Next-generation museum genomics: Phylogenetic relationships among palpimanoid spiders using sequence capture techniques (Araneae: Palpimanoidea)
Graphical abstract
Introduction
Spiders are important predators in terrestrial ecosystems, and with over 47,000 described species (World Spider Catalog, 2018) they are notable in terms of global ubiquity, diversity in behavior and ecology, and medical importance. Spiders rank as the seventh most diverse arthropod order, and total species diversity will likely triple as more species are discovered and named (Coddington and Levi, 1991). However, until very recently research in spider phylogenetics was hampered by a scarcity of genetic markers with few (e.g., six or less) available for phylogenetic inference using traditional Sanger sequencing approaches (e.g., Wheeler et al., 2016, Dimitrov et al., 2017). This small set of genetic markers has been mostly unsuccessful at resolving deeper relationships at the interfamilial level (Wheeler et al., 2016). Phylogenetic resolution has been further confounded by insufficient taxon sampling, which is complicated by the difficulty field biologists face in gathering genetic samples for taxonomically diverse organisms that are rare and elusive, or that live in remote, inaccessible areas. Natural history museums, with the goal of documenting the diversity of life, provide an additional source of genetic resources as they have amassed large collections over a long period of time, which include rare or extinct species and specimens from remote areas of the world. Historical museum specimens are invaluable for morphological and taxonomic research, but typically their DNA is degraded making traditional sequencing techniques difficult to impossible for many specimens (Wandeler et al., 2007).
Recent advances in Next-Generation Sequencing (NGS), specifically target capture, makes use of short fragment sizes typical of degraded DNA, opening up the possibility of gathering genomic data for phylogenetic analysis from existing museum specimens (Jones and Good, 2016). Target capture techniques have been successfully performed on dried (i.e., pinned insects or dried skins) museum specimens over 100 years old for birds (McCormack et al., 2016), mammals (Bi et al., 2013, Guschanski et al., 2013, Bailey et al., 2016), and insects (Blaimer et al., 2016). Genetic fragments have even been recovered from fluid preserved museum specimens (Ruane and Austin, 2017), including a lizard that was collected 145 years ago (McGuire et al., 2018). Target capture techniques have also been used to infer arachnid phylogeny (Starrett et al., 2017), including using some museum material (Hamilton et al., 2016, Hedin et al., 2018).
Arachnid specimens in museum collections are typically stored in 70–80% ethanol and at these concentrations at room temperature DNA degrades by oxidation and hydrolysis (Quicke et al., 1999, Vink et al., 2005). For such specimens with fragmented DNA, NGS target capture techniques are preferred to traditional techniques, such as Sanger sequencing of PCR amplified specific genes or gene fragments, because large amounts of data can be generated rapidly and at relatively low cost. Target capture techniques can gather data from numerous loci throughout the genome. This approach can dramatically advance research in spider systematics in two ways: first, the ability to sequence hundreds of regions throughout the genome may resolve deep interfamilial relationships more accurately; second, legacy and archival museum collections offer cheap and efficient ways to include rare lineages that currently are difficult to collect.
Palpimanoidea is an ancient spider lineage that has evolved some remarkable morphologies in the carapace and chelicerae (functionally equivalent to jaws or mandibles) compared to other spiders (Wood et al., 2012, Wood et al., 2016) (Fig. 1). Their strange morphology complements their unusual and highly specialized predatory behaviors: for example, mecysmaucheniids use ballistic trap-jaw strikes (Wood et al., 2016), and archaeids are specialists that attack other spiders at a distance with their long chelicerae (Wood et al., 2012). As a possible sister clade (Wood et al., 2012, Wheeler et al., 2016, Fernández et al., 2018) to the more modern, derived spiders, the Entelegynae, palpimanoids are an important group for understanding spider evolution. Palpimanoids are paleoendemics, and are currently mostly confined to the Southern Hemisphere, although there is an extensive fossil record from the Northern Hemisphere, with distribution patterns of some extant lineages likely relating to Gondwanan vicariance (Wood et al., 2013), . Phylogenetic analysis using both morphological data and four genetic markers strongly supports a monophyletic Palpimanoidea that consists of five families (Wood et al., 2012, Wood et al., 2013): Archaeidae, Huttoniidae, Mecysmaucheniidae, Palpimanidae, and Stenochilidae. Another analysis using only molecular data from six genetic markers (not all markers were successfully sequenced for all terminals) suggested a paraphyletic Palpimanoidea, although with weak branch support (Wheeler et al., 2016), but a recent phylogeny based on transcriptomic data strongly supported the monophyly of Palpimanoidea (Fernández et al., 2018). Phylogenetic relationships among palpimanoid families were weakly supported in these previous studies, corroborating that the standard genetic markers used in spider studies are not adequate for resolving deep relationships (Agnarsson et al., 2013), and although transcriptomic data helped confirm the monophyly of Palpimanoidea it did not help resolve internal nodes. Palpimanoids occur in many remote areas of the world that are hard to access, so that thorough taxon sampling for the group is difficult. However, there are many representatives of palpimanoids in museum collections, mostly collected over the last two decades, stored in 75–95% ethanol. Because palpimanoids are ancient spiders that were once more widespread, because of their phylogenetic placement as possible sister to a major clade of spider (Entelegynae), and because of their unusual predatory behaviors and morphology, they are an excellent group to study in order to examine phylogeny, trait evolution, biogeography, and diversification patterns in relictual lineages.
