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Cladistics

Cladistics 24 (2008) 708–722 10.1111/j.1096-0031.2008.00202.x

Family ties: molecular phylogeny of (Araneae: )

Suresh P. Benjamina,b* , Dimitar Dimitrova, Rosemary G. Gillespieb and Gustavo Hormigaa aThe George Washington University, Department of Biological Sciences, 2023 G Street NW, Washington DC, 20052, USA; bUniversity of California, Berkeley, Biology Division—ESPM, 201 Wellman Hall 3112, Berkeley, CA 94720-3112, USA Accepted 2 November 2007

Abstract

The first quantitative phylogenetic analysis of three sequenced genes (16S rRNA, cytochrome c oxidase subunit I, histone 3) of 25 genera of crab spiders and 11 outgroups supports the monophyly of Thomisidae. Four lineages within Thomisidae are recovered. They are informally named here as the Borboropactus , clade, clade and the clade, pending detailed morphology based cladistic work. The Thomisus clade is recovered as a strongly supported monophyletic group with a minimal genetic divergence. previously widely considered a subfamily of Thomisidae do not group within thomisids and is excluded from Thomisidae. However, Aphantochilinae previously generally considered as a separate family falls within the Thomisus clade and is included in Thomisidae. The recently proposed new family Borboropactidae is rejected, as it is paraphyletic. The Willi Hennig Society 2008.

Thomisidae Sundevall, 1833, commonly called crab Gabritschevsky, 1927; Comstock, 1948). vatia spiders are often cryptically colored sit-and-wait preda- (Clerck, 1757) has a remarkable ability to change color, tors that do not build capture webs (Fig. 1). Thomisidae which takes place during migration to flowers of is the sixth largest family. It includes 2062 different color from spring to the early part of summer described in 171 genera (Platnick, 2007) with (Comstock, 1948). Crab spiders are attracted by fra- many more species remaining to be described. They are grance components of flowers (Aldrich and Barros, mainly active during the day and ambush with 1995; Krell and Kraemer, 1998) and use visual and their well-adapted first and second legs (Homann, 1934; tactile cues for selecting flowers (Morse, 1988; Greco Comstock, 1948). Not surprisingly, they are an impor- and Kevan, 1994). They reach their ambush sites in a tant component of terrestrial ecosystems (Riechert, step-by-step process using several draglines and bal- 1974). As predators of agricultural pests, thomisids play looning events (Homann, 1934). an important part in natural pest control (Riechert and There are social crab spiders with maternal care in the Lockley, 1984; Nyffeler and Benz, 1987; Uetz et al., Eucalyptus forest of (Main, 1988; Evans, 1999). 1995). Mother ergandros Evans (1995), catch Some thomisids (e.g., Misumena, Diaea, and larger prey for their own offspring, but not for the Thomisus) possess the ability to change color and blend adopted offspring leading to large size and possibly into their habitat, in most cases flowers (Packard, 1905; better survival rates for the natural offspring (Evans, 1998). Mothers further increase survival of natural offspring by producing trophic oocytes used in a system *Corresponding author: of sacrificial care (Evans, 1998). E-mail address: [email protected] Present address: Department of Entomology, National Museum of Myrmecomorphism is known in a number of thomis- Natural History NHB 105, PO Box 37012, Smithsonian Institution, ids: Strigoplus albostriatus Simon, 1885, Washington, DC 20013-7012, USA. forticeps (O.P.-Cambridge, 1873), A. lineatipes

The Willi Hennig Society 2008 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 709

A B

CD

Fig. 1. Living thomisids sampled in this study. (A) ‘‘’’ sp. A, , Central Province; (B) Diaea placata, Sri Lanka, Western Province (not sampled); (C) taprobane, Sri Lanka, Central Province; (D) ‘‘’’ sp. B, Sri Lanka, Central Province. All photos by SPB.

