Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: Implications for classification, biogeography, and the evolution of web building behavior

Molecular Phylogenetics and Evolution 38 (2006) 583–602 www.elsevier.com/locate/ympev

Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: Implications for classification, biogeography, and the evolution of web building behavior Nicholas P. Murphy a, Volker W. Framenau b, Stephen C. Donnellan c, Mark S. Harvey b, Yung-Chul Park d, Andrew D. Austin a,* a

c

Centre for Evolutionary Biology and Biodiversity, and School of Earth and Environmental Sciences, The University of Adelaide, SA, 5005, Australia b Department of Terrestrial Invertebrates, Western Australian Museum, Locked Bag 49, Welshpool, Western Australia 6986, Australia Evolutionary Biology Unit, and Centre for Evolutionary Biology and Biodiversity, South Australian Museum, North Terrace, Adelaide, SA 5000, Australia d Department of Biology, College of Natural Science, Dongguk University, Seoul, South Korea Received 9 May 2005; revised 7 September 2005; accepted 7 September 2005

Abstract Current knowledge of the evolutionary relationships amongst the wolf spiders (Araneae: Lycosidae) is based on assessment of morphological similarity or phylogenetic analysis of a small number of taxa. In order to enhance the current understanding of lycosid relationships, phylogenies of 70 lycosid species were reconstructed by parsimony and Bayesian methods using three molecular markers; the mitochondrial genes 12S rRNA, NADH1, and the nuclear gene 28S rRNA. The resultant trees from the mitochondrial markers were used to assess the current taxonomic status of the Lycosidae and to assess the evolutionary history of sheet-web construction in the group. The results suggest that a number of genera are not monophyletic, including Lycosa, Arctosa, Alopecosa, and Artoria. At the subfamilial level, the status of Pardosinae needs to be re-assessed, and the position of a number of genera within their respective subfamilies is in doubt (e.g., Hippasa and Arctosa in Lycosinae and Xerolycosa, Aulonia and Hygrolycosa in Venoniinae). In addition, a major clade of strictly Australasian taxa may require the creation of a new subfamily. The analysis of sheet-web building in Lycosidae revealed that the interpretation of this trait as an ancestral state relies on two factors: (1) an asymmetrical model favoring the loss of sheet-webs and (2) that the suspended silken tube of Pirata is directly descended from sheet-web building. Paralogous copies of the nuclear 28S rRNA gene were sequenced, confounding the interpretation of the phylogenetic analysis and suggesting that a cautionary approach should be taken to the further use of this gene for lycosid phylogenetic analysis.
2006 Published by Elsevier Inc. Keywords: Lycosidae; Web building; 12S rRNA; NADH1; 28S rRNA paralogous copies; Biogeography

1. Introduction With nearly 2300 described species in over 100 genera (Platnick, 2005), the araneomorph spider family Lycosidae

*

Corresponding author. E-mail address: [email protected] (A.D. Austin).

1055-7903/$ - see front matter
2006 Published by Elsevier Inc. doi:10.1016/j.ympev.2005.09.004

(wolf spiders) belongs to one of the dominant spider groups in the world, both in diversity and in local abundance. Wolf spiders occur in significant numbers in virtually every terrestrial habitat and have managed to colonise some of the most inhospitable places on earth. Wolf spiders are readily identified by the characteristic arrangement of their eyes in three rows, the lack of a retrolateral tibial apophysis on the male pedipalp and their

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mobile brood care (Dondale, 1986). Females carry the egg-sac attached to their spinnerets (e.g., Townley and Tillinghast, 2003) and, subsequently, their young on the abdomen (e.g., Rovner et al., 1973). This mobile brood care appears to allow wolf spiders to colonise habitats that are otherwise inhospitable for spiders, particularly in areas that are prone to inundation, such as coastal and riparian habitats or the shores of inland lakes (Framenau et al., 2002; Manderbach and Framenau, 2001; McKay, 1974; Morse, 2002). Many species also disperse via ballooning, enabling wolf spiders to quickly recolonise disturbanceprone environments. For example, they are amongst the first animals to recolonise lava fields after volcanic eruptions (Crawford et al., 1995; Edwards and Thornton, 2001). Wolf spiders display a range of prey-capture strategies from web building species to burrow-dwellers and vagrants, which may have contributed to their ecological success. Whilst web building groups are generally postulated as being the most basal within the family (e.g., Job, 1974), this supposition has never been tested within a robust phylogenetic framework. There is morphological evidence that web building lycosids do not form a monophyletic clade since the members of the web building genera Sosippus, Venonia, Hippasa, and Aulonia vary substantially in their somatic and genital morphology. Zehethofer and Sturmbauer (1998) interpreted a variety of prey capture strategies on the basis of a molecular phylogeny of central European wolf spiders. Within their selection of taxa, tube building (genus Pirata) was ancestral, and vagrant hunting evolved twice independently, in Pardosa and in Xerolycosa. Permanently burrowing wolf spiders appear to mainly include species traditionally included in the subfamily Lycosinae. However, there are a variety of burrow modifications, such as trapdoors and palisade structures around the entrance of the burrow. The latter modification appears to be prevalent in a number of unrelated taxonomic groups, such as Nearctic Geolycosa and Hogna (Marshall et al., 2000; Shook, 1978) and the Australian Dingosa (listed as Pardosa in McKay, 1979, 1985) and Mainosa (Framenau, in press a). Wolf spiders have served extensively as model organisms addressing a number of evolutionary questions including aspects of both natural and sexual selection. The evolution of sexual dimorphism, such as size difference between males and females and differences in troph and locomotor morphology (Framenau, in press b; Walker and Rypstra, 2001, 2002, 2003), and the significance of secondary male sexual traits (e.g., Hebets, 2004; Stratton and Uetz, 1981, 1983, 1986; Uetz, 2000) have received considerable attention. However, a phylogenetic framework for the interpretation of sexual dimorphic structures and their function does not exist. Evolutionary research on wolf spiders is not only limited to sexual selection and mating behaviour. Lycosids also serve as model organisms addressing fundamental questions in relation to optimal foraging theory (Framenau

et al., 2000; Persons and Uetz, 1996, 1997) and territoriality (Marshall, 1995, 1996, 1999; Moya-Laran˜o et al., 2002). Most recently, it has been shown that mating preferences of female wolf spiders can be acquired through exposure as subadults to unrelated, sexually active males. This represents the first reported case stressing the potential importance of learning and memory of immatures on adult mate choice in arthropods (Hebets, 2003). In order to understand how these different traits evolved a clear picture of phylogenetic relationships amongst wolf spiders is required. 1.1. Classification and phylogenetic structure The monophyly of the Lycosidae is well supported (Dondale, 1986; Griswold, 1993), but relationships within the family are poorly understood and a stable subfamilial classification does not exist, as current schemes are almost exclusively based upon northern hemisphere taxa. Dondale (1986) recognised five subfamilies, Allocosinae, Lycosinae, Pardosinae, Sosippinae, and Venoniinae, and presented a phylogenetic hypothesis based upon the morphology of the male pedipalp (Fig. 1). He refuted Petrunkevitch (1928) Hippasinae by including Hippasa in the Lycosinae, and also Lehtinen and Hippa (1979) and Hippa and LehtinenÕs (1983) Zoicinae, which he considered a junior synonym of the Venoniinae. Later authors added the Evippinae and Wadicosinae (Zyuzin, 1985), Piratinae (Zyuzin, 1993), and Tricassinae (Alderweireldt and Joque´, 1993), but none of these authors has attempted to accommodate their new subfamilies into DondaleÕs (1986) rudimentary phylogenetic framework. The Lycosinae and Pardosinae represent the most species rich of the currently recognised subfamilies, and Dondale (1986) considered them to be sister taxa. However, Lehtinen (1978) and Zyuzin (1985) had earlier cast doubt on their independent status and regarded both groups as members of a single subfamily, Lycosinae. A molecular phylogeny based on the mitochondrial 12S rRNA gene had not resolved the relationship between Pardosa and the putatively lycosine genera Alopecosa and Trochosa as the three genera represented a trichotomy in the preferred phylogeny (Zehethofer and Sturmbauer, 1998). This study

Fig. 1. Hypothesis of lycosid subfamily relationships based on the morphology of the male pedipalp (Dondale, 1986).