This study uses such museum specimens, combined with recent target capture sequencing techniques, to examine phylogenetic relationships among palpimanoid spiders. We use the recently designed Ultra-Conservative-Elements (UCE) Arachnid bait-set (Faircloth, 2017, Starrett et al., 2017) and an exon-based bait-set that was designed from spider transcriptomes. The UCE method makes use of short, highly conserved DNA sequences that span major lineages (Siepel et al., 2005, Faircloth et al., 2012). Hundreds of independent UCE loci can be extracted from diverse taxa, with the regions flanking the UCE cores providing signal for phylogenetic inference at multiple taxonomic scales. Sequence variation in flanking regions increases with distance from the UCE core (Faircloth et al., 2012). This increasing variation makes UCE useful for reconstructing phylogenetic relationships across a variety of timescales, from recently diverged populations to distantly related groups (Faircloth et al., 2012, McCormack et al., 2012, Smith et al., 2013). An exon-based bait-set was designed for this study to complement the UCE data by providing additional markers for protein-coding regions within the genome that do not overlap with the UCE data. We used these two bait-sets to gather molecular sequences of lineages that spanned the modern spiders (Araneomorphae), but that focused on the palpimanoids. Fourteen museum specimens that were collected over the last two decades and stored in 75% ethanol were included in this study. We also perform an ancestral character state reconstruction to examine evolution of the tubular carapace in palpimanoids.
Section snippets
Taxa selection and DNA extraction
To examine relationships among palpimanoids we included 34 terminals representing the five palpimanoid families (number of genera included/total number of genera): Archaeidae (5/5), Huttoniidae (1/1), Mecysmaucheniidae (5/7), Palpimanidae (11/18), Stenochilidae (1/2). We included 14 additional non-palpimanoid Araneomorphae taxa representing 13 families that represent major clades within the Araneomorphae: Hypochilidae, Filistatidae, Pholcidae, Scytodidae, Segestriidae, Austrochilidae, Eresidae,
Results
The majority of DNA extractions were from specimens that had been collected directly into 95% ethanol, and upon completion of the field expedition, were stored at below freezing. Fourteen extractions were from museum specimens in 70–75% ethanol that were stored at room temperature (5 from the year 2000, 1 from 2003, 1 from 2005, 1 from 2010, and 5 from 2012). An additional seven museum specimens in 70–75% ethanol were extracted but were not sequenced for the phylogenetic analysis. Of these
Discussion
Palpimanoidea classification. Based on a total evidence analysis of morphological data and four molecular markers Palpimanoidea was restricted to the following five families (Wood et al., 2012): Archaeidae, Huttoniidae, Mecysmaucheniidae, Palpimanidae, and Stenochilidae. This study also placed Palpimanoidea as sister to the Entelegynae and recovered the well-supported relationships of (Archaeidae + Stenochilidae, both long branches) and (Palpimanidae + Huttoniidae) (other relationships among
Acknowledgements
Funding for this study came from an Exploratory Award (year 2016) from the Global Genome Initiative at the Smithsonian Institution National Museum of Natural History. NS received support by the Danish National Research Foundation grant DNRF96 to the Center for Macroecology, Evolution and Climate.
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