O.P.-Cambridge, 1901 and Aphantochilus rogersi that did not fit into the above categories were included O.P.-Cambridge, 1870, are known to be mimics. in Misumeninae and Philodrominae. Species groups A. forticeps is of the same color as the ant Oecophylla were then formed within these subfamilies based on eye smaragdina and bears on the posterior part of the pattern and shape of prosoma. abdomen a pair of eye-like spots that correspond to eyes Since Simon (1892) the understanding of generic of the ant (Shelford, 1902). The similarity of A. rogersii relationships has not changed greatly. Holm (1940), on to some spiny South American is striking, and the grounds of embryological studies, and Homann forms a further instance of myrmecomorphism (Oliveira (1975), on the grounds of eye morphology, excluded and Sazima, 1984). Philodromids from Thomisidae. Philodromids were Given their ecological significance and appealing later given family status (Ono, 1988), which has been adaptations one would expect to see a plethora of accepted since (but see Roberts, 1995). Separately, phylogenetic studies. However, no such studies of family status was proposed for some thomisids, Steph- thomisids exist. Moreover, understanding the exact anopidae (Pickard-Cambridge, 1871) and Aphantochi- taxonomic limits of this large family has always been lidae (Thorell, 1873). Levi (1982) placed the Thomisidae problematic. Thomisidae was proposed to accommo- in a superfamily along with Aphantochilidae, which was date spiders with legs generally extended sideways not accepted by Ono (1988). In the most current study (laterigrade), instead of being oriented towards the front Thomisidae was separated into seven subfamilies (Ono, or back as in most other spiders. Originally all spiders 1988): Stephanopinae, Thomisinae, Bominae, Stiphro- with laterigrade legs such as Sparassidae and Philodr- podinae, Dietinae, Strophiinae and Aphantochilinae omidae were included. Simon (1892) was the first to using characters proposed by Simon (1892). Recently propose a hypothesis of generic groups for all thomisid (Wunderlich, 2004b) proposed a new family ‘‘Borborop- genera recognized during his time. Although his actidae’’ to include parts of Thomisidae. Thus, to date, ‘‘groups’’ were neither evolutionary nor phylogenetic the monophyly of Thomisidae remains untested. in the modern sense, he provided some information on A great part of our knowledge of evolutionary history Thomisidae morphology and arguments supporting his is derived from phylogenies, reconstructed by sampling ideas. His Stephanopsinae contained spiders with che- and grouping characters. Harvey and Pagel (1991) liceral teeth; Aphantochilinae and Strophiinae con- illustrated the richness of evolutionary questions that tained species with modifications such as elongated can be approached with phylogenies. Our current maxillae related to their ant mimicking habits; Stiphro- understanding of relationships within Thomisidae is podinae included species with an enlarged tarsus; spiders largely based on Simon (1892) and modifications to his 710 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 ideas by different workers (Schick, 1965; Homann, 1975; (P. Lehtinen, pers. comm.). We have included six species Ono, 1988). However, they do not provide much of Salticidae, one species each of and information, partly because the family has never been in our study. As Philodromidae have been subject to quantitative phylogenetic analysis. Recent in the past included as a subfamily of Thomisidae, we taxonomic work on tropical Asian crab spiders (Tikader, include three philodromid taxa in our study. 1980; Barrion and Litsinger, 1995) added more confu- sion, illustrating the fragile systematic state of the DNA sequencing and alignment family. Here we present the first cladistic analysis of Thom- Genomic DNA was extracted from either fresh or isidae. This analysis of molecular data aims to test the ETOH-preserved leg tissue using the Qiagen DNeasy monophyly of Thomisidae, including the validity of Tissue Kits (Qiagen, Valencia, CA, USA). Otherwise ‘‘Borboropactidae’’ and the placement of some enig- intact spiders, preserved in alcohol have been deposited matic taxa such as Epidius and Cebrenninus, which share as voucher specimens (Table 1). Partial fragments of the characters such as the presence of a conductor and mitochondrial genes cytochrome c oxidase subunit I median apophysis with ‘‘Borboropactidae’’. Epidius was (COI) and 16S rRNA (16S) and the nuclear gene histone provisionally placed in Thomisidae owing to its unusual H3 (H3) were amplified using the following primer pairs: male palp (Benjamin, 2000). Owing to the considerable (COI) C1-J-1751 and C1-N-2191 (Simon et al., 1994) volatility in thomisid systematics and paucity of mor- (16S) LR-N-13398 (Simon et al., 1994) and LR-J-12864 phological information for a large number of taxa, we (Arnedo et al., 2004) and (H3) H3aF and H3aR (Colgan do not formally name any higher-level taxa here. et al., 1998). PCR products were purified using the However, we provide putative morphological synapo- QIAquick PCR Purification Kit (Qiagen) and sequenced morphies for most of the largest . The first author directly in both directions using an ABI 3730 automated is presently undertaking a detailed morphological revi- sequencer (Applied Biosystems, Foster City, CA, USA), sion of Thomisidae. in combination with the ABI PRISM Big Dye Termi- nator Cycle Sequencing Ready Reaction Kit. The chromatograms produced for each DNA sequence were Materials and methods edited in Sequencher 3.1 (Gene Codes, Ann Arbor, MI, USA). Ingroup taxa The protein-coding COI and H3 sequences were easily aligned unambiguously. For non-protein-coding se- We sampled 41 ingroup and 13 outgroup taxa for quences there are two basic approaches to multiple DNA sequencing (Table 1). Representatives of all major sequences alignment. The first are the static alignment thomisid subfamilies except for Stiphropodinae and methods with fixed homology statements, in which the Strophiinae are included. They are: Stephanopinae, alignment is independent of the phylogenetic analysis. Thomisinae, Bominae, Dietinae and Aphantochilinae. The second is the optimization alignment method where Additionally, several sequences of thomisids and out- alignment is an integral step of phylogenetic analysis groups available in GenBank were added to the anal- (Wheeler, 1996). We explored our data using both yses. Accession codes for all sequences are given in analytical philosophies. In all analyses gaps were treated Table 1. either as missing data (parsimony and Bayesian infer- ence) or as a fifth state (POY). Outgroup taxa Direct optimization Thomisids fall within the large clade (Coddington and Levi, 1991), which are characterized Direct optimization (Wheeler, 1996; Wheeler et al., by the loss of the unpaired tarsal claw. The monophyly 2006) was implemented with the software package POY of Dionycha has, however, been elusive. Furthermore, Version 3.0.11. Unlike analysis of statically aligned data, the phylogenetic structure within Dionycha has not been in POY alignments are tree dependent and as a result the fully explored [however, see Davila (2003) who included homology statements are dynamic. In all the analyses a small sample of Dionycha in her study of Ctenidae the protein-coding genes H3 and COI were treated as interrelationships]. Thus, the only character-based out- prealigned. This avoids the use of in ⁄del transformations group hypothesis for thomisids is by Loerbroks (1984); for these fragments and saves computing time without in a functional study of the male palp of M. vatia,he alteration of results (Giribet, 2001). To further reduce found characters to relate Salticidae to Thomisidae. computational time and to minimize the probability of Morphological comparisons of representatives of fam- unwanted artifacts in the alignment the 16S was ilies within Dionycha have suggested that Creugas divided into two homologous fragments (Giribet, (Corinnidae) might be the sister to thomisids 2001). Homolog partitions in 16S were defined based S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 711

