Pholcid spider molecular systematics revisited, with new insights into the biogeography and the evolution of the group

We analysed seven genetic markers sampled from 165 pholcids and 34 outgroups in order to test and improve the recently revised classification of the family. Our results are based on the largest and most comprehensive set of molecular data so far to study pholcid relationships. The data were analysed using parsimony, maximum-likelihood and Bayesian methods for phylogenetic reconstruction. We show that in several previously problematic cases molecular and morphological data are converging towards a single hypothesis. This is also the first study that explicitly addresses the age of pholcid diversification and intends to shed light on the factors that have shaped species diversity and distributions. Results from relaxed uncorrelated lognormal clock analyses suggest that the family is much older than revealed by the fossil record alone. The first pholcids appeared and diversified in the early Mesozoic about 207 Ma ago (185–228 Ma) before the breakup of the supercontinent Pangea. Vicariance events coupled with niche conservatism seem to have played an important role in setting distributional patterns of pholcids. Finally, our data provide further support for multiple convergent shifts in microhabitat preferences in several pholcid lineages. Our findings suggest that both adaptive and non-adaptive speciation may have played an important role in the diversification of pholcid lineages. The Willi Hennig Society 2012 Pholcids (Araneae: Pholcidae) have been singled out (e.g. Huber, 2000, 2011a; Bruvo-Madaric et al., 2005) for their often high abundance in tropical forests (e.g. Huber, 2000, 2003a), unusually high number of synanthropic species (e.g. Huber, 2000, 2011a), and the extensive knowledge that exists on their sexual behaviour and on the functional morphology of their copulatory organs (e.g. Huber, 1994, 1995, 1996a,b, 2002; Uhl et al., 1995; Uhl, 1998). Commonly known as daddy-longlegs spiders, they may be among the arthropods with the longest legs relative to their body size. Some synanthropic species such as Pholcus phalangioides have probably shared a roof with all of us on at least one occasion, yet many aspects of the biology and diversity of pholcids remain unknown. With more than 1200 described species (Huber, 2011a; Platnick, 2012) pholcids are among the most species-rich spider families. Generic revisions, however, often double the number of described taxa in their focal genera, suggesting that the actual number of species is much higher (e.g. Huber, 2005a, 2011a). Since the first cladistic analysis of Pholcidae (Huber, 2000), there have been numerous phylogenetic studies focusing on this group of haplogyne spiders (e.g. Huber, 2000, 2003a, 2005a,b, 2011a; Bruvo-Madaric et al., 2005; Astrin et al., 2007; Dimitrov and Ribera, 2007; Huber and Astrin, 2009) some of which primarily rely on molecular data (e.g. BruvoMadaric et al., 2005; Astrin et al., 2007; Dimitrov et al., 2008; Huber and Astrin, 2009; Huber et al., 2010). The evidence presented in these studies has resulted in a recent revision of the classification of the family (Huber, 2011b). Despite a few earlier attempts (Petrunkevitch, 1928; Mello-Leitão, 1946) to revise the original classification proposed by Simon (1893), his system has been *Corresponding author. E-mail address: [email protected] The Willi Hennig Society 2012 Cladistics 29 (2013) 132–146 Cladistics 10.1111/j.1096-0031.2012.00419.x used for more than a century with only a few amendments. Although a big step forward, Huber s revised classification was explicitly proposed as ‘‘... a working hypothesis ...’’ (Huber, 2011b, p. 221) as there are still numerous genera that have not been studied in a phylogenetic context or because cladistic analyses based on morphological data alone have not been able to unambiguously resolve their relationships. There are also several instances where generic limits continue to be contentious (e.g. Spermophora, Leptopholcus, Belisana, etc.) and several nodes within and among subfamilies remain ambiguous. The lack of a rigorous phylogenetic hypothesis has largely prevented higher level biogeographical and diversification research in pholcids. As a result, just a couple of island radiations have been studied in some detail (Dimitrov et al., 2008; Huber et al., 2010). Despite some fossils, most of which can be placed in extant genera (e.g. Huber and Wunderlich, 