DOI: 10.5061/dryad.6hdr7sr2w
title: Micro-endemic species of snails and amphipods show population genetic structure across very small geographic ranges

description: Understanding variation in population genetic structure, even across small
 distances and for species with extremely limited ranges, is critical for
 conservation planning and the development of effective management
 strategies for imperiled species. Organisms that occupy the same
 geographic extent can maintain different population structures, ranging
 from highly diverged to panmictic. Such differences can result from
 differences in biological characteristics such as dispersal ability or
 demographic history. We used microsatellite loci to evaluate population
 genetic structure and variation of four desert spring invertebrates having
 high to low dispersal ability: the lung snail Physa acuta, two species of
 gilled snails (Juturnia kosteri and Pyrgulopsis roswellensis; family
 Hydrobiidae) and the amphipod Gammarus desperatus. The study location
 represents entire species ranges for the micro-endemic hydrobiids and G.
 desperatus, while P. acuta is ubiquitous throughout much of North America.
 We found little evidence of significant population genetic structure for
 P. acuta and J. kosteri, but much more for P. roswellensis and G.
 desperatus. Our results demonstrate differences in habitat preference
 and/or dispersal ability between the species. While significant
 isolation-by-distance was detected in the two hydrobiids, dispersal is
 likely more limited in P. roswellensis than J. kosteri. This information
 provides insight into how gene flow shapes varying population genetic
 structure between species across small spatial scales (&lt;100 km2).
 Most importantly, our results suggest that conservation agencies should
 not consider these microendemic species to be composed of single
 populations, but rather, that management plans for such species should
 account for population genetic variation across the species’ ranges.
description: From
 2014-2016, we sampled 16 sites throughout the refuge for all four species,
 attempting to obtain 24 individuals of each species per site; however,
 presence and abundance varied among species and across sites. The sampled
 area includes the entire ranges of the hydrobiid snails and amphipods
 (Fig. 1). Each species was sampled throughout the refuge within a single
 sampling effort (i.e. all <em>Gammarus</em> and
 <em>Physa</em> specimens were collected in 2015, all
 hydrobiids in 2014) with the exception of Unit 5 sites (U5A, U5B, U5C)
 which were sampled in 2016. We sorted individuals while in the field;
 <em>P. roswellensis</em> and <em>J. kosteri,</em>
 were distinguished by operculum color using a hand lens or dissecting
 microscope. All specimens were stored in 95% ethanol until DNA was
 extracted using DNeasy kits (Qiagen, Inc.).  We used published
 microsatellite primers and methods to genotype 11 loci for
 <em>Physa</em>
 (Ansah, Inoue, Lang & Berg, 2014), 16 loci for
 <em>J.</em> <em>kosteri</em> and 20 loci for
 <em>P. roswellensis </em>(both described in Holste, Inoue,
 Lang & Berg, 2016) to assess genetic diversity of these
 species. For
 <em>G. desperatus</em>, we developed novel microsatellite
 primers using the Plant-Microbe Genomics Facility at Ohio State University
 to perform <em>de novo</em> pyrosequencing in
 1/4<sup>th</sup> of a picotiterplate using a Roche 454 FLX
 Titanium Genome Sequencer which produced 55,692 reads. From sequencing
 outputs, we used MSATCOMMANDER v1.0.8 (Faircloth, 2008) to identify
 putative microsatellite loci and PRIMER3 (Rozen & Skaletsky, 2000)
 to design primers on flanking regions. Tail sequences of M13R or CAG were
 added to the 5’-end of either the forward or reverse primer. We identified
 202 putative microsatellite markers and we were able to design primers for
 120 microsatellite loci. Trinucleotides were the most abundant repeat type
 (46.7% of all loci), followed by dinucleotides (42.5%), and
 tetranucleotides (10.8%). Twenty loci (Table S1, Supplementary
 Information) were consistently amplified and used for population-level
 analyses. These loci were amplified in 10 µL polymerase chain reactions
 (PCR) using Gotaq Master Mix (Promega Corporation), 0.2 µM of universal
 fluorescently labeled primer (M13R or CAG) including the non-tailed
 primer, 0.04 µM of tailed primer, and 10 ng of DNA. For each locus, we
 identified optimum annealing temperature using the following PCR
 conditions: initial denaturing at 95°C for 2 min; followed by 35 cycles at
 95°C for 30 s, annealing at 48-62°C (or 55-65°C as a higher temperature
 gradient, if needed) for 45s, extension at 72°C for 1 min; and final
 extension at 72°C for 30 min. For all species examined, fragment analyses were performed
 on an ABI Genetic Analyzer with LIZ600 size standard (Applied Biosystems,
 Inc.). We used PEAKSCANNER v1.0 (Applied Biosystems, Inc.) to score
 alleles and TANDEM v1.07 (Matschiner & Salzburger, 2009) to assign
 integers to DNA fragment sizes.