Table 1 Specimens sequenced for molecular analyses. Fragments successfully sequenced and GenBank accession number are given

Family Species Country Locality COI 16S H3 Depository Philodromidea Pagiopalus nigriventris USA Maui, HI EU168155 EU168142 ⁄ EU157106 USNM EU168143 Proernus stigmaticus USA Hawaii, HI EU168156 EU168144 EU157107 USNM sp. USA Berkeley, CA EU168157 EU168145 EU157108 USNM Salticidae Onomastus sp. A Sri Lanka Central Province EU168158 EU168146 NA USNM Onomastus sp. B Sri Lanka Central Province EU168159 EU168148 EU157109 USNM labiata NA NA AY297361 AY296653 NA NA Lyssomanes virides NA NA AY297360 AY296652 NA NA Neon nelli NA NA AF327988 AF327959 NA NA Helvetia cf. zonata NA NA AY297394 AY296685 NA NA Corinnidae Castianeira sp. NA NA AY297419 AY296710 NA NA Miturgidae sp. NA NA AY297421 NA NA NA Thomisidae Borboropactus cinerascens Bukit Timah NR NA EU168138 EU157126 MHNG Borboropactus sp. South Limpopo EU168187 EU168151 NA MHNG Epidius parvati Sri Lanka Western Province EU168163 EU168141 EU157114 MHNG Cebrenninus rugosus Chumphon Province EU168175 EU168139 EU157134 MHNG Cebrenninus rugosus Kelantan EU168177 EU168140 NA MHNG ‘‘Epicadinus’’ sp. Misiones NA EU168153 NA MACN-Ar ‘‘Stephanopis’’ sp. A Region IX EU168167 EU168137 EU157117 CAS ‘‘Stephanopis’’ sp. B Australia Tasmania EU168185 NA EU157138 USNM ‘‘Stephanopis’’ sp. C Australia Tasmania NA NA EU157137 USNM ‘‘Stephanopis’’ sp. D Australia Western Australia NA NA EU157139 USNM Pseudoporrhopis granum Fianarantsoa EU168170 EU168131 EU157120 CAS Oxytate taprobane Sri Lanka Central Province EU168161 EU168133 EU157112 MHNG Amyciaea forticeps Thailand Montha Tarn NA NA EU157135 MHNG ‘‘Lysiteles’’ sp. A Sri Lanka Central Province EU168184 EU168128 EU157133 MHNG ‘‘Lysiteles’’ sp. B Sri Lanka Central Province EU168183 EU168129 NA MHNG versicolor USA VA NA NA EU157136 USNM californicus USA CA EU168181 EU168115 EU157131 USNM Xysticus sp. USA CA EU168182 EU168116 EU157132 USNM ‘‘Monaeses’’ sp. A Sri Lanka Central Province EU168172 EU168117 EU157122 MHNG ‘‘Monaeses’’ sp. B Australia Western Australia EU168186 EU168150 NA CAS angulatus USA CA EU168179 EU168120 EU157110 USNM USA CA EU168180 EU168121 EU157111 USNM sp. Kien Gian Province NA EU168135 EU157127 MHNG Runcinia albostriata Sri Lanka Central Province EU168178 EU168125 EU157130 MHNG Runcinia albostriata Bago Division EU168165 EU168124 EU157116 CAS Runcinia acuminata Myanmar Bago Division EU168166 EU168126 NA CAS Diaea subdola Sri Lanka Central Province EU168174 EU168118 EU157124 MHNG Aphantochilus sp. Argentina Misiones NA EU168152 EU157140 MACN-Ar Diaea nr subdola Myanmar Yangon Division EU168169 EU168127 EU157119 CAS Cyriogonus sp. Madagascar Antananarivo EU168168 EU168130 EU157118 CAS Thomisus granulifrons Sri Lanka Western Province EU168162 EU168122 EU157113 MHNG Thomisus sp. A Sri Lanka Sabaragamuwa Province EU168164 NA EU157115 MHNG Thomisus sp. B Thailand Phuket Province EU168176 EU168123 EU157129 MHNG piger Sri Lanka Western Province EU168171 EU168134 EU157121 MHNG Haplotamarus sp. Sri Lanka Western Province EU168173 EU168132 EU157123 MHNG sp. Sri Lanka Central Province NA EU168119 EU157125 MHNG Boliscus sp. Malaysia Kelantan NA EU168136 EU157128 MHNG a semispinos NA NA DQ174382 DQ174339 NA NA Mecaphesa naevigera NA NA DQ174344 DQ174387 NA NA Xysticus sp. B NA NA AY297423 AY296714 NA NA on a preliminary static multiple alignment with ence between the 16S and the COI + H3 partitions ClustalW (Thompson et al., 1994) using the default was used (Wheeler and Hayashi, 1998). Congruence parameters. was measured using the incongruence length difference Sensitivity of the results to various parameters was (ILD) test (Mickevich and Farris, 1981). Tables 2 and explored using five different values of gap opening 3 contain the information about the parameter com- costs. As an objective criterion to select among binations investigated together with statistics from the different parameter combinations, maximum congru- analyses. All POY searches were run on a 712 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722

