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Detailed information on species delineation and population genetic structure is a prerequisite for designing effective restoration and conservation strategies for imperiled organisms. Phylogenomic and population genomic analyses based on genome-wide double digest restriction-site associated DNA sequencing (ddRAD-Seq) data has identified three allopatric lineages in the North American freshwater mussel genus Cyprogenia. Cyprogenia stegaria is restricted to the Eastern Highlands and displays little genetic structuring within this region. However, two allopatric lineages of C. aberti in the Ozark and Ouachita highlands exhibit substantial levels (mean uncorrected FST = 0.368) of genetic differentiation and each warrants recognition as a distinct evolutionary lineage. Lineages of Cyprogenia in the Ouachita and Ozark highlands are further subdivided reflecting structuring at the level of river systems. Species tree inference and species delimitation in a Bayesian framework using single nucleotide polymorphisms (SNP) data supported results from phylogenetic analyses, and supports three species of Cyprogenia over the currently recognized two species. A comparison of SNPs generated from both destructively and non-destructively collected samples revealed no significant difference in the SNP error rate, quality and amount of ddRAD sequence reads, indicating that nondestructive or trace samples can be effectively utilized to generate SNP data for organisms for which destructive sampling is not permitted.

The larvae of freshwater mussels in the order Unionoida are obligate parasites on fishes. Because adult mussels are infaunal and largely sessile, it is generally assumed that the majority of gene flow among mussel populations relies on the dispersal of larvae by their hosts. The objective of this study was to compare the genetic diversity and the degree of congruence between the population structures of two related freshwater mussels Leptodea leptodon and Leptodea fragilis and their fish host, Aplodinotus grunniens. Host specificity in parasites has been shown to result in greater congruence between the population structures of the two interacting species, and assessing the congruence of genetic structure of the endangered L. leptodon with its sister species L. fragilis and their sole host is an important step in understanding the impact of host dispersal on population structure. Analysis of microsatellite data indicated that despite its imperiled status, L. leptodon displayed greater genetic diversity than the more common L. fragilis. However, the population structures of all three species were incongruent even in the presence of substantial gene flow. Other factors such as habitat specificity may play a role in generating the differences in population structure observed. This study indicates that barriers to gene flow or lack of available host fish are not the cause of decline of the federally endangered L. leptodon, and suggests that alternative explanations should be considered.

Accurately identifying species is a crucial step for developing conservation strategies for freshwater mussels, one of the most imperiled faunas in North America. This study uses genetic data to re-examine species delineation in the genus Cyprogenia. Historically, Cyprogenia found west of the Mississippi River have been ascribed to Cyprogenia aberti (Conrad 1850), and those east of the Mississippi River were classified as Cyprogenia stegaria (Rafinesque 1820). Previous studies using mitochondrial DNA sequences indicated that C. aberti and C. stegaria were not reciprocally monophyletic groups, suggesting the need for systematic revision. We generated a novel dataset consisting of 10 microsatellite loci and combined it with sequence data from the mitochondrial ND1 gene for 223 Cyprogenia specimens. Bayesian analysis of the ND1 nucleotide sequences identified two divergent clades that differ by 15.9%. Members of these two clades occur sympatrically across most sampling locations. In contrast, microsatellite genotypes support recognition of three allopatric clusters defined by major hydrologic basins. The divergent mitochondrial lineages are highly correlated with the color of the conglutinate lures used by mussels to attract and infest host fishes, and tests for selection at the ND1 locus were positive. We infer that the incongruence between mtDNA and microsatellite data in Cyprogenia may be the result of a combination of incomplete lineage sorting and balancing selection on lure color. Our results provide further evidence that mitochondrial markers are not always neutral with respect to selection, and highlight the potential problems of relying on a single-locus-marker for delineating species.

