Research Article |
Corresponding author: Russell Minton ( minton001@gannon.edu ) Academic editor: Matthias Glaubrecht
© 2017 Russell Minton, Bethany McGregor, David Hayes, Christopher Paight, Kentaro Inoue.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Minton RL, McGregor BL, Hayes DM, Paight C, Inoue K (2017) Genetic structuring in the Pyramid Elimia, Elimia potosiensis (Gastropoda: Pleuroceridae), with implications for pleurocerid conservation. Zoosystematics and Evolution 93(2): 437-449. https://doi.org/10.3897/zse.93.14856
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The Interior Highlands, in southern North America, possesses a distinct fauna with numerous endemic species. Many freshwater taxa from this area exhibit genetic structuring consistent with biogeography, but this notion has not been explored in freshwater snails. Using mitochondrial 16S DNA sequences and ISSRs, we aimed to examine genetic structuring in the Pyramid Elimia, Elimia potosiensis, at various geographic scales. On a broad scale, maximum likelihood and network analyses of 16S data revealed a high diversity of mitotypes lacking biogeographic patterns across the range of E. potosiensis. On smaller geographic scales, ISSRs revealed significant population structure, even over the distance of a few hundred meters. Unlike other freshwater mollusks like mussels, E. potosiensis showed no evolutionary patterns relating to biogeography. The species does show population-level genetic structure, which may have implications in conservation efforts.
Freshwater snails, gastropods, Interior Highlands, phylogeny, population genetics
The Interior Highlands, separated by the Arkansas River Valley into the Ozark and Ouachita regions, is a major geographical feature of southern North America and includes the Ozark and Ouachita Mountains (
With nearly 170 recognized species (
We utilized two forms of genetic data for our study, mitochondrial DNA sequences at the species level and inter-simple sequence repeats (ISSRs) at the population level. The bulk of freshwater gastropod genetic studies employ one of two mitochondrial DNA fragments derived from either the 16S or cytochrome oxidase c subunit I gene. While these sequences may show utility in population and species-level studies, results are mixed in freshwater cerithioideans including pleurocerids (e.g.
We collected live E. potosiensis from six river drainages throughout the Ozark and Ouachita highlands (Figure
Taxon | Accession | Locality | Identifier | Reference |
---|---|---|---|---|
Elimia alabamensis | U73761 | Lydeard et al. 1997 | ||
E. caelatura | AF100988 | Holzangel and Lydeard 2000 | ||
E. catenaria | FJ471493 | Strong and Kohler 2009 | ||
E. clenchi | FJ471492 | Strong and Kohler 2009 | ||
E. comalensis | KU052563 | Salado Creek at Interstate 35, Salado, Bell County, TX | new | |
E. crenatella | U73762 | Lydeard et al. 1997 | ||
E. cylindracea | U73765 | Lydeard et al. 1997 | ||
E. doolyensis | DQ311118 | Lee et al. 2006 | ||
E. haysiana | U73763 | Lydeard et al. 1997 | ||
E. hydei | U73764 | Lydeard et al. 1997 | ||
E. interrupta | AY010521 | Lydeard et al. 2002 | ||
E. laqueata | KU052565 | Green River, KY | new | |
E. livescens 1 | DQ311116 | Lee et al. 2006 | ||
E. livescens 2 | KU052564 | French Creek, PA | new | |
E. melanoides | AF540003 | Minton et al. 2003 | ||
E. olivula | U73766 | Lydeard et al. 1997 | ||