2006; Penney, 2006; Wunderlich, 2008a; Dunlop et al., 2012), nothing is known about the origins of the family and its early history except that Pholcidae may be much older than the oldest known fossils (see comments on haplogyne spiders in Ayoub et al., 2007). A robust phylogenetic framework is therefore essential to test the limits and relationships of most pholcid subfamilies and many genera. To provide and improve such a framework, we analyse seven molecular markers sequenced from a large sample of pholcid species representing all major lineages within the family and 36 of the 85 extant genera (Platnick, 2012). In addition, we used information from the fossil record to calibrate the pholcid family tree and estimate the age of the main lineages within the family. The availability of a dated phylogeny also allows us to contextualize evolutionary events and address biogeographical and macroevolutionary questions. Materials and methods Taxon sampling and gene selection The in-group taxon sampling focused on maximizing the representation of pholcid lineages at the generic level. In a few cases in which monophyly of genera was uncertain, several species representing potentially nonmonophyletic groups were included. Particular effort has been made to include distinct Pholcus lineages, by far the largest pholcid genus, and closely related taxa in order to test the monophyly of Pholcus and its composition. Numerous trips to collect DNA-ready material of different pholcid lineages have been carried out as part of the present research. We have specifically targeted undersampled lineages and regions. In a few cases (e.g. Carapoia, Spermophora) additional effort to achieve high coverage at the species level has been made to address specific biogeography and ⁄or conservation questions. Outgroup taxa from all major spider lineages were added, with emphasis on haplogynes. Denser outgroup sampling has been shown to increase accuracy of the results (Dimitrov et al., 2012). In addition, some of the outgroups (e.g. Araneidae) were selected to facilitate the use of additional fossil calibration points for the molecular dating analyses. We have targeted seven molecular markers that represent both nuclear and mitochondrial ribosomal and protein-coding genes: 12S rRNA, 28S rRNA, 18S rRNA, 16S rRNA, cytochrome c oxidase subunit I, histone H3 and wingless. All of them have successfully been used in previous studies of phylogenetic relationships at different taxonomic levels in pholcids and other spider groups (e.g. Bruvo-Madaric et al., 2005; Astrin et al., 2006; Dimitrov et al., 2008, 2012; Álvarez-Padilla et al., 2009; Arnedo et al., 2009; Dimitrov and Hormiga, 2011). The bulk of character data used in the present study were newly generated as a result of current sampling efforts. In addition, both taxon and character sampling were further expanded by including additional sequence data from GenBank. The complete list of taxa and the sequence accession numbers are given in Supporting Information, Table S1. DNA extraction, amplification and sequencing Total genomic DNA was extracted from the prosoma (usually in females), opisthosoma (usually in males) or single whole individuals (in small specimens, for example juveniles) using the BioSprint96 magnetic bead extractor and corresponding extraction kits by Qiagen (Hilden, Germany). Extracted DNA is deposited at the Biobank of the ZFMK (Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany). PCR was carried out in total reaction mixes of 20 lL, including 1.2–2 lL of undiluted DNA template, 1.6 lL of each primer (10 pmol ⁄lL), 2 lL of ‘‘Q-Solution’’ and 9.5 lL of ‘‘Multiplex PCR Master Mix’’, containing hot start Taq DNA polymerase and buffers. The latter components are available in the ‘‘Multiplex PCR’’ kit from Qiagen. In spite of the kit s name, the PCR reactions were not multiplexed, but were run individually. The kit helped in minimizing PCR optimization. A list of primers used for amplification and sequencing is given in Table S2. Thermal cycling was performed on a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA), using the same ‘‘touch-down’’ PCR protocol for all genes: hot start Taq activation: 15 min at 95 C; first cycle set (15 repeats): 35 s denaturation at 94 C, 90 s annealing at 60 C ()1 C per cycle) and 90 s extension at 72 C; second cycle set (25 repeats): 35 s denaturation at 94 C, 90 s annealing at 50 C, and 90 s extension at 72 C. After enzymatic 133 D. Dimitrov et al. / Cladistics 29 (2013) 132–146