Table 2 matrices (elision matrix; Wheeler et al., 1995; Hedin and Statistics resulting from the exploration of the effect of a range of gap Maddison, 2001). In principle, an elision matrix should opening ⁄ gap extension cost on the data set used to generate the tree generally recover relationships that are robust to align- shown in Fig. 1 ment differences. This was done as suggested by Hedin Gap 16S COI + H3 Combined and Maddison (2001). cost length length length ILD Topological congruence between the results from the 1 1362 1834 3279 0.025312595 analysis of each particular cost scheme and the elision 2 1489 1834 3414 0.02665495 matrix was measured by the number of nodes in 3 1569 1834 3501 0.027992002 common between the consensus tree, which they pro- 4 1642 1834 3578 0.028507546 5 1709 1834 3649 0.029049055 duced and the average symmetric-difference distances between all trees from the individual and the elision matrices (Hedin and Maddison, 2001; Swofford, 2002). Based on these criteria the alignment built with gap Table 3 Statistics resulting from the exploration of the effect of a range of gap opening ⁄gap extension cost of 20 ⁄2 was selected and opening ⁄ gap extension cost on the data set used to generate the tree used in all further analyses. shown in Fig. 2 Phylogenetic analysis of static alignments Gap 16S COI + H3 Combined cost length length length ILD Both parsimony and likelihood methods were used as 1 1423 1772 1774 0.053893989 optimality criteria for the phylogenetic analyses. Bayes- 2 1539 1772 1798 0.019543974 3 1591 1772 1807 0.027191206 ian inference was preferred over maximum likelihood 4 1631 1772 1809 0.035430839 because performing Bayesian analysis is computationally 5 1671 1772 1828 0.041481069 less demanding than traditional likelihood methods. Manipulations of data matrices and trees were performed with MacClade (Maddison and Maddison, 2001), computer cluster at The George Washington PAUP* (Swofford, 2002) and WinClada (Nixon, 2002). University. Initial trees were built using an approximation short- Parsimony cut (command –approxbuild). Thirty replicates of random addition of sequences were performed (com- Parsimony searches were conducted using TNT versus mand –replicates 30) holding up to 300 trees 1.0 (Goloboff et al, 2003a). All multistate characters (command –holdmaxtrees 300). Tree fusing and were treated as non-additive (Fitch, 1971). In all tree drifting (TBR) strategies were used for tree search- parsimony analyses, heuristic searches under the ‘‘tra- ing (commands –tbr –treefuse –drifttbr). In ditional search’’ option were performed using 1000 order to increase the search efficiency and guarantee that replicates, holding 10 trees per replicate to a maximum POY will save only disparate trees the command of 10 000 trees. Clade support was assessed by means of -fitchtrees was used. To reduce error from heuristic non-parametric bootstrap analysis (Felsenstein, 1985) as operations suboptimal trees with 1% length distance implemented in TNT with 1000 pseudoreplicates of from the shortest trees were submitted to a further heuristic searches with 100 interactions of random round of TBR (command –checkslop 10). Indices addition of taxa and holding 10 trees per interaction. and topology specific implied alignments were saved The same parameters were used to perform a jackknife using the commands –indices and –implied analysis (Farris et al., 1996). Additionally, Poisson alignment. weighting bootstrap and symmetric re-sampling support values (Goloboff et al., 2003b) were calculated using the Static alignments same search parameters. These methods try to avoid errors where high support values are found for groups Static alignments were built using ClustalW (Thomp- not supported by the original data (Goloboff et al., son et al., 1994) with the following gap opening ⁄gap 2003b). While bootstrap and Poisson weighting boot- extension costs: 8 ⁄2, 8 ⁄4, 15 ⁄6.66 (default), 20 ⁄2, 24 ⁄4 strap re-sample characters by building a matrix of the and 24 ⁄6 and transition weight fixed to 0.5. Owing to the same size as the original, jackknife and symmetric re- lack of an a priori basis for deciding on one alignment sampling removes part of the characters, constructing a over the other and to avoid having to use all six smaller matrix where character replacement does not alignments, we adopted phylogenetic congruence to occur (Goloboff et al., 2003b). In both cases the select an ‘‘optimal’’ alignment. Phylogenetic congruence analyses were performed using the default setting in works by comparing phylogenetic results from each TNT (probability of character removal in the pseudore- alignment matrix with results of a concatenation of all plicates was set to 0.36). S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 713