Anthropogenic development of floodplains and alteration to natural hydrological regimes have resulted in extensive loss of off-channel habitat. Interest has grown in restoring these habitats as an effective conservation strategy for numerous aquatic species. This study developed a process to reproducibly identify areas of former stream meanders to assist future off-channel restoration site selections. Three watersheds in Iowa and Minnesota where off-channel restorations are currently being conducted to aid the conservation of the Topeka Shiner (Notropis topeka) were selected as the study area. Floodplain depressions were identified with LiDAR-derived digital elevation models, and their morphologic and topographic characteristics were described. Classification tree models were developed to distinguish relic streams and oxbows from other landscape features. All models demonstrated a strong ability to distinguish between target and non-target features with area under the receiver operator curve (AUC) values ≥ 0.82 and correct classification rates ≥ 0.88. Solidity, concavity, and mean height above channel metrics were among the first splits in all trees. To compensate for the noise associated with the final model designation, features were ranked by their conditional probability. The results of this study will provide conservation managers with an improved process to identify candidate restoration sites.

Assessing genetic variation in species of conservation concern is critical for developing sound recovery strategies. In this study, we compared sympatric populations of two related species, the endangered Quadrula fragosa (Conrad, 1836) and its common relative Amphinaias pustulosa (Lea, 1831), using standard genetic parameters such as allelic richness, heterozygosity, and effective population size. Our primary aim was to determine if a small population size and isolation from conspecifics had negatively affected the genetic diversity of this population of Q. fragosa. By comparing the endangered species to a related and sympatric, common species we can assess the rare species for genetic effects associated with reduced population size, and in addition, develop management targets for what a recovered Q. fragosa population looks like genetically. Examination of eight microsatellite loci indicated that Quadrula fragosa exhibited reduced genetic variation when compared to A. pustulosa at all measures,...

The middle and lower portions of the Yangtze River basin is the most species-rich region for freshwater mussels in Asia. The management and conservation of the taxa in this region has been greatly hampered by the lack of a well-developed phylogeny and species-level taxonomic framework. In this study, we tested the utility of two mitochondrial genes commonly used as DNA barcodes: the first subunit of the cytochrome oxidase c gene (COI) and the first subunit of the NADH dehydrogenase gene (ND1) for 34 putative species representing 15 genera, and also generated phylogenetic hypotheses for Chinese unionids based on the combined dataset of the two mitochondrial genes. The results showed that both loci performed well as barcodes for species identification, but the ND1 sequences provided better resolution when compared to COI. Based on the two-locus dataset, Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses indicated 3 of the 15 genera of Chinese freshwater mussels examined were polyphyletic. Additionally, the analyses placed the 15 genera into 3 subfamilies: Unioninae (Aculamprotula, Cuneopsis, Nodularia and Schistodesmus), Gonideninae (Lamprotula, Solenaia and Ptychorhychus) and Anodontinae (Cristaria, Arconaia, Acuticosta, Lanceolaria, Anemina and Sinoanodonta). Our results contradict previous taxonomic classification that placed the genera Arconaia, Acuticosta and Lanceolaria in the Unioninae. This study represents one of the first attempts to develop a molecular phylogenetic framework for the Chinese members of the Unionidae and will provide a basis for future research on the evolution, ecology, and conservation of Chinese freshwater mussels.

The Topeka shiner Notropis topeka is native to Iowa, Kansas, Minnesota, Missouri, Nebraska, and South Dakota and has been federally listed as endangered since 1998. Our goals were to determine the present distribution and qualitative status of Topeka shiners throughout its current range in Iowa and characterize the extent of decline in relation to its historic distribution. We compared the current (2016–2017) distribution to distributions portrayed in three earlier time periods. In 2016–2017 Topeka shiners were found in 12 of 20 HUC10 watersheds where they occurred historically. Their status was classified as stable in 21% of the HUC10 watersheds, possibly stable in 25%, possibly recovering in 8%, at risk in 33%, and possibly extirpated in 13% of the watersheds. The increasing trend in percent decline evident in earlier time periods reversed, going from 68% in 2010–11 to 40% in the most recent surveys. Following decades of decline, the status of Topeka shiners in Iowa appears to be improving. One potential reason for the reversal in the distributional decline of Topeka shiners in Iowa is the increasing number of oxbow restorations. Until a standardized monitoring program is established for Iowa, periodic status assessments such as this will be necessary to chronicle progress toward conserving this endangered fish species.

