .noi ؉ Dyspnoi)) is more parsimonious in some molecu- amber fossil beds, as well as in other fossil beds lar data analyses. Relationships within the four main clades are also addressed. Evolution of some morpho- Background in Opilionid Systematics logical characters is discussed, and plesiomorphic states of these characters are evaluated using molecu- The first studies on the systematics of the Opiliones lar data outgroup polarization. Finally, Martens’ hy- were developed in the last century. Sørensen (1873) pothesis of opilionid evolution is assessed in relation divided the order Opiliones into two families named to our results. ௠ 1999 Academic Press Gonyleptidae and Opilionidae. Thorell (1876) changed these families into taxonomic suborders, naming them SO. Laniatores and SO. Palpatores. Simon (1879) re- INTRODUCTION named these two suborders Mecostethi (ϭLaniatores) and Plagiostethi (ϭPalpatores) and described the new The order Opiliones, commonly known as harvest- suborder Cyphophthalmi. Later, Hansen and Sørensen men, is a cosmopolitan group of arachnids. Opiliones (1904) divided Palpatores into two tribes: Dyspnoi and are often found in disturbed habitats as well as in Eupnoi. This grouping of Opiliones into three subor- forests, under stones, in caves, on the trunks of trees, ders and two tribes of Palpatores was perpetuated by on the soil, in forest litter, in soil crevices, and some- Roewer (1923; and successive supplements) in his monumental Compendium of the World Opilionid Fauna. 1 Present address: Department of Invertebrates, American Mu- Subsequently, Silhavy´ (1961) raised the Dyspnoi and seum of Natural History, Central Park West at 79th Street, New York, NY 10024. E-mail: [email protected]. Eupnoi to subordinal rank and also split Laniatores 2 To whom correspondance should be addressed. E-mail: into two more suborders, Oncopodomorphi and Gonyl- [email protected]. eptomorphi, leading to the division of the order Opil- 296 1055-7903/99 $30.00 Copyright ௠ 1999 by Academic Press All rights of reproduction in any form reserved. TOTAL EVIDENCE PHYLOGENY OF THE OPILIONES 297 iones into five suborders. This proposal has not been the Ischyropsalidoidea). One year later, Martens (1976) widely followed. presented an opilionid cladogram, in which the tribe The only author who considered the Opiliones para- ‘‘Dyspnoi,’’ the most problematic group of Palpatores, phyletic was Savory (1977), who divided them into two was divided into two taxa at the superfamily level: the different orders; the Cyphophthalmi and the Troguloidea and the Ischyropsalidoidea, which to- Phalangida, the latter including Laniatores and Palpa- gether were not considered a monophyletic clade. Thus, tores. This proposal by Savory was rebutted by Shear the former Palpatores became a paraphyletic clade, (1980), who did not agree with the cyphophthalmid whose members were arranged in four superfamilies: ‘‘uniqueness’’ listed by Savory. Caddoidea, Phalangioidea, Troguloidea, and Ischyrop- Despite the existence of these alternative phyloge- salidoidea. Further studies along these lines have been netic hypotheses, the relationships among the main conducted by several authors (Gruber, 1978; Martens, opilionid groups have received relatively little examina- 1980, 1986; Martens et al., 1981; Shear and Gruber, 1983). tion using modern systematic methods until Martens As a summary of the systematics of the Opiliones, the and co-workers presented their analyses derived from existence of three principal groups (Cyphophthalmi, Lania- genitalic characters (Martens, 1976, 1980, 1986; Mar- tores, and Palpatores) has been commonly accepted, except tens et al., 1981), basically from the anatomy of the by Martens and co-workers. The evident difficulties emerge ovipositor. These characters led Martens to consider when establishing their phylogenetic relationships. Palpatores a paraphyletic group, the representatives of The present work is a phylogenetic approach to the which had successively split at different times from the arachnid order Opiliones, based on cladistic analyses of precursors of the modern Cyphophthalmi. Conse- molecular and morphological data. The molecular data quently, he proposed to unite the Cyphophthalmi and matrix has complete 18S rDNA sequences and partial the resulting paraphyletic Palpatores in a new clade 28S rDNA sequences (D3 region) from 15 species of named ‘‘Cyphopalpatores,’’ based on their long, seg- Opiliones, representing the three suborders: 3 species mented ovipositor with a bifurcate tip (Fig. 1). of Cyphophthalmi; 6 species of Laniatores representing Shear (1980) discussed the presence of a second the three main lines, Gonyleptoidea, Oncopodoidea, important character shared by Cyphophthalmi and and Travunioidea; and 6 species of Palpatores represent- Palpatores, the number of midgut diverticula (Dumi- ing the Dyspnoi (Ischyropsalidoidea and Troguloidea) trescu, 1975a,b). The classification system of Martens and the Eupnoi (Phalangioidea and Caddoidea) (see was also adopted by some modern authors working on Table 1). Sequences from other arachnids were used to Opiliones taxonomy (Crawford, 1992; Cokendolpher test the monophyly of the Opiliones, and two Merosto- and Lee, 1993). mata (Limulus polyphemus and Carcinoscorpius rotun- Within the suborder Palpatores, Shear (1975) pro- dicaudatus) were used as outgroup for all the Arach- posed the use of superfamily names to replace the old nids. A data matrix of 45 morphological and ecological names ‘‘Eupnoi’’ and ‘‘Dyspnoi,’’ and so Eupnoi was split characters was developed for the 15 opilionid species. into Caddoidea and Phalangioidea. The Dyspnoi was The aim of this work was to test higher categories not rearranged, although it was renamed Troguloidea used in opilionid systematics and to solve the internal (including both the currently accepted Troguloidea and relationships of the order, testing the classical morpho- logical classifications with the topologies obtained from molecular data. Furthermore, the cladistic analysis based on total evidence (combined morphological ϩ molecular data) was used to provide answers about the plesiomorphic/ synapomorphic state of some of the morphological charac- ters used by different authors, with the aim of finding the most coherent solution for these characters according to present-day knowledge of the Opiliones. MATERIALS AND METHODS Biological Samples Fifteen opilionid species were collected and frozen at Ϫ80°C or preserved in absolute ethanol for the molecu- lar study. The material used for the morphological study was preserved in 70% ethanol. An effort was made in the taxonomic sampling to analyze the maxi- FIG. 1. Cladogram proposed by Martens (1976, 1980, 1986) and mum morphological diversity inside each clade, which Martens et al. (1981) for the phylogenetic relationships within the is also assumed to reflect the maximum genetic diver- order Opiliones. sity. A checklist of the sampled species and their 298 GIRIBET ET AL. TABLE 1 Sample Preparation for the Molecular Study Taxa Used in this Study with the Accession Codes Genomic DNA samples were obtained from fresh, from GenBank frozen, or ethanol-preserved