In addition to the parsimony analysis with equal Direct optimization character weights, analyses using implied weighting were conducted in TNT. Implied weighting was used to build The results of the analysis with the combination of trees using differential character weighting (Goloboff, gap opening ⁄gap extension cost of 2 ⁄1 produced implied 1993a). It is superior to the successive weighting as it alignments with maximum congruence among partitions provides an optimality criterion (maximum fit) to build (Tables 2 and 3). One of the two most parsimonious trees and weight characters (Goloboff, 1993b). This trees (L ¼ 3377) shown in Fig. 2, recovers a monophy- helps to avoid iterative searches from preliminary trees, letic Thomisidae. The strict consensus of the two most which were one of the most criticized aspects of the parsimonious trees is given in Fig. 3. Thomisids are successive weighting method [Swofford and Olsen rendered possibly paraphyletic in the strict consensus, as (1991), but see Steel (1994) and Farris (2001)]. Fit is it unites Cesonia sp. (Gnaphosidae) and Hibana sp. calculated as a function in which concavity depends on () in a basal tricotomy with ingroup taxa the constant K. With the increase of K the penalization (Fig. 3). However, this arrangement of Cesonia sp. and against homoplasy decreases (Goloboff, 1993b). Implied Hibana sp. within thomisids is not supported and is very weighting analyses, for K-values from 1 to 100 were sensitive to taxon sampling. Adding sequence fragments done in TNT for 500 replicates. of four taxa (one , two Mecaphesa and one Xysticus species; see Table 1) and ⁄or excluding the Bayesian inference (likelihood approach) outgroup taxon Portia labiata (Thorell, 1887) recovers a monophyletic Thomisidae, which excludes Cesonia MrBayes 3.4 (Ronquist and Huelsenbeck, 2003) was and Hibana (Fig. 4). The analysis of this modified used to estimate topologies and to calculate posterior matrix found six most parsimonious trees (L ¼ 3279). probabilities of inferred clades. The best-fit model of The combination of gap opening ⁄gap extension cost of sequence evolution was selected using Modeltest 3.6 1 ⁄1 resulted in higher overall congruence for this matrix. using the Akaike information criterion (Posada and The strict consensus of this analysis is shown in Fig. 4. Crandall, 1998). Four replicate analyses were run In all analyses under dynamic optimization Anyphaeni- simultaneously from random starting trees using default dae (represented by Hibana) plus Gnaphosidae (repre- priors. Metropolis-coupled MCMC (one cold and three sented by Cesonia) is recovered as the closest relative of heated) were run for 10 million generations. Topologies Thomisidae. were sampled at intervals of 1000 generations within each chain. Average likelihood scores (–ln) were exam- Parsimony analyses with static homology ined to assess convergence. Sampled trees were plotted against generations in order to determine point of Analyses of the static alignment under equal weights stability. Trees under the stability value were discarded found a single most parsimonious tree (L ¼ 3234, CI ¼ as ‘‘burn-in’’. There is some disagreement on the 0.32, RI ¼ 0.477) shown in Fig. 5. Thomisids form a reliability of the posterior probability, which has been monophyletic family. The main lineages within Thom- criticized by Suzuki et al. (2002) for overestimating isidae and their relationships are the same as in the trees statistical confidence when concatenated gene sequences resulting from dynamic optimization. However, only the are used. However, others have countered: Wilcox et al. Epidius clade and to a lesser extent the Thomisus clade (2002) based on simulations have shown that Bayesian are well supported. Hibana sp., but not Cesonia sp. support values represent much better estimates of appears as closest relative to Thomisidae. phylogenetic accuracy than do non-parametric boot- Parsimony analyses under the criterion of implied strap support values. However, when model misspecifi- weights constantly recover the monophyly of Thomis- cations are present (which is absent in simulation-based idae. Relationships within Thomisidae are as in uweight- studies) inflation of posterior probabilities may occur. ed parsimony and dynamic optimization analyses supporting the existence of the same four main lineages. The only difference refers to the composition of the Results Stephanopis clade, which was always recovered as monophyletic, but for the majority of the concavities it The lengths of the sequenced fragments excluding the also included Epicadinus sp. (Fig. 6). primers were as follows: 16S, 430 bp; H3, 328 bp; and COI, 557 bp. All sequences have been deposited in Bayesian inference GenBank and their acquisition numbers are listed in Table 1. The ILD test (Farris et al., 1994), done in The best fitting model for the fragment of 16S was NONA, did not find significant incongruence between GTR + I + C; for H3 it was HKY + I + C and for the three gene fragments. Thus, all three gene fragments COI it was GTR + I + C. The results of the first were combined into a single matrix. 150 000 generations were discarded as burn-in. The 714 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722

Fig. 2. One of the two most parsimonious trees (L ¼ 3377) found under direct optimization. recovers a monophyletic Thomisidae. Gap opening ⁄ gap extension cost of 2 ⁄ 1. Jackknife values greater than 60 are shown above the branches. resulting tree is shown in Fig. 7. Results practically Discussion mirror those from the implied weight parsimony anal- yses and are highly congruent with the tree topologies Monophyly of Thomisidae found by the unweighted parsimony and direct optimi- zation analyses. Thomisidae is again monophyletic and The of Thomisidae is challenging. Almost includes the same four main lineages as in all previous all genera await revision. Despite past attempts (Simon, analyses. The Borboropactus clade is sister to all other 1892; Petrunkevitch, 1928; Roewer, 1954; Ono, 1988; thomisids. Support for thomisid monophyly is higher Lehtinen, 2005), the monophyly of Thomisidae was than in other analyses. Most of the main groups within never established. Our study, based on molecular data Thomisidae and their relationships are also well sup- provides strong support for the monophyly of Thomis- ported. Further, the Bayesian inference results suggest idae for the first time. Monophyly of Thomisidae is that Anyphaenidae (represented by Hibana sp.) is the supported by three molecular synapomorphies in this sister lineage of the family Thomisidae. study, all from the 16S gene fragment. They are three S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 715