The phylogenetic relationships of species of the mussel genus Ptychobranchus Simpson, 1900 were examined using the mitochondrial DNA sequences of the ND1 and 16S gene regions. A total of 31 individuals representing the five species are included in this analysis. Outgroups were drawn from other unionid genera previously shown to be closely related to Ptychobranchus. Phylogenetic analysis was conducted using Bayesian methods applying several data-partitioning strategies. The results of all analyses support the monophyly of Ptychobranchus, and the interrelationships of its constituent species are consistent across all analyses. Ptychobranchus occidentalis (Conrad, 1836) is recovered as paraphyletic with P. fasciolaris (Rafinesque, 1820). Molecular analyses indicate that Ptychobranchus jonesi van der Schalie, 1934 is a member of the genus, and character state reconstruction predicts that it should possess the complex conglutinate that is found in other species of Ptychobranchus.

The family Margaritiferidae is a small but widely distributed group within the Unionoida, or freshwater mussels, whose taxonomy and systematics has been the subject of numerous publications. Despite several efforts, there is no consensus on which characters reliably diagnose this family. Herein, we present the results of a phylogenetic analysis of the most comprehensive data set for Margaritiferidae in terms of taxa and phylogenetic markers assembled to date, including eleven out of the twelve margaritiferid species currently considered valid. In addition, we review the fossil record of the family and attempt to integrate fossil and DNA sequence data to provide a diagnosis of Margaritiferidae, identify its origin and biogeographic patterns, and determine the systematic relationships of its constituent species and their taxonomic affinities. We assembled a molecular data set comprised of five markers: COI, 16S, 28S, 18S and histone 3 for a total of 59 specimens representing eleven species of Margaritifera. Our results indicate that the family Margaritiferidae is a monophyletic group comprised of the single genus Margaritifera, which includes the following 12 species: M. dahurica, M. margaritifera, M. monodonta, M. middendorffi, M. laevis, M. marrianae, M. hembeli, M. falcata, M. laosensis, M. auricularia and M. marocana plus the unstudied M. homsensis. Estimates of divergence times using fossil calibrations or mean substitution rates produced dramatically different results. Divergence estimates based on the fossil calibrations were 10 times higher than those obtained applying the mean substitution rates. The current distribution of the family implies dispersal across marine or brackish waters by their host fish, leaving a fossil record on four continents that dates to the Mesozoic. Margaritiferidae appear to be derived from putative ancestor in the Silesunionidae, with a likely origin in Asia. We suggest that Margaritiferidae had spread along the Tethys margins and crossed the Atlantic already in the Late Triassic or Early Jurassic. Further dispersal events, in the Late Cretaceous or Eocene, may be linked to salinity-depleted coastal waters or freshwater layering., This study was partially funded by the Spanish project of the Ministry of Economy and Competitiveness CTM2014-57949-R.

Atyid freshwater shrimps are globally distributed and form an important part of freshwater ecosystems, particularly in the tropics and subtropics. Despite their widespread distribution and ecological importance, their phylogenetic relationships are largely unresolved. Here we present the first comprehensive molecular phylogeny of the Atyidae investigating the evolutionary relationships among 32 of the 42 genera using mitochondrial and nuclear markers. Our data indicate that the established classification of the Atyidae is in need of substantial taxonomic revision at all taxonomic levels. We suggest a new suprageneric systematization of atyids and discuss problematic issues at the generic level, particularly in the most speciose genus, Caridina. Molecular clock based divergence time estimates for atyids vary widely, but invariably support the assumption that atyids are an ancient freshwater lineage with an origin in the mid-Cretaceous at the very latest. Atyid distribution patterns are the result of instances of both long-distance dispersal and vicariance, depending largely on the reproductive mode of taxa. From an evolutionary perspective, the high frequency of independent origin of both a complete (landlocked) freshwater life cycle and a cave-dwelling mode of life is remarkable and unparalleled among crustaceans.