E. potosiensis | KT988910 | Alum Fork Saline River, AR 34.67310N, 92.79920W | 2 | new |
KT988911 | ||||
KT988965 | Illinois River, AR 36.10320N, 94.34500W | 14 | new | |
KT988932 | War Eagle Creek, AR 36.12100N, 93.69340W | 45 | new | |
KT988962 | ||||
KT988940 | Otter Creek, AR 36.22380N, 92.25190W | 72 | new | |
KT988923 | Blanchard Springs, AR 35.95680N, 92.13960W | 111 | new | |
KT988950 | ||||
KT988951 | ||||
KT988956 | Spring River, AR 36.31530N, 91.49080W | 123 | new | |
KT988957 | ||||
KT988964 | Mill Creek Spring, AR 36.05720N, 91.60890W | 149 | new | |
KT988916 | Mammoth Spring, AR 36.49580N, 91.53320W | 154-1 | new | |
KT988922 | ||||
KT988943 | ||||
KT988944 | ||||
KT988945 | ||||
KT988933 | Warm Fork Spring, AR 36.49580N, 91.53320W | 154-2 | new | |
KT988934 | ||||
KT988929 | ||||
KT988954 | Mulberry Fork Little Red River, AR 35.74210N, 92.33380W | 165 | new | |
KT988955 | ||||
KT988939 | Mulberry River, AR 35.62275N, 93.91023W | 186 | new | |
KT988958 | Cossatot River, AR 34.37950N, 94.23680W | 221 | new | |
KT988967 | ||||
KT988963 | Tributary to South Fork Ouachita River, AR 34.51870N, 93.75940W | 260 | new | |
KT998966 | Saline River, AR 34.58710N, 92.60480W | 270 | new | |
KT988968 | ||||
KT988953 | Big Creek, MO 37.29375N, 90.62775W | M2 | new | |
KT988927 | Big River, MO 37.81483N, 90.77049W | M3 | new | |
KT988941 | Brenton Creek, MO 37.93847N, 90.79239W | M4 | new | |
KT988952 | ||||
KT988921 | Meramac River, MO 37.57011N, 91.30293W | M5 | new | |
KT988938 | ||||
KT988942 | ||||
KT988918 | Trek Creek, MO 37.95693N, 91.89561W | M6 | new | |
KT988919 | ||||
KT988925 | ||||
KT988949 | Little Piney River, MO 37.91063N, 91.90381W | M7 | new | |
KT988960 | ||||
KT988924 | Mill Creek, MO 37.87296N, 91.92921W | M8 | new | |
KT988928 | ||||
E. potosiensis | KT988947 | Clifty Creek, MO 38.04173N, 91.96089W | M10 | new |
KT988948 | ||||
KT988914 | North ForkWhite River, MO 36.66723N, 92.28129W | M14 | new | |
KT988915 | ||||
KT988926 | ||||
KT988931 | James River, MO 37.19040N, 93.12660W | M16 | new | |
KT988937 | Indian Creek, MO 36.79320N, 94.24380W | M19 | new | |
KT988930 | Spring River, MO 37.11560N, 93.89420W | M20 | new | |
KT988961 | Clear Creek, MO 37.30831N, 93.50060W | M22 | new | |
KT988970 | ||||
KT988959 | Niangua River, MO 37.51970N, 92.98420W | M24 | new | |
KT988969 | ||||
KT988917 | Big Piney River, MO 37.32720N, 92.00210W | M27 | new | |
KT988946 | ||||
KT988935 | Current River, MO 37.27990N, 91.40600W | M31 | new | |
KT988936 | ||||
KT988912 | Sallisaw Creek, OK 35.57660N, 94.83047W | OK | new | |
KT988913 | ||||
KT988920 | ||||
E. showalteri | U73767 | Lydeard et al. 1997 | ||
E. virginica 1 | DQ311117 | Lee et al. 2006 | ||
E. virginica 2 | AF100989 | Holzangel and Lydeard 2000 | ||
Io fluvialis | AF100999 | Holzangel and Lydeard 2000 | ||
Juga plicifera | AF101004 | Holzangel and Lydeard 2000 | ||
Leptoxis ampla 1 | U73768 | Lydeard et al. 1997 | ||
Leptoxis ampla 2 | KF680604 | unpublished | ||
Le. crassa anthonyi | AF101001 | Holzangel and Lydeard 2000 | ||
Le. dilatata | DQ311122 | Lee et al. 2006 | ||
Le. foremani | KF680592 | unpublished | ||
Le. picta 1 | KF680596 | unpublished | ||
Le. picta 2 | U73769 | Lydeard et al. 1997 | ||
Le. plicata | U73770 | Lydeard et al. 1997 | ||
Le. praerosa | AF101002 | Holzangel and Lydeard 2000 | ||
Le. taeniata 1 | U73771 | Lydeard et al. 1997 | ||