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was hampered by the use of only 27 European species from five genera. More recently, a molecular data set also using 12S rRNA for 30 species (Vink et al., 2002) demonstrated that whilst some Australasian species (e.g., Lycosa godeffroyi and three species of Venatrix) nested neatly with an existing subfamily (Lycosinae), others such as species of Anoteropsis and Artoria formed separate clades that poorly reflect existing subfamilies. This preliminary research has cast considerable doubt on any subfamilial system that does not adequately sample taxa outside the Holarctic region, and highlights the necessity for including exemplars from the southern continents to better understand Lycosidae systematics. The concept of the genus Lycosa is central to phylogenetic problems within the family, as it has been used as a ‘‘dumping ground’’ for wolf spiders that could not be satisfactorily placed in other genera. However, there is now ample morphological evidence (e.g., Zyuzin and Logunov, 2000) that the majority of Lycosa species are unrelated to the type species, L. tarantula from the Mediterranean region, and should be removed to other genera. This process has been largely completed for the North American fauna where a series of revisions has defined several genera and transferred numerous species from Lycosa (e.g., Dondale and Redner, 1978, 1979, 1983a,b, 1990). Amongst the Australasian fauna, several species have been recently transferred from Lycosa (Framenau, 2002, 2005; Framenau, in press a, in press c, in press; Framenau and Vink, 2001; Vink, 2002). While it is clear that no Australasian wolf spider possesses the defining features of Lycosa, these species await critical study to determine their true systematic position. 1.2. Aims Employing sequences for multiple genes and a more comprehensive sampling comprising 70 species from more than 25 genera, representing most biogeographic regions, we aimed to develop a more robust phylogenetic hypothesis for the Lycosidae. In particular, we were interested in: (1) testing the monophyly of and relationships among currently recognised subfamilies, determining the subfamilial placement of several genera, including a number of key taxa from Australia and New Zealand that, on morphological grounds, could not be satisfactorily placed; (2) examining whether sheet-web-spinning behaviour is an ancestral trait within the family and how burrowing behaviour may have evolved; (3) determining whether specific higher-level clades are restricted to the southern continents (as indicated by the preliminary study of Vink et al. (2002)).

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nised lycosid subfamilies as specimens of the small groups Allocosinae, Tricassinae and Wadicosinae were not available to us. We attempted to acquire material of the type species of each genus but this was not always possible. Importantly, we were able to sequence L. tarantula, the type species of Lycosa. Included in the study were multiple species of Piratinae (Aulonia, Hygrolycosa, and Pirata), Sosippinae (Aglaoctenus and Sosippus), Pardosinae (10 species of Pardosa from the Palaearctic and Africa), Venoniinae (Allotrochosina, Anomalosa, and Venonia) and Lycosinae (34 species worldwide from Alopecosa, Geolycosa, Hippasa, Hogna, Lycosa, Pavocosa, Rabidosa, Trochosa, Varacosa, and Venatrix). A number of undescribed genera were also included; these were classified to subfamily where possible (Table 1). Some Australasian genera that we were unable to assign to existing subfamilies (Anoteropsis, Artoria, Notocosa, Tetralycosa, Trochosa expolita, and three undescribed genera) were also included. The Evippinae were represented by a single species (Xerolycosa). After field collection, the entire body of smaller specimens and dissected legs of larger specimens were placed in 100% ethanol. They were generally refrigerated prior to DNA extraction. Voucher specimens of most taxa have been deposited in the Western Australian Museum collections. As we aimed to test hypotheses on a variety of prey-capture strategies including the ancestral state of the sheetweb, we included representatives of all genera that are known to construct sheet-webs (Aulonia, Hippasa, Sosippus, and Venonia), as well as those that build simple suspended silken tubes (some Pirata). Taxon sampling also included vagrant hunters (Artoria, Hygrolycosa, Pardosa, some Pirata, and Xerolycosa), and burrow dwellers (Geolycosa, Lycosa s. str., and ‘‘New Genus 6’’). The geographic representation of species was as follows: 31 species from the Palaearctic (mainly from central Europe and Korea), 27 species from Australasia (including three from New Zealand), nine species from the Americas (six from the Nearctic and three from the Neotropics), and three species from Africa. The outgroup taxa used for the study were an undescribed Australian Dolomedes (Pisauridae) and two representatives of the Miturgidae (Mituliodon tarantulinus and Miturga gilva), since both families are close relatives to the Lycosidae within the superfamily Lycosoidea (Griswold, 1993; Raven and Stumkat, 2005). The nursery web of Dolomedes is interpreted as a reduced sheet web and was coded as such in our analysis. Mituliodon tarantulinus is a vagrant hunter (Raven and Stumkat, 2003), whereas M. gilva, (as do many other Australian Miturga), constructs sheet webs under rocks and logs (MSH, personal observation).

2. Materials and methods 2.2. DNA extraction, amplification, and sequencing 2.1. Taxonomic sampling In total 70 lycosid species were utilised in the study (Table 1). We sampled all but three of the currently recog-

Genomic DNA was extracted from ethanol preserved tissue using the Puregene DNA Purification Kit (Gentra Systems). Partial sequences were obtained from an approx-

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Table 1 Lycosidae employed in this study including prey capture strategy and distributions for each species Subfamily and species

Prey capture/burrow

Distribution

Lycosinae Allocosa hasselti (L. Koch, 1877) Alopecosa albostriata (Grube, 1861) Alopecosa cinnameopilosa (Schenkel, 1963) Alopecosa kochi (Keyserling, 1877) Alopecosa licenti (Schenkel, 1953) Alopecosa moriutii Tanaka, 1985 Alopecosa pulverulenta (Clerck, 1757) *Arctosa cinerea (Fabricius, 1977) Arctosa ebicha Yaginuma, 1960 Arctosa kwangreungensis Paik & Tanaka, 1986 Arctosa maculata (Hahn, 1822) Arctosa sp. n. Arctosa stigmosa (Thorell, 1875) Geolycosa missouriensis (Banks, 1895) Hippasa holmerae Thorell, 1895 Hogna crispipes (L. Koch, 1877) Lycosa bicolor Hogg, 1905 Lycosa clara L. Koch, 1877 Lycosa coelestis L. Koch, 1878 Lycosa erythrognatha Lucas, 1836 Lycosa godeffroyi L. Koch, 1865 Lycosa leuckarti (Thorell, 1870) Lycosa castanea Hogg, 1905 Lycosa suzukii Yaginuma, 1960 *Lycosa tarantula (Linnaeus, 1758) New Genus 3 sp. New Genus 4 sp. New Genus 5 sp. New Genus 6 sp. *Pavocosa gallopavo (Mello-Leita˜o, 1941) Rabidosa punctulata (Hentz, 1844) *Rabidosa rabida (Walckenaer, 1837) Trochosa expolita (L. Koch, 1877) *Trochosa ruricola (De Geer, 1778) Trochosa terricola Thorell, 1856 *Varacosa avara (Keyserling, 1877) *Venatrix funesta (C.L. Koch, 1847) Venatrix konei (Berland, 1924)

b ? v b ? ? v bb v v bb ? bb bt w v b v ? b b b b ? b ? ? ? bd b v v v bb bb ? b v

NE Australia E Palaearctic E Palaearctic Nearctic E Palaearctic E Palaearctic Palaearctic Palaearctic, N. Africa Asia Asia W Palaearctic Africa Palaearctic Nearctic E Palaearctic Australia, Oceania Australia Australia E Palaearctic South America Australia Australia Australia E Palaearctic W Palaearctic Africa Africa Africa E Australia Neotropical Nearctic Nearctic Australia Holarctic Holarctic Nearctic Australia Australia, Oceania