For groups of animals with limited or unstable taxonomic resolution, congeneric phylogeographical sampling represents a way to potentially increase resolution of species limits and internal branches during phylogenetic inference. We investigated species limits and whether there was better support for hypothesized relationships among Micropterus (black basses) using wide geographical sampling. Bayesian and maximum likelihood estimates of Micropterus phylogeny including 205 mitochondrial DNA cytochrome b (1140 bp) sequences (150 haplotypes) representing eight extant species and one subspecies recovered eight well-supported clades. Haplotypes from fish identified as Micropterus coosae, Micropterus henshalli, Micropterus punctulatus, Micropterus salmoides, and Micropterus treculii created eight forms of broad-sense polyphyly in the tree, which we hypothesized were the result of incomplete lineage sorting and hybridization-mediated introgression (natural and anthropogenic, i.e. associated with stocking). Our findings mostly agree with hypothesized relationships. However, they provide a more complex view of Micropterus biodiversity, highlighting population-level processes. Our data also provide a useful guide for expanding character sampling (nuclear loci and morphology) to evaluate the history, distinctiveness, and geographical distributions of genetic lineages within and among black basses. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 346–363.

This study examines the role that historical events have played in the diversification of members of the genus Ambloplites by estimating divergence times of clades within the genus. DNA sequences of the mitochondrial cytochrome b gene were used to develop phylogenetic hypotheses for Ambloplites using maximum parsimony and Bayesian methods. The timing of diversification within and between species of Ambloplites was estimated using nonparametric rate-smoothing and penalized likelihood methods calibrated by fossil dates. Monophyly of three of the four species was supported. The ages of the most recent common ancestors of species of Ambloplites estimated in this study confirm earlier estimates that in most instances, their origins occurred prior to the Pleistocene. A comparison of estimated divergence times of lineages to sea level fluctuations indicates a correspondence to extremely high or low sea stands throughout the Neogene Period. Populations of Ambloplites in the Ozarks and Ouachita highlands ...

Freshwater bivalves of the order Unioniformes represent the largest bivalve radiation in freshwater. The unioniform radiation is unique in the class Bivalvia because it has an obligate parasitic larval stage on the gills or fins of fish; it is divided into 6 families, 181 genera, and ;800 species. These families are distributed across 6 of the 7 continents and represent the most endangered group of freshwater animals alive today. North American unioniform bivalves have been the subject of study and illustration since Martin Lister, 1686, and over the past 320 y, significant gains have been made in our understanding of the evolutionary history and systematics of these animals. Here, the current state of unioniform systematics and evolution is summarized, and suggestions for future research themes are proposed. Advancement in the areas of systematics and evolutionary relationships within the Unioniformes will require a resurgence of survey work and reevaluation of all taxa, especially outside of North America and Western Europe. This work will require collection of animals for shell morphology, comparative anatomy, and molecular analyses. Along with reexamination of described taxa, a renewed emphasis on the natural history, host-fish relationships, ecology, and physiology of these animals is needed. Traditional conchological and anatomical characters should be reevaluated, new character suites should be added, and new morphometric methods should be applied. The fossil record of freshwater bivalves should be carefully reviewed, and phylogenetic hypotheses including fossil taxa must be developed. We will have to expand our set of molecular tools to include or develop additional markers, such as single-copy nuclear genes and microsatellites. Examination of double uniparental inheritance of mitochondrial deoxyribonucleic acid (DNA) is providing new insights into the evolution of this order. Mitochondrial gene order differs among genera but is still to be explored. Expansion of our understanding of the evolutionary relationships and history of unioniform bivalves will provide a solid foundation to study the zoogeography of these rather sessile, obligate freshwater organisms. The unique natural history of unioniform bivalves provides a fertile area for testing and developing evolutionary theories, and, as our understanding of the systematics of these animals improves, a better understanding of the evolution of this expansive radiation in freshwater will develop.