Le. taeniata 2 | KF680600 | unpublished | ||
Le. virgata | AF101000 | Holzangel and Lydeard 2000 | ||
Lithasia armigera | AF100998 | Holzangel and Lydeard 2000 | ||
Li. duttoniana | AF100997 | Holzangel and Lydeard 2000 | ||
Li. geniculata fuliginosa | AF100996 | Holzangel and Lydeard 2000 | ||
Li. geniculata geniculata | AF100995 | Holzangel and Lydeard 2000 | ||
Pleurocera acuta 1 | AF100994 | Holzangel and Lydeard 2000 | ||
P. acuta 2 | MF357697 | Mulberry Fork Little Red River, AR 35.74210N, 92.33380W | new | |
P. acuta 3 | MF357698 | Warm Fork Spring, AR 36.49580N, 91.53320W | new | |
P. annulifera | U73772 | Lydeard et al. 1997 | ||
P. prasinata 1 | U73774 | Lydeard et al. 1997 | ||
P. prasinata 2 | U73773 | Lydeard et al. 1997 | ||
P. pyrenella 1 | AF100990 | Holzangel and Lydeard 2000 | ||
P. pyrenella 2 | DQ311123 | Lee et al. 2006 | ||
P. pyrenella 3 | KT164352 |
|
||
P. uncialis hastata | AF100993 | Holzangel and Lydeard 2000 | ||
P. vestita | U73775 | Holzangel and Lydeard 2000 | ||
P. walkeri | AF100992 | Holzangel and Lydeard 2000 |
We employed ISSRs as population markers on two different spatial scales. In our first study, herein referred to as the ‘small’ study, ten individuals from each of 12 E. potosiensis populations were collected (Figure
We followed the ‘small’ study with a second ‘large’ study. We collected 50 E. potosiensis from each of four localities (Figure
We isolated genomic DNA as before, purified it on silica filters (UltraClean PCR Clean-up Kit, MoBio), and diluted it with sterile water to 50 ng/μl concentration. We amplified ISSRs by PCR in 50 μl volumes with the GoTaq PCR Core System I reagents (Promega) at the manufacturer’s recommended concentrations. The cycling profile consisted of an initial denaturation cycle of 95°C for three minutes, 30 cycles of 95°C for 30s, annealing for 30s, and 72°C for 60s, followed by a ten-minute extension period at 72°C. We used one primer in each ISSR reaction (Table
Sequences, annealing temperatures (ºC), and number of bands produced for ISSR primers used in each study.
‘small’ study | ‘large study’ | ||||
---|---|---|---|---|---|
Primer sequence | Annealing temperature | number of bands | Primer sequence | Annealing temperature | number of bands |
5’-(AG)8T | 50º | 6 | 5’-(AC)8C | 53º | 47 |
5’-AC8 | 49º | 2 | 5’-(CCA)5 | 54º | 27 |
5’-BHB(GA)7 | 49º | 1 | 5’-(CA)7RG | 53º | 26 |
5’-RY(CA)7 | 49º | 2 | |||
5’-CA7 | 54º | 7 | |||
5’-WB(GACA)4 | 50º | 4 |
We analyzed our data matrices using GenAlEx 6.052 (
Within E. potosiensis we observed nucleotide diversity π = 0.066 and Tajima’s D = 0.894 (p [D > 0.894] = 0.38) using aligned sequences prior to processing with Gblocks. Our parsimony network (Figure
In the ‘small’ study, we were able to bin and identify 22 markers. There were 110 unique genotypes identified among the 120 total individual snails. A genotype accumulation curve suggested that the minimum number of markers needed to discriminate individuals was 19, so our analyses were close to the detection limit for those markers. Simpson diversity for individual loci ranged from 0.033 to 0.500, while evenness ranged from 0.383 to 1. Nei’s gene diversity by population ranged from 0.248 (B3) to 0.394 (C3). AMOVA (Table
Results of the ‘small’ study AMOVA. Regions were spring (S populations), and creek above (C) and below (B) confluence with the spring.