Pardosinae Pardosa astrigera L. Koch, 1878 Pardosa brevivulva Tanaka, 1975 Pardosa californica Keyserling, 1887 Pardosa crassipalpis Purcell, 1903 Pardosa foveolata Purcell, 1903 Pardosa hedini Schenkel, 1936 Pardosa isago Tanaka, 1977 Pardosa laura Karsch, 1879 Pardosa lugubris (Walckenaer, 1802) Pardosa palustris (Linnaeus, 1758)

v v v v v v v v v v

E Palaearctic Asia Nearctic Africa Africa E Palaearctic E Palaearctic E Palaearctic Holarctic Asia

Piratinae Pirata hygrophilus Thorell, 1872 Pirata piratoides (Bo¨senberg & Strand, 1906) Pirata subpiraticus (Bo¨senberg & Strand, 1906) Pirata uliginosus(Thorell, 1856)

t t t t

Palaearctic Asia Asia W Palaearctic

Sosippinae *Aglaoctenus lagotis (Holmberg, 1876) Sosippus placidus Brady, 1972

w w

Neotropic Nearctic

Venoninae Allotrochosina karri Vink, 2001 *Allotrochosina schauinslandi (Simon, 1899) *Anomalosa kochi (Simon, 1898)

? v v

W Australia New Zealand E Australia

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Table 1 (continued) Subfamily and species

Prey capture/burrow

Distribution

*Aulonia albimana (Walckenaer, 1805) *Hygrolycosa rubrofasciata (Ohlert, 1865) Venonia micarioides (L. Koch, 1877) *Xerolycosa nemoralis (Westring, 1861)

w v w v

Palaearctic Palaearctic Australia Palaearctic

unplaced Anoteropsis adumbrata (Urquhart, 1887) Artoria flavimana Simon, 1909 Artoria howquaensis Framenau, 2002 Artoria separata Vink, 2002 New Genus 1 sp. New Genus 2 sp. *Notocosa bellicosa (Goyen, 1888) *Tetralycosa oraria (L. Koch, 1876)

v v v v v ? bt bb

New Zealand Australia Australia New Zealand Australia N Australia New Zealand Australia

Outgroups Miturgidae *Mituliodon tarantulinus (L. Koch, 1873) Miturga gilva L. Koch, 1872

v w

Australia Australia

Pisauridae Dolomedes sp. n.

w

Australia

Subfamily classification follows Dondale (1986) and Zyuzin (1993). An asterisk in front of the species name indicates that this species represents the type species of the genus. Prey capture strategy/burrow: b, permanent burrow; bd, brrow with trapdoor; bt, burrow with turret; bb, temporary burrow (with broodcare); w, web building; t, suspended tubular hide out; v, vagrant; ?, insufficient biological information.

imately 400 base pairs (bp) product obtained by polymerase chain reaction (PCR) amplification of the mitochondrial 12S rDNA gene using the primers12St-L (Croom et al., 1991) and 12Sbi-H (Simon et al., 1994). Amplification of an approximately 600 bp segment of mitochondrial protein coding NADH1 was undertaken using the primers TL-1-N12718 (Hedin, 1997) and M510 (50 -ATACTAATTCKG ATTCKCCTTC-30 ) (designed in this study from complete NADH1 sequences of Heptathela hangzhouensis [GenBank Accession No. NC005924] and Habronattus oregonensis [GenBank Accession No. NC005942]). Amplification of an approximately 800 bp fragment of the nuclear 28S rDNA gene, spanning the D2 and D3 segments, was implemented using the primers 28S ‘‘O’’ and 28S ‘‘C’’ (Hedin and Maddison, 2001). Internal nested primers were designed directly from lycosid sequences obtained in this study to amplify problematic taxa with the first set of primers (G700 50 -TGCGGACCTCCACCAGAGTTTCT-30 and G701 50 -ACTGCTCAGAGGTAAACGGGAGG-30 ). PCR amplifications were carried out in 25 lL containing PCR buffer, 0.2 mM of each dNTP, 0.4 lM of each primer, 2 mM MgCl2, 0.5 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems) and 25–100 ng of genomic DNA. Nested PCR was undertaken for some samples for the 28S rDNA gene using the primers G700 and G701 following the above PCR amplification protocol substituting the genomic DNA for 2 lL of a 1/100–1/1000 dilution of the initial PCR product obtained using the 28S ‘‘O’’ and 28S ‘‘C’’ primers. Thermocycling conditions were: an initial denaturation step of 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, an annealing temperature of 50 C for 30 s, and an extension temperature of 72 C for 30 s. This was then followed by an additional extension of 72 C

for 3 min. PCR products were purified using the UltracleanTM PCR Clean-up Kit (MOBIO Laboratories). Sequencing reactions were performed using ABI Big Dye Terminator Chemistry, fragments were resolved on an ABI 3700 sequencer. 2.3. Sequence alignment and analysis Alignment of 12S rDNA was initially undertaken using the secondary structure model developed by Hickson et al. (1996) to infer the position of stem and loop regions. Clustal X (Thompson et al., 1997) was used for the subsequent alignment of the more quickly evolving loop regions. A number of gap opening/gap extension schemes (gap to change costs 1:1 1:2 1:5 1:10) were implemented and regions of uncertain alignment that varied markedly between different alignment schemes were deleted (Gatesy et al., 1993). The alignment of the 28S rRNA gene was undertaken using the same procedure as 12S rDNA with the secondary structure based on the model proposed by Hedin and Maddison (2001). The NADH1 gene was aligned by eye and the amino acid sequence was translated with reference to Habronattus oregonensis (GenBank Accession No. NC005942) as a test of the presence of nuclear paralogues, e.g., stop codons. As significant heterogeneity in base composition can lead to erroneous grouping of unrelated taxa with similar composition (Lockhart et al., 1994), homogeneity v2 analysis of each gene was undertaken using PAUP* 4.0b10 (Swofford, 2000) to identify significant differences in base composition. The presence of nucleotide saturation, in particular at the third codon position of NADH1, was examined by plotting observed transitions and transversions

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against the overall nucleotide divergence. Further examination of the nucleotide properties of 12S rRNA and 28S rRNA and the separate codon positions of NADH1 was undertaken using Bayesian analyses implemented in MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2001). MrBayes was used to estimate base frequencies, transition matrices, proportion of invariant sites and C shape parameter for each partition (Schwarz et al., 2004). Analyses were run across four chains for two million generations sampling every 100 generations, and stationarity was determined from an examination of log likelihoods. Multiple runs were performed to assess that parameters were not considerably different at stationarity. The combination of data partitions during phylogenetic analyses should increase phylogenetic accuracy, provided the partitions share a common evolutionary history. The presence of significant heterogeneity between partitions (genes), assessed using the approach outlined by Wiens (1998) comparing the support in different topologies derived from the different genes, was implemented using Bayesian posterior probabilities to determine heterogeneity based on strongly supported conflicting clades (Reeder, 2003). A further method of assessing congruence was implemented using the Approximately Unbiased (AU) test (Shimodaira, 2002). The optimal topology for each individual data partition was tested against the alternative topologies for the other genes when and the combined gene topology. Maximum parsimony (MP) analyses were performed in PAUP* using the heuristic search algorithm with 100 random sequence addition replicates to eliminate any bias from taxon ordering in the datasets. Gaps were treated as missing data and two weighting schemes were analysed; characters were either weighted equally, or transition substitutions at the third codon position in NADH1 were ignored. Support for nodes on the MP trees was assessed from 1000 non-parametric bootstrap pseudoreplications and from Bremer support indices calculated using PRAP v 1.0 (Muller, 2004). One major advantage of maximum likelihood (ML) and Bayesian methods of phylogenetic reconstruction is the ability to circumvent the problem of multiple substitutions at a site. Bayesian analysis was preferred in this study over ML methods; one of the major advantages of Bayesian analyses is the ability to allow models to be applied to separate data partitions whilst searching for optimal trees that describe the entire data set. Bayesian analysis is also much less computationally intense, allowing much more comprehensive analyses to be performed. Bayesian phylogenetic analyses were performed using MrBayes. Likelihood ratio tests and the Akaike information criterion were performed using Modeltest (Posada and Crandall, 1998) and both showed that the GTR+I+C model is the most suitable for analysis of all genes. This model was therefore used for each partition and the model parameters were unlinked and estimated separately for each partition, including each codon position in NADH1. MrBayes analyses were run