Complete mitochondrial DNA cytochrome b gene sequences from 56 specimens representing all 12 species of Lepomis were used to examine phylogenetic relationships within the genus. Results supported the monophyly of Lepomis and all previously recognized subgenera, except Eupomotis, but there was no support for previously proposed relationships among subgenera. Seven species were recovered as monophyletic lineages, while five species (L. auritus, L. macrochirus, L. marginatus, L. miniatus, and L. symmetricus) were recovered as either poly- or paraphyletic or were placed as parts of unresolved polytomies with other species. Parametric bootstrapping tests rejected monophyly for only two of the five species (L. auritus and L. symmetricus). Without additional data, including increased geographic sampling and a comparable nuclear gene phylogeny, it is not possible to determine whether the failure to support monophyly for these two species reflects the presence of cryptic species or results from hybridization.

We isolated and characterized a total of 16 microsatellite loci in Leptodea leptodon. Loci were screened in 24 individuals from across the species current range in Missouri. The number of alleles per locus ranged from 3 to 17, observed heterozygosity ranged from 0.208 to 1.000, and the probability of identity values ranged from 0.014 to 0.67. These new loci will be used for conducting investigations into the genetic structure and diversity of extant populations of this federally endangered species.

The phylogenetic relationships of genera of the family Centrarchidae and its affinities to the Elassomatidae were examined using the mitochondrial DNA sequences of the cytochrome b gene. A total of 32 individuals representing 16 species from nine genera of centrarchids were sequenced. Outgroup were drawn from other perciform families including several families previously proposed to be closely related to the Centrarchidae. Phylogenetic analysis was conducted using the optimality criteria of maximum parsimony with two weighting schemes and using the maximum likelihood method. A priori and a posteriori alternative hypotheses of relationships within the Centrarchidae were investigated using the Shimodaira and Hasegawa Test. The results of all analyses support the monophyly of the Centrarchidae, although the interrelationships of centrarchid genera differed between analyses. Elassoma was never recovered as the sister taxon to the Centrarchidae; however, the sister relationship of these taxa could not...

Controlled propagation, augmentation, and reintroduction (PAR) of rare and endangered aquatic organisms has become a priority action for recovery and delisting, and in many cases is an action of “last resort” to either restore or maintain existing populations. The guiding principle of PAR efforts should be to avoid harming existing populations of congeneric or nontarget species and also minimize risks to extant populations and habitats. Controlled PAR of freshwater mussels should not be a long-term management strategy conducted in perpetuity and should not be used as a substitute for recovery tasks such as habitat restoration or addressing the causes of endangerment. The determination to pursue controlled PAR for freshwater mussels should follow a thorough evaluation of the status of existing wild populations, an agreement that PAR in the historic range is needed, and a conclusion that suitable habitat for long-term success is present. The primary purpose of any efforts to augment or reintroduce animals should be to establish free-ranging wild populations. Concomitant with this goal is the distinct possibility that these activities can represent appreciable genetic or ecological risks to resident animals, both nontarget taxa and wild conspecifics. To maintain the integrity of the fauna, communities, and ecosystems it is imperative that these risks be carefully considered before conducting controlled PAR. In this paper we pose several questions that we believe are important to consider before initiating PAR of freshwater mussels. We also recommend actions, some already used at individual facilities or by agencies, that we believe will aid in developing a more uniform approach to controlled PAR and safeguarding the ecological and genetic integrity of freshwater mussel communities.

