DOI: 10.5061/dryad.c866t1g62
title: ddRAD-seq alignment data for Unionid mussels

description: North American watersheds contain the world’s highest diversity of
 freshwater mussels (Unionoida), and up to 40 species can co-occur in a
 single riffle. They collectively exhibit little evidence for ecological
 differentiation during the long-lived, benthic phase of their life
 cycle.  In contrast, their brief parasitic larval phase
 involves the infection of a wide diversity of fish hosts. Gravid female
 mussels have evolved multiple methods for increasing the probability of
 infecting a host fish. Some species use a passive broadcast strategy:
 placing high numbers of larvae in the water column and relying on chance
 encounters with potential hosts for infection. Most species have a
 proactive strategy that entails the use of prey-mimetic lures to change
 the behavior of the hosts, i.e., eliciting a feeding
 response through which they become infected. Gravid females collectively
 produce two main lure types: a mantle tissue lure (on the female’s body)
 and a brood lure, containing infective larvae, that she releases into the
 external environment. In this study, we used a phylogenomic approach
 (ddRAD-seq) to place the diversity of infection strategies used by 54
 North American lampsiline mussels into an evolutionary context. Ancestral
 state reconstruction recovered evidence for the early evolution of mantle
 lures in this clade, with brood lures and broadcast infection strategies
 both being independently derived twice. The most common infection
 strategy, occurring in our largest ingroup clade, is a mixed one in which
 mimetic mantle lures are apparently the predominant
 infection mechanism, but gravid females also release simple,
 non-mimetic brood lures at the end of the season. This mixed
 infection strategy clade shows some evidence of an increase in
 diversification rate and most members use bass
 (Micropterus & Ambloplites spp.) as
 their predominant fish hosts. Broad linkage between infection
 strategies and predominant fish host genera is also seen in other
 lampsiline clades: worm-like mantle lures of Toxolasma
 spp. with sunfish (Lepomis spp.); insect larvae-like brood lures
 (Ptychobranchus spp.), or mantle lures
 (Medionidus spp., Obivariaspp.), or mantle
 lures combined with host capture (Epioblasma spp.) with a
 spectrum of darter (Etheostoma & Percinaspp.)
 and sculpin (Cottus spp.) hosts, and tethered brood lures
 (Hamiota spp.) with bass
 (Micropterus & Ambloplites spp.). Our
 phylogenetic results confirm that discrete lampsiline
 mussel clades exhibit considerable specialization in the primary fish host
 clades their larvae parasitize, and in the host infection strategies they
 employ to do so. They are also consistent with
 the hypothesis that larval resource partitioning of fish
 hosts is an important factor in maintaining species
 diversity in mussel assemblages.  We conclude that
 taking their larval ecology and host-infection mechanisms into account,
 lampsiline mussels may be legitimately viewed as a cryptic adaptive
 radiation.
description: Unionid tissue samples were collected from various museums and
 the field. Genomic DNA was extracted from tissue samples using the E.Z.N.A. Mollusk DNA kit (Omega Bio-Tek, Norcross, GA) according to the manufacturer’s instructions and then stored at -80°C. The quality and quantity of DNA extractions were assessed using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA) and ddRADseq libraries were prepared following the protocols of Peterson et al. (2012). We then used 200 ng of DNA for each library prep. This involved digestion with Eco-RI-HF and MseI (New England Biolabs, Ipswich, MA) restriction enzymes, followed by isolating 294-394 bp fragments using a Pippen Prep (Sage Science, Beverly, MA) following the manufacturer’s instructions. Prepared ddRADseq libraries then were submitted to the University of Michigan’s DNA sequencing core and run in three different lanes using 150 bp paired-end sequencing on an Illumina HiSeq 2500. The alignment-clustering algorithm in ipyrad v.0.7.17 (Eaton, 2014; Eaton & Overcast, 2020) was used to identify homologous ddRADseq tags. Ipyrad is capable of detecting insertions and deletions among homologous loci which increases the number of loci recovered at deeper evolutionary scales compared to alternative methods of genomic clustering (Eaton, 2014). Demultiplexing was performed by sorting sequences by barcode, allowing for zero barcode mismatches (parameter 15 setting 0) and a maximum of five low-quality bases (parameter 9). Restriction sites, barcodes, and Illumina adapters were trimmed from the raw sequence reads (parameter 16 setting 2) and bases with low-quality scores (Phred-score &lt;20, parameter 10 setting 33) were replaced with an N designation. Sequences were discarded if they contained more than 5 N’s (parameter 19). Reads were clustered and aligned within each sample at two different similarity thresholds, 85 and 90% and clusters with a depth &lt; 6 were discarded (parameters 11 and 12). We also varied the number of individuals required to share a locus from ~25% (N = 27) to ~46% (N = 50). Ipyrad output files were used for further downstream analyses.