across four chains for two million generations sampling every 100 generations, and stationarity was determined from an examination of log likelihoods and model parameters. Trees recovered prior to stationarity being reached were discarded and Bayesian posterior probabilities of each bipartition, representing the percentage of times each node was recovered, were calculated from a 50% majority rule consensus of the remaining trees. Multiple runs were performed to assess that all parameters were not considerably different at stationarity based on alternate prior probabilities. Ancestral states and transitions between sheet-web building and non-sheet-web building lycosids were reconstructed on the MP and Bayesian phylogenies using ML methods implemented in Mesquite (Maddison and Maddison, 2003a). Both symmetrical (Mk1 model: equal forward and backward rates of transitions) and asymmetrical (Asymm. 2 param model: unequal forward and backward rates of transitions) models were used to estimate the loss/gain of sheet-web building as a dichotomous variable using the Ancestral States Module of Mesquite (Maddison and Maddison, 2003b). Rates of change were estimated from the data using both character distribution and tree (including branch length information). ML methods of ancestral character state reconstruction are valuable because they use branch length information and quantify uncertainty in character state reconstruction (Pagel, 1999). An ancestral state at a given node was considered significant and preferred over the other if its likelihood value was higher by at least two log units than the likelihood value of the other ancestral state (likelihood decision threshold values [T] set to two by default in Mesquite). The ancestral state of sheet-web building was examined across two separate data sets, one coding the genus Pirata as sheet-web building, the second coding Pirata as nonsheet-web building. The alternative coding of Pirata is based on their construction of a suspended silken tube, which may or may not be a reduced form of a sheet-web. 3. Results All sequences have been deposited in GenBank (Accession Nos. DQ019640–DQ019823). The alignment of 12S rRNA introduced gaps at 14 positions, no regions varied between alignments and were deemed unalignable. Gaps were introduced at 33 positions in the alignment of 28S rRNA, while six regions consisting of approximately 70 bp (between bases 93–112, 304–315, 383–393, 598– 607–628–633, 642–653 in the original alignment) were deemed unalignable and removed from further analysis. Of the 316 bp analysed for 12S rRNA, 231 sites were variable and 180 were parsimony informative; 311 sites of the 626 bp analysed for 28S rRNA were variable and 245 parsimony informative, whilst 356 sites of 554 bp analysed for NADH1 were variable and 308 were parsimony informative. Among the 554 bp of NADH1 analysed, 65% of changes were synonymous (11% 1st base substitutions,

N.P. Murphy et al. / Molecular Phylogenetics and Evolution 38 (2006) 583–602

89% 3rd base substitutions) and 35% of changes were nonsynonymous (49% 1st base substitutions, 25.5% 2nd base substitutions, 25.5% 3rd base substitutions). Overall 52% of variation in the NADH1 gene was explained by synonymous changes occurring at the 3rd codon position. No significant difference in base composition across taxa was detected for any of the genes (12S rRNA: v2 = 75.14, df = 216, P > 0.05, 28S rRNA: v2 = 46.48, df = 159, P > 0.05 (less taxa were examined for the 28S rRNA gene as described below), NADH1: v2 = 163.03, df = 216, P > 0.05). However, analysis of separate codon positions in the NADH1 gene showed that the 3rd base position contained significant heterogeneity in base composition (v2 = 376.32, df = 216, P < 0.001), whilst there was no significant difference at the other two base positions (1st pos. v2 = 83.37, df = 216, P > 0.05; 2nd pos v2 = 24.03, df = 216, P > 0.05). The results of Bayesian estimates for GTR substitutions matrices, invariant sites and C-shapes are shown in Table 2. Of particular interest were the differences between the codon positions, which were not unexTable 2 Substitution rate matrices, proportion of invariant sites, and C-shapes (a), estimate using Bayesian analyses for 1nt, 2nt, and 3nt NADH1 partitions and for 12S rRNA and 28S rRNA Codon position

C

G

T

P(inv)

a

1st position NADH1 A 1.208 C G

6.490 1.014

3.752 21.973 1

0.374

0.783

2nd position NADH1 A 1.874 C G

2.206 5.838

1.651 4.141 1

0.436

0.731

3rd position NADH1 A 0.081 C G

28.728 1.449

0.685 34.478 1

0.013

0.550

0.528

7.350 0.123

0.672 0.941 1

0.087

0.574

1.002

3.187 0.3621

1.461 1.836 1

0.236

0.885

12S rRNA A C G 28S rRNA A C G

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pected given the different constraints on these positions for synonymous/non-synonymous substitutions. Saturation plots of the different partitions indicated that the NADH1 3rd codon positions might be influenced by saturation, whilst all other partitions appear to be unaffected (data not shown). A comparison of the topologies and Bayesian support between the gene regions suggested a degree of phylogenetic congruence. The 12S rRNA tree, in general, was unresolved and had little support for deeper nodes; however, the majority of supported clades were congruent with the NADH1 tree (trees not shown, available from authors on request). The AU test (Table 3) revealed that the tree topology from the combined analysis was not a significantly worse representation of the nucleotide data than either of the 12S rRNA and NADH1 genes. Based on these results, the decision was made to combine the two genes for further analysis. The MP analysis for the combined data with all sites weighted equally gave 12 most parsimonious trees. A strict consensus of these trees revealed a very unresolved tree that contained little non-parametric bootstrap support. The analysis of the combined data without NADH1 3rd base transitions yielded 16 most parsimonious trees. Despite the larger number of trees, the strict consensus of these trees gave a much more resolved tree with the only differentiation between the individual MP trees being found within clades E1 and E5, both of which exhibited high bootstrap support. Non-parametric bootstrap and Bremer support for a number of groups on this tree was very strong (Fig. 2). Each of the multiple Bayesian runs resulted in very similar estimates for substitution parameters and identical topologies (Fig. 3). The Bayesian and MP analyses produced the same major clades, however, they differed in the relationships between these major clades. None of the major differences between the two analyses were supported by high posterior probabilities or bootstraps. Generally, congenerics included in the analysis were found to be monophyletic, however a number were not, and some were quite disparate. Arctosa ebicha and A. kwangreungensis appear to be distinct (in clade F with Aglaoctenus lagotis and Sosippus placidus) from the remaining Arctosa species in clade D (including the type of the genus, A. cinerea), suggesting that their systematic position should be reanalysed. Alopecosa cinnameopilosa is not found with-

Table 3 Results of AU congruence tests (Shimodaira, 2002) for comparisons of topologies from analysis of alternate DNA data partitions Data partition

Tree

12S rRNA

12S rRNA Combined NADH1*

Ti
6618.0379
39.5
68.3

NADH1

NADH1 Combined 12S rRNA*


13668.0290 23.6 431.6

AU

SE

PF

0.952 0.069 0.004


0.004
0.005
0.001

0.219 0.193 0.949

0.934 0.066 4.00E
06


0.004
0.004 0

0.462 0.462 1

Ti, best log-likelihood score for data partition or difference relative to it; AU, P value from the approximately unbiased test; SE, standard error of AU; PF, P value for the breakdown of asymptotic theory (*significant difference in log-likelihood scores at 0.05).