Molecular Ecology Resources Primer Development Consortium: et al., This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

During a recent survey a small population of Quadrula mitchelli (Simpson, 1895), a species thought to have been extinct, was discovered in Texas. In total, 7 live individuals were collected from the Guadalupe River near Gonzales, Gonzales County, Texas. Our finding represents the only known population for this species in Texas and the first record of live specimens in over 30 y, which is significant because this species is currently under review for protection under the Endangered Species Act.

The majority of Middle American cichlids are placed in the informal assemblage ‘ Cichlasoma. ’ The group is divided into eight sections which appear to be based primarily on trophic morphology. Although several members of ‘ Cichlasoma ’ have been used in ecomorphological, behavioral, and biogeographic studies, no phylogenetic hypotheses for the group exist. In an attempt to develop a better understanding of the phylogenetic relationships of ‘cichlasomine’ cichlids, we examined the evolution of the trophic specialization, substratum-sifting, in two sections, ‘ Cichlasoma ( Thorichthys )’ and ‘ C. ( Amphilophus ),’ to determine whether the trait reflects common ancestry. We sequenced the complete mitochondrial cytochrome b gene for 19 cichlids representing six sections of ‘ Cichlasoma, ’ and representatives of other Neotropical Cichlidae. Additional cichlid, and noncichlid outgroup sequences were included for a total of 22 taxa. The molecular phylogeny supports the recognition of the section ‘ C. ( Thoricthys )’ as a natural group, and we place those cichlids in the genus Thorichthys. The phylogeny also depicts ‘ C. ( Amphilophus )’ as paraphyletic, with substratum-sifters and generalized predators forming separate nonsister clades. We recommend that the substratum-sifting clade of the section ‘ C. ( Amphilophus )’ be placed in the resurrected genus Astatheros. The generalized predator clade of ‘ C. ( Amphilophus )’ contains only two species, ‘ C. (A.) citrinellum ’ and ‘ C. (A.) labiatum, ’ which we place in the genus Amphilophus. The phylogenetic hypotheses generated indicate that the substratum-sifting genera Thorichthys and Amphilophus do not share a common ancestor. Reconstruction of the evolution of substratum-sifting is equivocal, requiring either the independent evolution of the trait on two separate occasions or its presence in a more inclusive clade and subsequent loss in nonsubstratum sifting species.

The objective of this chapter is to test the phylogenetic utility of the mitochondrial cytochrome b gene by analyzing the relationships of monophyletic Actinopterygii or ray-finned fishes at different hierarchic levels. Modern actinopterygians are the most diverse of all vertebrate groups and include more than 25,000 species. In order to assess the utility of the cytochrome b gene, actinopterygian fishes representing a diverse array of taxa and divergence times were selected. Facts and figures supporting phylogenetic hypothesis of actinopterygian fishes, including only the taxa, are examined based on morphological data from various sources. Although this chapter focuses on the cytochrome b gene, this study serves as a model for further studies that examines the utility of other genes. It lists the actinopterygian species examined in this study and their current classification. The mitochondrial cytochrome b gene sequences for the remaining 18 ray-finned fishes were retrieved from GenBank. The results of Cytochrome b sequence variation, base compositional bias, and amino acids differences are very important from the analytical point of view as is the assessment of the phylogenetic utility of the mitochondrial cytochrome b gene arrived at by examining taxonomic congruence between molecular- and morphological-based phylogenetic hypotheses.

A survey of the fishes of the Black Warrior River was undertaken to determine fish host(s) of the federally threatened inflated heelsplitter, Potamilus inflatus. Seven hundred-twenty individual fishes representing 30 species were examined; mussel glochidia were found on 10 individual fishes representing nine species. Potamilus inflatus glochidia were only found infesting one freshwater drum (Aplodinotus grunniens), which is concordant with previous findings for the genus Potamilus. The morphology of P inflatus glochidia is described and compared to P purpuratus.