Jennifer E. Buhay

Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.

Subterranean phylogeography of freshwater crayfishes shows extensive gene flow and surprisingly large population sizes

Subterranean animals are currently viewed as highly imperiled, precariously avoiding extinction in an extreme environment of darkness. This assumption is based on a hypothesis that the reduction in visual systems and morphology common in cave faunas reflects a genetic inability to adapt and persist coupled with the perception of a habitat that is limited, disconnected, and fragile. Accordingly, 95% of cave fauna in the United States are presumed endangered due to surface environmental degradation and limited geographic distributions. Our study explores the subterranean phylogeography of stygobitic crayfishes in the southeastern United States, a global hotspot of groundwater biodiversity, using extensive geographic sampling and molecular data. Despite their endangered status, our results show that subterranean crayfish species have attained moderate to high levels of genetic diversity over their evolutionary histories with large population sizes and extensive gene flow among karst systems. We then compare the subterranean population histories to those of common surface stream‐dwelling crayfishes. Our results show recent drastic declines in genetic variability in the surface crayfish and suggest that these species also warrant conservation attention.

Avoidance of extinction through nonexistence: the use of museum specimens and molecular genetics to determine the taxonomic status of an endangered freshwater crayfish

We investigated the endangered status and taxonomic status of the freshwater crayfish Procambarus ferrugineus, a crayfish species considered for the candidate list of the Endangered Species Act. This species has a narrow distribution from central Arkansas, USA and is codistributed with its presumed sister species, Procambarus liberorum. We sampled extensively throughout the ranges of both primary burrowing species and collected mitochondrial DNA from a hypervariable fragment of the 16S gene from 109 individuals across 22 sites. We also collected data from a variable region of the 12S gene from a subset of the resulting 16S haplotypes. Due to our inability to sample what we considered P. ferrugineus in the field, we included museum specimens from the United States Natural History Museum of both P. ferrugineus and P. liberorum. Analyses of the resulting data suggested that these two species are indeed the same and we therefore synonymize them under the name of priority—P. liberorum. Additionally, our sampling discovered three new cryptic species from southwestern Arkansas all from the genus Procambarus. Nested clade phylogeographic analysis coupled with population genetic analyses suggested that P. liberorum has had three rounds of range expansion throughout the inferred evolutionary history. Using IUCN Red List criteria for conservation assessment, we conclude that the species P. liberorum should be considered stable, but with special concern because of habitat fragmentation and urbanization, small restricted range, and a moderate level of genetic diversity. Procambarus reimeri should be considered endangered due to its limited geographic range and the potential for a decline in suitable habitat. The three potentially newly discovered species should be considered data deficient until more information is obtained on their distributional limits and habitat requirements. Our study highlights the importance of thorough geographic and taxonomic sampling coupled with the utility of collecting data from museum specimens to reach robust taxonomic and conservation conclusions for endangered species.

Taxonomic Revision of Cave Crayfishes in the Genus Orconectes, Subgenus Orconectes (Decapoda: Cambaridae) along the Cumberland Plateau, Including a Description of a New Species, Orconectes Barri

Abstract Historically, three obligate cave-dwelling species of Orconectes comprise an assemblage along the Cumberland Plateau of the southeastern United States, including Orconectes australis (with two subspecies, australis and packardi), O. incomptus, and O. sheltae. Using genetic data from three mitochondrial genes (cytochrome oxidase subunit I (COI), 12S, and 16S), we present evidence for four cryptic lineages excluding O. sheltae. The subspecies of Orconectes australis represent distinct taxonomic units with non-sister phylogenetic relationships and will be each recognized as separate species. Orconectes (Orconectes) barri is a new species of subterranean crayfish with a small distribution around the Kentucky - Tennessee state border in Mississippian limestone. This species was uncovered during a previous thoroughly-sampled phylogeographic survey of the southern Appalachians. Additionally, we assign genetic barcodes (COI sequences) and conservation status to each species for management direction and identification of newly discovered populations.

Taxonomic Revision of Cave Crayfish in the Genus Cambarus, Subgenus Aviticambarus (Decapoda: Cambaridae) with Descriptions of Two New Species, C. Speleocoopi and C. Laconensis, Endemic to Alabama, U.S.A.

Abstract As historically recognized, three stygobitic species of the subgenus Aviticambarus, genus Cambarus inhabit Mississippian limestone caves along the southern edge of the Southern Appalachians and Highland Rim in southeastern Tennessee and Northern Alabama, U.S.A. These include Cambarus hamulatus, C. jonesi, and C. veitchorum. All stygobitic members of the genus Procambarus inhabit caves in Florida, Cuba, and Mexico with exception of P. pecki (in the monotypic subgenus Remoticambarus), which exists in only three caves with C. jonesi in Northwestern Alabama. It was hypothesized that Procambarus pecki was derived from a primitive Procambarus stock that gave rise to the genera Cambarus and Orconectes based on the morphological shapes of the gonopods. Excluding the unsampled rare C. veitchorum, here we present 16S rDNA phylogenetic evidence, contrary to former morphological-based inferences, for the recognition of five distinct Aviticambarus lineages including P. pecki. Cambarus laconensis is a new species restricted to one locality in Northern Alabama along the southern border of the Highland Rim. Cambarus speleocoopi is also a new species of subterranean crayfish restricted to Marshall County, Alabama. These two cryptic species, with distributions that do not overlap any other stygobitic species, were discovered during a previous phylogeographic survey of cave crayfishes in the Southern Appalachians. For cave crayfishes in particular, similar morphology owing to convergent evolution in replicate subterranean environments, obscures phylogenetic relationships and cryptic stygobitic lineages.