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N.P. Murphy et al. / Molecular Phylogenetics and Evolution 38 (2006) 583–602

Fig. 2. One of 16 maximum parsimony trees resulting from analysis of combined 12S rRNA and NADH 1 (excluding 3rd codon positions) mtDNA genes. Variation between the 16 trees occurred only within clades E1 and E4. Numbers above branches represent non-parametric bootstrap support; numbers below branches represent Bremer support indices. Designated clades (e.g., C2) represent groups found in both the MP and Bayesian (Fig. 3) analyses.

in the major Alopecosa clade nor does it appear to be the sister to the remaining Alopecosa. Its position is in conflict between the Bayesian analyses where it groups with the Pardosa clade and the MP analysis where it is found in weak support with L. tarantula. A number of species currently placed in Lycosa s. l. (viz. L. bicolor, L. clara, L. coelestis, L. erythrognatha, L. godeffroyi, L. leuckarti, L. castanea, and L. suzukii) do not form a clade, nor do

any appear to be closely affiliated with the type of the genus, L. tarantula. In fact, only four ‘‘Lycosa’’ species are associated, albeit in two separate groups in clade E5 (L. bicolor + L. castanea and L. godeffroyi + L. leuckarti). The three Artoria species, whilst found in the same clade (C1) were not monophyletic. Similarly, the Northern Hemisphere Trochosa ruricola and T. terricola (in clade E3) are split by the African ‘‘New Genus 3’’, while T. expo-

N.P. Murphy et al. / Molecular Phylogenetics and Evolution 38 (2006) 583–602

591

Fig. 3. Bayesian phylogenetic analysis of combined 12S rRNA and NADH 1 mtDNA genes. Tree produced from 2 · 106 generations using the GTR+I+C model of sequence evolution unlinked across all partitions, including each codon position. Numbers on branches indicate posterior probabilities, only branches receiving posterior probabilities greater than 0.50 are resolved.

lita is not closely affiliated with other Trochosa, but belongs to the Australasian clade C1. The representatives of the Lycosinae generally formed a monophyletic group (clade E). The exception to this was Arctosa (clade D), the position of which is unclear. Bayesian analysis places clade D in a reasonably well-supported basal position to the rest of the Lycosinae. MP analysis, however, supports a closer relationship with the clades containing species from Venoniinae (clade A1) and Piratinae (clade A2). Within clade E (putative Lycosinae as it contains the type species L. tarantula) there are a number of

well-supported groups (clades E1–E4, E6) common between Bayesian and MP analyses. Included in this group are all species belonging to Pardosa (Pardosinae) (clade E4) along with a group containing a number of Australian species (H. crispipes, L. bicolor, L. castanea, L. godeffroyi, L. leuckarti, A. hasselti, L. clara, and ‘‘New Genus 6’’) and the South American P. gallopavo (clade E5). This clade does not have high support from either MP or Bayesian analyses, however a clade of the Australian species is supported by Bayesian analysis with a posterior probability of 93%.

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The other major clade supported by these analyses is the grouping of a number of Australian and New Zealand species (clade C). This group exhibits very high posterior probabilities (98%), but it is not supported by a high bootstrap value (<50%). Relationships within this clade are somewhat ambiguous. Bayesian analyses suggest two major clades, one with very strong posterior probabilities (100%) containing A. flavimana, A. adumbrata, A. separata, N. bellicosa, A. howquaensis, and T. expolita (clade C1) and another with posterior probabilities of 78% containing ‘‘New Genus 1,’’ ‘‘New Genus 2,’’ and Tetralycosa oraria (clade C2). The MP analyses suggest a slightly different arrangement without support, inferring a close relationship between ‘‘New Genus 1’’ and T. expolita. The other consistent and strongly supported grouping contains two Venoniinae species, Aulonia albimana and Hygrolycosa rubrofasciata (clade B). The relationships of these taxa within the lycosids are also unclear, with MP analyses suggesting that they are the sister lineage to all other lycosids, whilst Bayesian suggests a close relationship with clades A1 and A2. Again, neither analysis strongly supports these positions. The other taxon with unclear evolutionary affinities is Hippasa holmerae; MP analyses placed this species as the sister to the other lycosids, with the exception of A. albimana and H. rubrofasciata, whilst Bayesian analyses suggest a closer affinity with Arctosa (clade D) and the putative Lycosinae (Clade E). Based on Bayesian and MP analyses, a number of a posteriori, alternative phylogenetic hypotheses containing topological constraints were tested against the Bayesian tree (Table 4). The subfamilies and genera shown to be non-monophyletic had their monophyly enforced to examine whether this created significantly worse representations of the data. The enforced monophyly of the genus Lycosa and Trochosa both resulted in significantly worse trees, while the enforcement of Lycosinae with the exclusion of Pardosinae also resulted in a significantly inferior tree. Other constraints tested were not significantly different and there was no significant difference between the Bayesian and MP trees.

3.1. Evolution of sheet-web building Maximum likelihood ancestral state reconstruction of sheet-web building was undertaken on both the MP and Bayesian topologies with alternate coding for sheet-web building in Pirata (Figs. 4A–D). A summary of the results from the reconstructions is found in Table 4. The unconstrained estimates of transition rates for the asymmetrical model for all analyses undertaken are biased towards a loss of web building (Table 5). A likelihood ratio test indicates that the asymmetric model is significantly more likely than the symmetric model across the MP tree, however it is not significantly more likely across the Bayesian tree. The main differences found in this analysis appear to be due to the differences in the Mk1 and Asymm. 2 param models. The Mk1 (symmetrical) model consistently suggests a non-sheet-web ancestry for the entire lycosid clade and that sheet-web building has been regained independently at least four times. The inclusion of Pirata as a sheet-web group (Figs. 4B and D, left trees) had no effect on the outcome when using the Mk1 model and this analysis suggests that Pirata does not have a sheet-web ancestry. Conversely, the Asymm. 2 param (asymmetrical) model largely suggested that sheet-web building was an ancestral trait in the Lycosidae (Figs. 4A–D, right trees), and that the trait has been lost independently a number of times. One exception was the Bayesian tree with Pirata coded as non sheet-web building (Fig. 4C), on which the asymmetrical model suggested that sheet-webs are not an ancestral lycosid trait and have been regained independently a number of times. The coding of Pirata as sheet-web building had little effect on the outcome of the asymmetrical model of ancestral states across the MP tree, however, it did cause a drastic difference in the ancestral states on the Bayesian tree. The inclusion of Pirata as sheet-web building changed from an ancestral state of non-sheet-web building and a general trend of independent evolution of sheet-webs, to an ancestral sheet-web state and a trend of independent loss of the trait. The Asymm. 2 param reconstruction of Pirata as a sheet-web builder on both the MP and Bayesian trees indi-

Table 4 Results of AU tests (Shimodaira, 2002) for comparison between topologies resulting from combined Bayesian and MP analyses, and comparson between optimal Bayesian topology and alternate constrained topologies (see Table 1 for species included in each alternate topology) Topology

Ti

AU

SE

PF

Bayesian tree MP tree Monophyly of Monophyly of Monophyly of Monophyly of Monophyly of Monophyly of


20485.0466
13.2
9.4
11.4
20.5
45.4
75.8
84.4

0.693 0.426 0.497 0.404 0.199 0.049 0.003 0.004


0.009
0.012
0.012
0.012
0.011
0.006
0.002
0.002

0.190 0.130 0.685 0.230 0.321 0.230 0.431 0.425

Alopecosa Artoria Arctosa Lycosa* Lycosinae (no Pardosinae)* Trochosa*

Ti, best log-likelihood score for data partition or difference relative to it; AU, P value from the approximately unbiased test; SE, standard error of AU; PF, P value for the breakdown of asymptotic theory (*significant difference in log-likelihood scores at 0.05).

N.P. Murphy et al. / Molecular Phylogenetics and Evolution 38 (2006) 583–602

cates sheet-webs to be an ancestral state in this group. It should be noted that the inclusion of Pirata as a sheetweb building taxon dramatically increased the difference between the forward and backward rates of transition across both the MP and Bayesian trees. Knowledge of the different prey capture strategies is unavailable for all the lycosid species examined, however, the results demonstrate that few taxa sharing any of these traits form well-supported monophyletic groups.