Fig. 3. The strict consensus of the two most parsimonious trees found under direct optimization. Gap opening ⁄ gap extension cost of 2 ⁄ 1. Jackknife values greater than 60 are shown above the branches. changes to T optimized as synapomorphies at the and second, Philodromidae was considered a subfamily aligned positions 181, 183, 230, based on the preferred of Thomisidae (Simon, 1892). Philodromids are still topology from POY (Fig. 2). considered by some contemporary arachnologist as Of the 11 outgroup taxa of the families Philodromi- derived thomisids (Tikader, 1980; Roberts, 1995). In dae, Salticidae, Miturgidae, Corinnidae, Gnaphosidae this study they do not group within Thomisidae, and and Anyphaenidae included in our study, Hibana sp. may fall at the root of Dionycha. In contrast, Cheir- (Anyphaenidae) or Hibana sp. plus Cesonia sp. (Gnaph- acanthium sp. is placed closer to the root of Thomisidae. osidae) were placed as sister to Thomisidae. This was Cheiracanthium was previously a member of Clubioni- unexpected for two reasons: first, Salticidae was previ- dae, a family also proposed as sister to Thomisidae ously considered sister to Thomisidae (Loerbroks, 1984) (Ono, 1988). 716 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722

Fig. 4. The strict consensus with gap opening ⁄ gap extension cost of 1 ⁄ 1 under direct optimization: with the addition of the ingroups Misumenoides sp., two Mecaphsesa and one Xysticus species and excluding the outgroup Portia labiata. See text for details. Jackknife values greater than 60 are shown above the branches.

All phylogenetic analyses independent of gap open- families: Stephanopinae, Thomisinae, Bominae, ing ⁄gap extension cost or taxon sampling, recover four Stiphropodinae, Dietinae, Strophiinae and Aphantoc- well-supported lineages within Thomisidae. They are hilinae (Ono, 1988). The current study shows these informally named here as the Borboropactus clade, groupings to be paraphyletic and should thus be Epidius clade, Stephanopis clade and the Thomisus treated with caution. clade, pending detailed morphology-based cladistic The following morphological synapomorphies are work. The Borboropactus clade is sister to all remaining proposed to define Thomisidae: thomisids. All ‘‘derived’’ thomisids are grouped in 1 Legs 1 and 2 longer and stronger then legs 3 and 4 the Thomisus clade. Epidius and Stephanopis clades (Ono, 1988; Wunderlich, 2004b; Jocque´and Dippenaar- are more closely related to each other than to the Schoeman, 2006). However, this might not be obvious previous two groups. None of these clades recovered for some African genera such as Thomisops Karsch, in our study correspond strictly to any currently 1879 (Dippenaar-Schoeman, 1989). accepted subfamily groupings. In the most current 2 Lateral eyes on tubercles, larger and much more study Thomisidae was separated into seven sub- developed than the median eyes (Ono, 1988). S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 717

Fig. 5. The single most parsimonious tree (L ¼ 3234, CI ¼ 0.32, RI ¼ 0.477): static alignment, analyzed under unweighted parsimony. Support values are show as follows: above branches Bootstrap ⁄ Poisson Bootstrap; below branches Jackknife ⁄ Symmetric resampling. Support values bellow 50 are omitted.

3 Presence of a group of setae instead of a colulus the Malagasy endemic Geraesta Simon, 1889 should be (Homann, 1975). included in this clade based on the three characters given below (Benjamin unpublished data). Borboropactus clade The following morphological synapomorphies might define the Borboropactus clade: (1) flexibly attached cup- Borboropactus Simon, 1884 is one of the few thomisid shaped median apophysis (Fig. 8A,B); (2) hyaline con- genera represented in amber. It was dubbed a ‘‘relict’’ ductor (Fig. 8A–C); and (3) epigynal teeth (Fig. 8D–E). and elevated to family rank (Wunderlich, 2004a,b). This Wunderlich (2004b, figs 4–8) mentions a specialized study shows that there is no phylogenetic justification gland or ‘‘tarsal pit organ’’ of tarsi of legs 1–4 as for such a family (unless of course Thomisidae is split to synapmorphic for ‘‘Borboropactidae’’. However, this four families). Borboropactidae would be paraphyletic sensory organ appears to be autapomorphic for as it includes the Epidius clade. As Wunderlich (2004b) Borboropactus, as it is absent in Geraesta (Benjamin himself mentions Borboropactus shares characters such unpublished data). Furthermore, his ‘‘Borboropactidae’’ as the presence of a conductor and median apophysis should include the genera Cebreninnus, Cupa, Epidius with other thomisids such as Epidius and Cebrenninus and Stephanopis, which then is essentially Simon’s (Benjamin, 2000; Benjamin unpublished data). Further, ‘‘Stephanopinae’’. Homann (1934) suggested that 718 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722