Source | d.f. | Φ | % total variation | p value |
---|---|---|---|---|
Within populations | 108 | 0.054 | 95.86 | 0.014 |
Between populations within regions | 10 | 0.007 | 0.23 | 0.274 |
Between regions | 1 | 0.047 | 3.91 | 0.005 |
In the ‘large’ study, we were able to bin and identify a total of 100 markers. Each of the 200 snails possessed its own unique genotype. A genotype accumulation curve suggested that a minimum of 31 polymorphic markers was needed to discriminate individuals, so our dataset of 98 was sufficient for further analysis. Simpson diversity for individual loci ranged from 0.303 to 0.500, while evenness ranged from 0.376 to 1. Nei’s gene diversity for the populations ranged from 0.254 for ARK1 to 0.328 for ARK3. An AMOVA (Table
Results of the ‘large’ study AMOVA. Regions were either Arkansas or Oklahoma.
Source | d.f. | Φ | % total variation | p value |
---|---|---|---|---|
Within populations | 196 | 0.332 | 66.80 | <0.01 |
Between populations within regions | 2 | 0.153 | 12.10 | <0.01 |
Between regions | 1 | 0.211 | 21.10 | <0.01 |
Freshwater taxa from the same region tend to share evolutionary history, resulting in replicated patterns of biogeography and speciation (
Our analyses of mitochondrial 16S sequences suggested no genetic structuring. Mitochondrial haplotypes did not group together in our TCS network, and we recovered E. potosiensis in four well-supported clades instead of a single clade. However, we could not reject the monophyly of E. potosiensis based on our data. No statistical support existed for genetic structure within populations, within river drainages, or within populations or drainages in each of the four clades. We observed no evidence of heteroplasmy to suggest nuclear analogs of mitochondrial sequences (“numts”) in our data, nor did we find any biogeographic groupings of sequences that would suggest cryptic taxa. What we did observe, however, was a pattern where E. potosiensis sequences comprised multiple well-supported divergent clades. As mentioned previously, this is frequently observed in freshwater snails including pleurocerids using mitochondrial markers.
Four nominal morphospecies comprise the modern notion of E. potosiensis (
While we saw no broad scale genetic patterns in E. potosiensis, our two ISSR studies suggested population-level genetic structuring. Our ‘small’ study of ten individuals from each of 12 populations represented a pilot study as well as our first effort with ISSRs. In it, we were able to characterize 22 bands visualized on agarose gels using six primers. The AMOVA suggested that most of the genetic variation was within each population, but that a small (
Two issues we identified in our ‘small’ study were the low number of bands resolved by our primers and the small sample sizes from each population. Our band total for six primers was comparable to the number generated by a single primer in other studies, and is likely indicative of two factors. First, choice of our primer sequences was probably not ideal, employing dinucleotide repeats and/or degenerate 5’-bases ahead of the repeated portion. Results from the literature suggest that trinucleotide repeats and 3’-degenerate anchors after the repeats increase resolution and repeatability (
Our study also highlighted the need for novel and useful genetic markers for pleurocerid conservation. Nearly 80% of all pleurocerids are imperiled (
We thank Rachelle Amundson, Erin Basiger, Alan Christian, Dan Graham, John Harris, Ashley Meyer, and Bill Posey for their assistance throughout the study. The Louisiana Board of Regents EPSCoR Pilot Funding for New Research program, Arkansas Game and Fish Commission, USDA Ouachita National Forest, Environmental Sciences Program at Arkansas State University, Arkansas Biosciences Institute, Judd Hill Foundation, and Conchologists of America funded various portions of the research.