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3.2. Biogeography Due to the global nature of this study, and the limit to the number of exemplar taxa included, a broad-brush approach has been taken with respect to the designation of biogeographic regions (see Table 1). Mapping broad biogeographic zones (Table 1) onto the Bayesian phylogenetic tree (Fig. 5) shows that no monophyletic groupings of all species from a single biogeographic region are evident, however there is

Fig. 4. Maximum likelihood ancestral state reconstructions of sheet-web building employing (left tree) symmetrical (equal rates of loss and gain) and (right tree) asymmetrical (unequal rates of loss and gain) likelihood models. Trees shown are: (A) MP tree: Pirata coded as non sheet-web building; (B) MP tree: Pirata coded as sheet-web building; (C) Bayesian tree: Pirata coded as non sheet-web building; and (D) Bayesian tree: Pirata coded as sheet-web building. Pie diagrams (circles) at nodes indicate the likelihood of sheet-web building (white) or non sheet-web building (black).

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Fig. 4. (continued)

strong support for two separate Australian/New Zealand clades (clade A2 and clades C1/C2), whilst another Australian clade (clade E5, minus P. gallopavo) is also evident. Interestingly, within the putative Lycosinae clade (clade E) the Australian genus Venatrix (clade E2), does not form a clade with the other species of Australian species of Lycosinae (clade E5). 3.3. 28S rRNA phylogeny Preliminary analysis indicated a major difference in the topology of the 28S rRNA (Fig. 6) trees when com-

pared with the trees generated from the mitochondrial data (Figs. 2 and 3). Differences between genes, in particular between mitochondrial and nuclear genes, are not uncommon and in the majority of cases are due to gene tree/species tree differences, given that the gene tree topology has been recovered correctly. Closer examination of the 28S rRNA tree revealed some major discrepancies in the placement of taxa that cannot be explained by morphological convergences. The largest problems occurred in the splitting of a major clade (clade E, Fig. 6); in particular this split divides a number of genera (Pardosa and Rabidosa) and enforces a number of

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Table 5 Summary of maximum likelihood ancestral state reconstruction of sheet-web building Topology and codinga

Model

Lycosidae ancestral node statusb

No. of times WB lostc

No. of times WB re-evolvedc

X 21 d

MP (P-no)

Symm Asymm

Non web Web

0 5

4 1

4.873*

Symm Asymm

Non web Web

0 7

5 0

7.785*

Symm Asymm

Non web Non web

0 0

4 4

0.516

Symm Asymm

Non web Web

0 7

5 0

0.6905

MP P-yes Bayesian (P-no) Bayesian (P-yes)

Forward ratee

Backward ratee

9.969 · 10
3

5.894 · 10
4

7.782 · 10
3

9.847 · 10
9

0.946

0.310

1.426

0.043

a

Topology tested (P-no = Pirata coded as non-web building, P-yes = Pirata coded as web building). b The status of web building at the ancestral node of Lycosidae. c The number of times web building (WB) has either been lost or regained for each analyses. d Likelihood ratio test of the difference between the symmetrical and asymmetrical model of ancestral state reconstruction for each topology and coding (*significant difference in log likelihood). e The forward and backward rates as estimated by the asymmetrical model.

implausible relationships. Clearly related species in regard to somatic and genitalic characters that are separated by this split include the North American Rabidosa rabida and R. punctulata, the Australian species Lycosa castanea and L. bicolor (both part of the peculiar and clearly monophyletic ‘‘bicolor-group’’ (McKay, 1973, 1975)), and Pardosa californica and P. crassipalpis. The 28S rRNA tree also causes a major split in the genus Allotrochosina, with Allotrochosina schauinslandi being placed in clade E instead of as sister to Allotrochosina karri. Notably, these relationships are all strongly supported by the combined 12S rRNA/NADH 1 analysis. The characteristics of the 28S rRNA gene do not appear to be atypical (e.g., base homogeneity, saturation, secondary structure), and long-branch attraction does not appear to be an issue as the MP and Bayesian trees are similar (MP tree not shown). The most plausible explanation is the amplification of paralogous 28S rRNA gene copies. The presence of multiple copies in the 28S rRNA gene in lycosids is implied, not only by the substantial incongruence, but also by several species exhibiting uninterpretable sequence chromatograms. Samples displaying incomprehensible chromatograms followed a similar pattern whereby the first’ 100 bp (read from either primer) were easily readable and match closely all other sequences, then multiple peaks per site would be evident rendering the rest of the sequence uninterpretable. Closer examination of the phylogeny produced from the 28S rRNA sequences presents further evidence for the amplification of paralogous genes. As mentioned above, the major conflict between this tree and that produced from the combined analysis is the presence of an implausible split in clade E (putative Lycosinae), effectively forming two groups (Fig. 6, i and ii) in this clade. Both of these groups follow a similar pattern of relationships to each other and to that of the combined analysis, with basal Nearctic and Palaearctic species and more recently

derived Australian species from clade E4. The presence of A. schauinslandi (not a Lycosinae) within the group ii sequences and the relatively longer branch lengths within this group suggests that group i may contain orthologous 28S rRNA sequences, whilst group ii contains one or more paralogous 28S rRNA sequences. Due to the uncertainty of whether orthologous regions were being compared amongst 28S rRNA sequences, no further analysis of this gene was undertaken. 4. Discussion 4.1. Lycosid classification This study provides a novel phylogenetic framework for the Lycosidae at the subfamily level, since there is support for three clades that we consider represent unrecognised subfamilies; one clade including Aulonia and Hygrolycosa which were formerly placed in both Venoniinae (Dondale, 1986), and Piratinae (Zyuzin, 1993), a second clade represented by the traditionally lycosine genus Arctosa, and a third clade including Australasian genera such as Artoria, Anoteropsis and Tetralycosa (Fig. 4). Our analysis confirms Dondale’s (1986) hypothesis for the relationships of taxa traditionally included in the subfamilies Lycosinae, Pardosinae and Venoniinae (Fig. 1), but his classification of the Sosippinae as sister to all remaining Lycosidae is not supported. The Bayesian tree contains a weakly resolved trichotomy separating Evippinae, Piratinae/Venoniinae and Aulonia/Hygrolycosa that generally agrees with the subfamily classification suggested by Zyuzin (1993). The separate placement of Aulonia and Hygrolycosa, however, renders his concept of the Piratinae paraphyletic. Zyuzin (1993) recognised some deviation from his diagnosis of this subfamily in relation to the shape of the cephalothorax (Aulonia) and the arrangement of the eyes (Hygrolycosa).

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Fig. 5. Prey capture strategies (including web building) and biogeographic designations mapped onto the Bayesian phylogeny. Symbols for prey capture strategies are: web building; s vagrant; d permanent burrowing; burrow with trapdoor; burrow with turret; temporary burrow (broodcare); suspended tubular hideout. Clades circled designate those containing Australian and New Zealand species.

The Sosippinae and an unnamed Australasian clade (C1/C2) represent distinct subfamilies supported by ample morphological evidence. The genital structure in both clades, in particular of the male pedipalp is unique within the Lycosidae. Sosippus has three very distinctive tegular apophyses on the male pedipalp that do not appear to be homologous to any structure in other lycosids (e.g., Sierwald, 2000). The male pedipalp of representatives of the Australasian clade bears a unique basal apophysis on the embolus that had been argued previously to justify subfamily status for this group of spiders (e.g., Framenau, 2002; Framenau et al., in press; Vink, 2002). Current generic concepts within this Australasian clade are not supported by our molecular study, in particular for Artoria, which appears to be paraphyletic.

The arrangement of the three most derived clades questions the validity of the Pardosinae and intimates two additional subfamilies. Dondale (1986) included both Hippasa and Arctosa in the Lycosinae based on two synapomorphic characters of the male pedipalp that defined this subfamily. Subsequently, Zyuzin (1993) supported this concept, however, earlier molecular studies placed Arctosa as sister to all other Lycosinae and Pardosinae combined (Vink et al., 2002; Zehethofer and Sturmbauer, 1998). Based on the Bayesian phylogeny, we could accept this idea and include both genera in a broad concept of Lycosinae. Alternatively, given that both Hippasa and Arctosa are basal to the major lycosine clade in the Bayesian analysis and not at all affiliated with this clade or each other in the MP analysis, they could be regarded as representatives of distinct subfamilies.