Fig. 6. Majority rule consensus tree from the trees found by the implied weight analyses with K ranging from 4 to 100. Results with K-values below 4 were excluded from the consensus as suggested by Goloboff (1993b, 1995). thomisids should be placed in as they have a course through the tegulum (Schick, 1965; Ono, 1988; grate-shaped tapetum. Interestingly, Borboropactus has Benjamin, 2000); (5) conductor absent (Benjamin, 2000; epigynal teeth as do some Lycosoidea (Fig. 7D,E; Schick, 1965); and (6) median apophysis absent. Griswold, 1993). However, Borboropactus has a canoe- Within the Thomisus clade, although there is some shaped tapetum and not a grate-shaped tapetum phylogenetic structure, none of the groupings are well (Benjamin unpublished data). This apparent character supported. This might be due to either an inadequate conflict needs to be addressed with a broader taxon taxon sample, a suboptimal character sample (shorter or sample in future studies. less informative gene fragments) or both. On the other hand it might well be due to paraphyletic genera. This is Monophyly of the Epidius and Stephanopis clades most likely the case. The apparent non-monophyly of several thomisid genera is not surprising, and was No morphological characters supporting these two suspected earlier (Benjamin, 2001, 2002; Lehtinen, clades are known. This is because Stephanopis and 2005). The study of generic relationships within the related genera are poorly studied. It is clear that Thomisus clade will require additional data. Stephanopis is paraphyletic and badly in need of revision. Preliminary data indicate that Cebrenninus might be monophyletic. Epidius is the only genus that is Future studies well defined, due to its elongated tibia (Benjamin, 2000). Although our hypothesis of thomisid relationships Monophyly of the Thomisus clade forms the basis for future phylogenetic studies, it also emphasizes that little progress has been made in Our study revealed the Thomisus clade, morpholog- understanding the phylogeny of this key dionychan ically the most homogeneous, to be monophyletic family since Simon’s seminal work almost a century ago. (Figs 1–5). It is defined by several morphological The confusion in thomisid systematics is probably due synapomorphies: (1) scopula hairs circular in cross- to the emphasis placed on single character systems, such section (Homann, 1975); (2) bulbus subequal in length as male genital morphology, relative size of eyes or the and width (Benjamin, 2000); (3) tegulum disc shaped presence of cheliceral teeth. Thomisid genitalia are (Ono, 1988); (4) sperm duct follows a circular peripheral relatively simple and uniform, thus less informative. S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 719

Fig. 7. Tree recovered from the Bayesian analysis. Clade posterior probabilities are reported. Values <95% are omitted.

Further, it also may be due to the placement of undue work, and many more are available in museums for emphasis on taxonomic work based on Holarctic fauna morphological studies. Unfortunately, some enigmatic as in Loerbroks (1984), ignoring the fact that thomisid genera, such as Heterogriffus Platnick, 1976 and Stiph- diversity is biased towards tropical habitats, following ropella Lawrence, 1952 (Stiphropodinae) or the typical latitudinal gradient. Furthermore, cladistic Simon, 1895 (Strophiinae) are known only from a few methodology was not used in previous studies. museum specimens. There is an immediate need to Addition of more taxa and morphology to future include them in future molecular and morphological studies is vital for understand evolutionary relationships studies. within the four clades named here. Species of most Finally, thomisid outgroup relationships need atten- described genera could be collected easily for molecular tion. The phylogenetic relationships of Dionycha 720 S. P. Benjamin et al. / Cladistics 24 (2008) 708–722

ABCDE

Fig. 8. Borboropactus cinerascens. Male palp: (A) ventral view; (B) prolateral view; (C) retrolteral view. Female: (D) epigynum, ventral view; (E) vulva, ventral view. Scale bars: 1 mm (D,E), 2 mm (A–C). Abbreviations: C conductor; E embolus; ET epigynal teeth; MA median apophysis; RTA retrolateral tibial apophysis.