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597

Fig. 6. Bayesian phylogenetic analysis of 28S rRNA data. Tree produced from 2 · 106 generations using the GTR+I+C model of sequence evolution. Designated clades (e.g., E4) represent groups found also in the combined 12S rRNA and NADH 1 trees. Group i and group ii represents the two groups of 28S rRNA sequences amplified in this study. Superscript 1 = genera split between two groups. Superscript 2 = not part of clade E in combined 12S rRNA and NADH 1 analyses.

A phylogenetic concept accepting Hippasa and Arctosa as members of separate subfamilies would see the re-validation of the Hippasinae. This subfamily was established by Petrunkevitch (1928), following SimonÕs (1898) ‘‘Hippaseae’’ to include the web building lycosid genera with long posterior spinnerets (Aglaoctenus, Hippasa, and Sosippus), but was later synonymised with the Lycosinae due to the similar pedipalp structure of Hippasa with other lycosines (Dondale, 1986). In contrast, an ‘‘arctosine’’ clade would represent a new subfamily. Pardosinae loses its validity as all species of Pardosa nest as a monophyletic clade within the traditional Lycosinae. This arrangement supports previous interpretations that did not accept the Pardosinae as a distinguishable subfamily (Lehtinen, 1967; Zyuzin, 1985). However, the pedipalp morphology of the traditional Pardosinae does not reflect Dondale’s (1986) concept of the Lycosinae since the median apophysis is generally directed apically in Pardosa and allied genera, such as Acantholycosa, Mongolicosa, Pyreno-

cosa, and Sibiricosa (e.g., Marusik et al., 2004). Given that both Arctosa and Hippasa, which exhibit the ‘‘lycosine pedipalp morphology,’’ fall outside the Lycosinae clade, the direction of the median apophysis of the male pedipalp does not appear to be a synapomorphy for the Lycosinae. More detailed studies are required to resolve the relationships within this clade. Using the morphology of male and female genitalia, the Lycosinae have traditionally been divided into two groups, the Lycosa-group and the Trochosa-group (sensu Dondale, 1986). Similarly, Zyuzin (1993) distinguished the tribes Lycosini and Trochosini, in addition to his Hippasini represented only by the web building genus Hippasa. Our phylogenies do not support any of these groups. In contrast, clades within the Lycosinae appear to reflect geographic regions, rather than existing recognised morphological parameters. For example, representatives that must be regarded as ‘‘typical’’ members of the Trochosini based on morphological grounds (pedipalp with simple triangular

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median apophysis carrying a ventrally directed hook, sickle-shaped terminal apophyses and emboli, inverted T-shaped epigyne) are not monophyletic but are found in a number of separate, sometimes well supported clades within the Lycosinae (clade E): Trochosa (clade E3), Hogna crispipes (clade E5), Geolycosa and Rabidosa (clade E6), and Varacosa. There are a number of classification issues at the generic level (e.g., Artoria, Alopecosa, and Arctosa); the most striking of these is the non-monophyly of Lycosa. None of the Lycosa species included in this study form a monophyletic clade with the type of the genus (L. tarantula), confirming the status of this genus as a ‘‘dumping ground’’ for unclassifiable Lycosinae. More effort is needed to properly classify these and other ‘‘Lycosa,’’ particularly those found outside of the biogeographic region of L. tarantula. Similarly, Trochosa is rendered non-monophyletic in this study, most notably by the Australian T. expolita, but also by the undescribed ‘‘New Genus 3 sp.’’ splitting T. ruricola and T. terricola. There are two possibilities with this latter association; either ‘‘New Genus 3’’ belongs to Trochosa, or either of T. ruricola and T. terricola does not. The two Trochosa are morphologically quite close, therefore it would appear that ‘‘New Genus 3’’ also belongs to this genus. Obviously, further examination of a greater number of specimens of ‘‘New Genus 3’’ is required to accurately place this taxon. The Australasian H. crispipes was recently transferred from Geolycosa based on the similarity of the male pedipalp with the type of this genus, H. radiata from the Palaearctic (Framenau et al., in press). Given that H. crispipes is nested well within a truly Australasian clade that also includes species with very different somatic and genital characters, a close affinity with H. radiata does not seem likely. A common theme occurring as a result of this study is that there appears to be an over reliance on genital morphology for the classification of lycosids. In wolf spiders, phylogenetic information derived purely from genital morphology alone is not reliable for inferring relationships and it appears that some genitalic characteristics may have evolved convergently (see also Brady and McKinley, 1994). 4.2. The ancestral state of sheet-webs in lycosidae Despite the inclusion of all web building genera, the results of maximum likelihood ancestral character state reconstructions of sheet-web building in lycosids are inconclusive. The existence of sheet-web building as an ancestral trait in lycosids appears to be contingent on two factors; an asymmetrical model favouring the loss of sheet-webs; and the interpretation that the suspended silken tube of Pirata is directly descended from sheet-web building. An evolutionary model favouring the loss of sheet-web building as opposed to the trait being ‘‘re-evolved’’ in lycosids appears to be a valid assumption. There is a general consensus that a loss of web building in spiders follows a trajectory from complex to reduced to absent (Agnarsson, 2004; Benjamin and Zschokke, 2003; Griswold et al., 1998) and the behav-

ioural complexity of web building in general suggests that once lost it would be unlikely to be regained in the same form. However, non sheet-web building lycosids have not lost the ability to produce silk as many species line burrows with silk and all lycosids produce a silk egg sac, suggesting that the genetic mechanisms for creating webs may still be present. There is no doubt that the web building lycosids do not form a monophyletic group, thus if web building has been regained from non sheet-web ancestry then this would be a remarkable example of convergent evolution. Lycosid sheet-webs generally follow a typical pattern incorporating a funnel as retreat, however the nature of sheetweb construction and the morphological traits associated with it (e.g., specialised long posterior spinnerets) need greater examination in order to ascertain their evolutionary origin in lycosids. Of great importance is the evolutionary origin of the reduced tube-shaped retreat of Pirata. The inclusion of Pirata as a sheet-web building taxon dramatically affects the interpretation of the ancestral state of this trait. Both the MP and Bayesian tree indicated that there was some independent re-evolution of sheet-web building when Pirata was coded as a non-sheet-web builder. If further molecular and morphological information were to resolve the Bayesian tree as the correct tree, then the web building status of Pirata carries an enormous weight, as the change of state of Pirata effectively determined whether web building was an ancestral trait for Lycosidae as a whole (Figs. 4B and C). Intuitively, the suspended tube of Pirata appears to be a radically reduced web building adaptation, although a much closer investigation of behavioural and biological mechanisms is needed to properly understand the origins of this unique construction. Burrowing behavior occurs principally within the traditional Lycosinae (clade E) (Fig. 5). However, the low resolution of this part of the phylogeny and the absence of biological data for many taxa do not allow an evolutionary interpretation of the excavation of permanent retreats. Other unrelated lycosids, in particular those adapted to live in inhospitable environments, also excavate permanent burrows, e.g., some salt lake dwelling species in Tetralycosa (e.g., Framenau et al., in press; Hudson and Adams, 1996). Therefore, similar to the acquisition of vagrant behavior (Zehethofer and Sturmbauer, 1998), burrowing must have evolved a number of times within the Lycosidae. Our phylogeny does not allow inference of the evolutionary transitions between sheet-web building to true burrowing. Burrowing may have evolved directly from the sheet-web, such as to place the sheet-web funnel into the ground, or indirectly via a vagrant stage. The presence of burrowing lycosids in clades of otherwise truly vagrant species (e.g., Tetralycosa in clade C) and the larger number of vagrant species with close affinities to sheet-web builders seems to support the second hypothesis. Our phylogeny confirms an interesting behavioural reversal on a higher systematic scale in spiders. Burrowing in mygalomorphs is generally regarded as the ancestral