(a clade that includes 15 families in addition to Thomis- Benjamin, S.P., 2000. Epidius parvati sp. n., a new species of the genus idae) have got to be resolved. Understanding the Epidius from Sri Lanka (Araneae: Thomisidae). Bull. Br. Arachnol. placement of Thomisidae within Dionycha is a necessary Soc. 11, 284–288. Benjamin, S.P., 2001. The genus Oxytate L. Koch 1878 from Sri first step to resolve this major issue in spider evolution. Lanka, with description of Oxytate taprobana sp. n. (Araneae: Thomisidae). J. South Asian Nat. Hist. 5, 153–158. Benjamin, S.P., 2002. Smodicinodes schwendingeri sp. n. from Thailand Acknowledgments and the first male of Smodicinodes Ono, 1993, with notes on the phylogenetic relationships in the tribe Smodicini (Araneae: Thom- isidae). Rev. Suisse Zool. 109, 3–8. Thanks to the following individuals for spider spec- Coddington, J.A., Levi, H.W., 1991. Systematics and evolution of imens: Fernando Alvarez, Cristian Grismado, Charles spiders (Araneae). Annu. Rev. Ecol. Syst. 22, 565–592. Griswold, Charles Haddad, Stephen Lew, Lara Lop- Colgan, D.J., MacLauchlan, A., Wilson, G.D.F., Livingston, S.P., ardo, Martin Ramirez and Peter Schwendinger. Jona- Edgecombe, G.D., Macaranas, J., Cassis, G., Gray, M.R. 1998. than Coddington improved earlier drafts of this Histone H3 and U2 snRNA DNA sequences and arthropodmo- lecular evolution. Aust. J. Zool. 46, 419–437. manuscript. We thank Fernando Alvarez for analytical Comstock, J.H., 1948. The Spider Book. Comstock Publishing Co., support and Charles Griswold and Pekka Lethinen for Inc., Ithaca, New York. discussion. A. H. Sumanasena (Department of Wild Life Davila, D.S., 2003. Higher-level relationships of the spider family Conservation, Colombo) provided a research permit to Ctenidae (Araneae: Ctenoidea). Bull. Am. Mus. Nat. Hist. 274, 1– 86. collect in Sri Lanka. Funding for this research has been Dippenaar-Schoeman, A.S., 1989. The crab-spiders of Africa (Ara- provided by grants from the US National Science neae: Thomisidae). 8. The genus Thomisops Karsch, 1879. Phyto- Foundation (DEB-0328644 to G. Hormiga and phylactica, 21, 319–330. G. Giribet and EAR-0228699 to W. Wheeler, J. Evans, T.A., 1995. Two new species of social crab spiders of the genus Coddington, G. Hormiga, L. Prendini and P. Sierwald) Diaea from Eastern Australia, their natural history and distribu- tion. Rec. West. Aust. Mus. 52(Suppl.), 151–158. and by a REF grant from The George Washington Evans, T.A., 1998. Offspring recognition by mother crab spiders University to GH. Additional funding to SPB came with extreme maternal care. Proc. R. Soc. Lond. B, 265, 129– from the Schlinger Foundation, the University of 134. California, Berkeley and a Smithsonian Institution Farris, J.S., 2001. Support weighting. Cladistics 17, 389–394. postdoctoral fellowship. Farris, J.S., Albert, V.A., Kallersjo, M., Lipscomb, D., Kluge, A., 1996. Parsimony jackknifing outperforms neighbor-joining. Cla- distics 12, 99–124. Farris, J.S., Ka¨llersjo¨, M., Kluge, A.G., Bult, C., 1994. Testing References significance of incongruence. Cladistics 10, 315–319. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach Aldrich, J.R., Barros, T.M., 1995. Chemical attraction of male crab using the bootstrap. Evolution 39, 783–791. spiders (Araneae, Thomisidae) and kleptoparasitic flies (Diptera, Fitch, W.M., 1971. Towards defining the course of evolution: and Chloropidae). J. Arachnol. 23, 212–214. minimal change for a specific tree topology. Syst. Zool. 20, 406– Arnedo, M.A., Coddington, J., Agnarsson, I., Gillespie, R.G., 2004. 416. From a comb to a tree: phylogenetic relationships of the comb- Gabritschevsky, E., 1927. Experiments on color changes and regen- footed spiders (Araneae, ) inferred from nuclear and eration in the crab-Spider, . J. Exp. Zool. 47, 251– mitochondrial genes. Mol. Phylogenet. Evol. 31, 225–245. 266. Barrion, A.T., Litsinger J.A., 1995. Riceland Spiders of South and Giribet, G., 2001. Exploring the behavior of POY, a program for direct Southeast Asia. CAB International, Wallingford, UK. optimization of molecular data. Cladistics 17, 60–70. S. P. Benjamin et al. / Cladistics 24 (2008) 708–722 721

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Wunderlich, J., 2004b. The new spider (Araneae) family Borboropac- Conductor: a sclerite of the male copulatory palp that accompanies tidae from the tropics and fossil in baltic amber. Beitr. Araneol. 3, and supports the embolus; a hyaline conductor is a conductor that is 1737–1746. transparent and very thin (Fig. 8C). Colulus: short median protuberance in front of spinnerets, consid- ered a modification of the spinning plate or cribelum. Embolus: embolus is the intromitted part of the male copulatory Appendix 1 palp (Fig. 8B). Epigynum: a usually chitinous section on the ventral side of the Taxonomic changes, Fig. 7(A–E). abdomen of adult female spiders on which the genital openings are Genus: Borboropactus Simon, 1884. found. Epigynes of some thomisids have lateral sclerotized projections Borboropactus cinerascens (Doleschall, 1859). termed teeth (Fig. 7D,E). B. bangkongeus Barrion and Litsinger (1995). New synonomy. Male palp: modified tarsus of the palp in male spiders. It is a B. mindoroensis Barrion and Litsinger (1995). New synonomy. copulatory organ of great complexity and is probably the most B. umaasaeus Barrion and Litsinger (1995). New synonomy. important character system in spiders (Fig. 7A,B). B. mindoroensis is a juvenile female; penultimate female thomisids Median apophysis: an apophysis of the male palpal bulb (Fig. 8B). some times have partly sclerotized genitalia. B. bangkongeus and Scopula hairs: specialized hairs found on the tip of legs in some B. umaasaeus are one and the same species. Their illustrations (Barrion thomisids. Thought to improve grip. and Litsinger, 1995) leave no doubt that they are synonymous with Tegulum: part of the male copulatory palp that houses the sperm B. cinerascens. Vouchers are deposited at the Natural History Museum duct. of Geneva.

Appendix 2

Glossary of terms

Terms used in the discussion taken from Jocque´and Dippenaar- Schoeman (2006).