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state in spiders (Coddington and Levi, 1991; Main, 1976). Subsequently, the ancestral araneomorph spiders have evolved suspended prey capture webs, for example, the elaborate orb-webs of araneoid spiders and the sheet-webs found in some lycosids (Coddington and Levi, 1991). Whilst some groups evolved from a sheet-web to a vagrant life style, burrowing behaviour was re-established in more derived lycosids. This includes some intriguing parallel behavioral adaptations such as the construction of turrets and trapdoors that can also be found in mygalomorphs (Main, 1976). 4.3. Biogeography Although this study makes an important contribution to understanding the relationships among lycosids, there is a disproportionate representation of the Australian taxa. Therefore, a detailed biogeographic analysis of the lycosids is not possible at this time, however, several interesting points have emerged. Based on our analysis there are a number of separate Australasian clades, suggesting at least four independent origins for the Australian lycosids. Within the more basal nodes, there are two distinct, strongly supported clades containing both Australian and New Zealand species, hinting at a Gondwanan origin for both of these groups. However, more work is required to examine the timing of diversification within these groups. Within the Lycosinae, most of the Australian taxa form a monophyletic clade (E5) with the exception of two species of Venatrix. The affinity of the South American P. gallopavo to this Australasian clade E5 also suggests a Gondwanan origin of this group. In contrast, Venatrix may have invaded Australia subsequently following its approach to Southeast Asia during the Tertiary (e.g., Hopper et al., 1996). Species of Venatrix are also found in the Philippines and Palau (Framenau, in press c). A diversification of the genus in Australia is mainly restricted to the east coast, with putative basal species restricted to Queensland but not the ancient bioregions in AustraliaÕs south west (Framenau and Vink, 2001). Given that wolf spiders occur in virtually every terrestrial habitat, and are found across every continent (except Antarctica), clearly many more taxa need to be included to decipher their origins and explain their current distributions. However, the northern hemisphere lycosids do not appear to form geographically based clades to the same extent as the Australian species, suggesting that dispersal has played a significant role in modern day northern hemisphere lycosid distributions. This is not surprising taking into account the strong tendency for ballooning in juvenile wolf spiders (e.g., Greenstone, 1982; Richter, 1970) and given the more recent connections between the Nearctic and Palaearctic and longer-term isolation of the Australasian fauna. The inclusion of more taxa will also allow the age of this spider group to be more closely examined. The virtual absence of a fossil records for the Lycosidae (e.g., Penney,

599

2001) ensures that accurate dating of wolf spiders is reliant on molecular clock data. In order to accurately calibrate a lycosid molecular clock, species or groups split by a known geological event need to be included in future phylogenetic studies. A molecular clock will then allow the timing of biogeographical lineages to be examined, such as whether the clades containing Australian and New Zealand species have been shaped pre- or post-Gondwanan separation. 4.4. 28s rRNA evolution in lycosidae The nuclear rRNA (nrRNA) gene cluster generally consists of a tandomly repeated array of three rRNA genes, 18S, 5.8S, and 28S RNA separated by internal transcribed spacers (ITS), and is thought to exist in multiple copies throughout most metazoan genomes. The number of copies of the nrRNA array can range from one in the protozoan Tetrahymena (Yao and Gall, 1977) to thousands in some plants (Appels et al., 1980). Despite the numerous copies, sequences of nrRNA genes are generally thought to become homogeneous due to the process of concerted evolution (Hillis and Dixon, 1991), a complicated process involving gene conversion (Hillis and Dixon, 1991) and unequal crossing-over (Coen and Dover, 1983). Although the homogenisation of nrRNA genes throughout the genome is generally accepted with confidence, there are a number of cases where departures from concerted evolution have occurred, producing intragenomic heterogeneous nrRNA copies. Intra-individual variation in nrRNA gene arrays has been reported across a wide range of taxa, not only in 28S rRNA but also in 18S rRNA and ITS-1 (e.g., Harris and Crandall, 2000; Krieger and Fuerst, 2002; Marquez et al., 2003; Vogler and Desalle, 1994). One of the more striking examples is that of lake sturgeons that have a minimum of 17 different 18S rRNA gene sequences per individual, with divergence levels ranging from 0.3 to 4% and the most divergent sequence being almost identical to 18S rRNA sequences from a different genus (Krieger and Fuerst, 2002). Therefore, the occurrence of copies of 28S rRNA with different sequences within the Lycosidae, as suggested by the 28S rRNA phylogenetic tree, is not a novel phenomenon. The existence of multiple paralogous copies of nrRNA genes has serious ramifications for their usefulness for reconstructing phylogenetic relationships. Given that intragenomic nrRNA variation has been found across a broad spectrum of taxa, from plants to vertebrates, any study using these genes (or the internal transcribed spacers) should be wary about their ability to accurately trace evolutionary history. Measures such as cloning and a thorough examination of the molecular characteristics should be implemented in order to ascertain that orthologous genes of interest are being compared. Considering that the nrRNA genes exist as a multiple gene array, the existence of paralogous copies of 28S rRNA also has implications for the use of 18S rRNA for higher level phylogenetics and also ITS for lower level phylogenetic population studies.

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More work is required to confirm that paralogous 28S rRNA genes have indeed been amplified in lycosids. Further work should involve sequencing of multiple clones from individuals, identifying the number of and divergence among paralogous genes, identifying the extent of paralogous 28S rRNA genes in lycosids and more distant relatives, and determining whether paralogous copies are functional or pseudogenes. Acknowledgments We thank Gail Stratton, Sam Marshall, Maggie Hodge, ´ lvares, DaCor Vink, Phil Sirvid, Paolo Tongiorgi, E´der A quin Li, Joseph Koh, and Ansie Dippenaar-Shoeman for providing fresh wolf spider material from different parts of the world. The study would not have been possible without this invaluable support. In particular, we are grateful to Jung-Sun Yoo for providing material and life history information on Korean wolf spiders, and Frederick Hendrickx for his expert advice during a field trip collecting lycosids after the International Congress of Arachnology in 2004. Many more individuals assisted VWF and MSH during collection trips for this study, in particular Melissa, Sharon and Jeff Thomas, Terry Oroumis, Eileen Hebets, Greta Binford, Julianne Waldock, Robert Raven, Barbara Baehr, and Randolf Manderbach. Thank you to Gonzalo Giribet and two anonymous reviewers for comments on improving this paper. This study was supported by grants from the Australian Biological Resource Study to MSH and ADA, and the Australian Research Council and The University of Adelaide to ADA and SCD. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.ympev.2005.09.004. References Alderweireldt, M., Joque´, R., 1993. A redescription of Tricassa deserticola Simon, 1910, representing the Tricassinae, a new subfamily of wolf spiders (Araneae, Lycosidae). Belg. J. Zool. 123, 27–38. Agnarsson, I., 2004. Morphological phylogeny of cobweb spiders and their relatives (Araneae, Araneoidea, Theridiidae). Zool. J. Linn. Soc. 141, 447–626. Appels, R., Gerlach, W.L., Dennis, E.S., Swift, H., Peacock, W.J., 1980. Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals. Chromosoma 78, 293–311. Benjamin, S.P., Zschokke, S., 2003. Webs of theridiid spiders: construction, structure and evolution. Biol. J. Linn. Soc. 78, 293–306. Brady, A.R., McKinley, K.S., 1994. Nearctic species of the wolf spider genus Rabidosa (Araneae, Lycosidae). J. Arachnol. 22, 138–160. Coddington, J.A., Levi, H.W., 1991. Systematics and evolution of spiders (Araneae). Ann. Rev. Ecol. Syst. 22, 565–592. Coen, E.S., Dover, G.A., 1983. Unequal exchanges and the co-evolution of X and Y rDNA arrays in D. melanogaster. Cell 73, 849–855. Crawford, R.L., Sugg, P.M., Edwards, J.S., 1995. Spider arrival and primary establishment on terrain depopulated by volcanic eruption at Mount St. Helens Washington. Am. Midl. Nat. 133, 60–75.

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