DNA barcoding reveals taxonomic conflicts in the Herichthys bartoni species group (Pisces: Cichlidae)

Fishes of the genus Herichthys are the only representatives of the family Cichlidae to have colonized the Neartic region. In this study, we used DNA barcode of 64 individuals of the Herichthys bartoni species group to test the monophyly of the species and the efficiency of this tool to discriminate among species. The Bayesian phylogenetic tree and the neighbour joining (NJ) tree obtained from the Kimura two‐parameter model (K2P) give similar topologies. DNA barcoding resolution was very poor (25%). Additionally, the low levels of genetic divergence among taxa preclude the use of threshold values as has been suggested in earlier studies.     

Aggressive interactions between the invasive Rio Grande cichlid (Herichthys cyanoguttatus) and native bluegill (Lepomis macrochirus), with notes on redspotted sunfish (Lepomis miniatus)

The Rio Grande cichlid (Herichthys cyanoguttatus) has been established in the Greater New Orleans Metropolitan area for at least 20 years, and its effect on native fishes is unknown. Behavioral trials were performed to determine if aggressive interactions occur between invasive H. cyanoguttatus and native bluegill (Lepomis macrochirus). When defending a territory as the resident, L. macrochirus were markedly aggressive, averaging 11.6 aggressive actions per 10-min behavioral trial. In contrast, L. macrochirus were extremely passive as invaders, with 0.5 aggressive actions per trial. Herichthys cyanoguttatus were equally aggressive as residents and as invaders, averaging 4.9 and 6.0 aggressive actions per trial, respectively. Herichthys cyanoguttatus interacted aggressively with native species whether they held territory or not, indicating that this invasive species may have fundamentally different strategies of aggression compared with native L. macrochirus. These differences may explain the continued success of H. cyanoguttatus as an invasive fish in southeastern Louisiana.     

Do relaxed selection and habitat temperature facilitate biased mitogenomic introgression in a narrowly endemic fish?

Introgression might be exceptionally common during the evolution of narrowly endemic species. For instance, in the springs of the small and isolated Cuatro Ciénegas Valley, the mitogenome of the cichlid fish Herichthys cyanoguttatus could be rapidly introgressing into populations of the trophically polymorphic H. minckleyi. We used a combination of genetic and environmental data to examine the factors associated with this mitochondrial introgression. A reduced representation library of over 6220 single nucleotide polymorphisms (SNPs) from the nuclear genome showed that mitochondrial introgression into H. minckleyi is biased relative to the amount of nuclear introgression. SNP assignment probabilities also indicated that cichlids with more hybrid ancestry are not more commonly female providing no support for asymmetric backcrossing or hybrid‐induced sex‐ratio distortion in generating the bias in mitochondrial introgression. Smaller effective population size in H. minckleyi inferred from the SNPs coupled with sequences of all 13 mitochondrial proteins suggests that relaxed selection on the mitogenome could be facilitating the introgression of “H. cyanoguttatus” haplotypes. Additionally, we showed that springs with colder temperatures had greater amounts of mitochondrial introgression from H. cyanoguttatus. Relaxed selection in H. minckleyi coupled with temperature‐related molecular adaptation could be facilitating mitogenomic introgression into H. minckleyi.     

Introgressive hybridization in a trophically polymorphic cichlid

Trophically polymorphic species could represent lineages that are rapidly diverging along an ecological axis or could phenotypically mark the collapse of species through introgressive hybridization. We investigated patterns of introgression between the trophically polymorphic cichlid fish Herichthys minckleyi and its relative H. cyanoguttatus using a combination of population genetics and species tree analyses. We first examined the distribution of mitochondrial haplotypes within the alternative H. minckleyi pharyngeal jaw morphotypes that are endemic to the small desert valley of Cuatro Ciénegas. We recovered two clusters of mitochondrial haplotypes. The first contained a number of slightly differentiated cytochrome b (cytb) haplotypes that showed some phylogeographic signal and were present in both jaw morphotypes. The other haplotype was monomorphic, highly differentiated from the other cluster, present in equal frequencies in the morphotypes, and identical to H. cyanoguttatus haplotypes found outside Cuatro Ciénegas. Then, we investigated whether H. minckleyi individuals with the H. cyanoguttatus cytb were more evolutionarily similar to H. cyanoguttatus or other H. minckleyi using a species tree analysis of 84 nuclear loci. Both H. minckleyi pharyngeal morphotypes, regardless of their cytb haplotype, were quite distinct from H. cyanoguttatus. However, hybridization could be blurring subdivision within H. minckleyi as the alternative jaw morphotypes were not genetically distinct from one another. Accounting for introgression from H. cyanoguttatus will be essential to understand the evolution of the trophically polymorphic cichlid H. minckleyi.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Genome‐wide single‐nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species—The case of the Ancylus fluviatilis species complex

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high‐resolution genome‐wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome‐wide data for species delimitation in such groups.     

Phylogeography and lineage‐specific patterns of genetic diversity and molecular evolution in a group of North American skinks

Geography influences the evolutionary trajectory of species by mediating opportunities for hybridization, gene flow, demographic shifts and adaptation. We sought to understand how geography and introgression can generate species‐specific patterns of genetic diversity by examining phylogeographical relationships in the North American skink species Plestiodon multivirgatus and P. tetragrammus (Squamata: Scincidae). Using a multilocus dataset (three mitochondrial genes, four nuclear genes; a total of 3455 bp) we discovered mito‐nuclear discordance, consistent with mtDNA introgression. We further tested for evidence of species‐wide mtDNA introgression by using comparisons of genetic diversity, selection tests and extended Bayesian skyline analyses. Our findings suggest that P. multivirgatus acquired its mitochondrial genome from P. tetragrammus after their initial divergence. This putative species‐wide mitochondrial capture was further evidenced by statistically indistinguishable substitution rates between mtDNA and nDNA in P. multivirgatus. This rate discrepancy was observed in P. multivirgatus but not P. tetragrammus, which has important implications for studies that combine mtDNA and nDNA sequences when inferring time since divergence between taxa. Our findings suggest that by facilitating opportunities for interspecific introgression, geography can alter the course of molecular evolution between recently diverged lineages.     

The role of head shape and trophic variation in the diversification of the genus Herichthys in sympatry and allopatry

In the present study we evaluated the putative cases of sympatric speciation in the genus Herichthys by studying the variation in head shape using principal component analysis, phylomorphospace and reconstructions of the ancestral states of feeding preferences. Herichthys includes both allopatric and sympatric sister species, as well as sympatric unrelated species and thus offers great potential for evolutionary studies of putatively sympatric speciation. Herichthys is the northernmost group of cichlids in America and one of the most ecologically disparate genera within Middle American cichlids. Fifteen anatomical points were recorded on the heads of 293 specimens of the 11 species recognized within the genus. The results show that in spite of having wide variation in consumed diets, most species of Herichthys are close in morphospace. However, morphological variation was great among the two pairs of sympatric sister species in agreement with the suggested sympatric model of speciation.     

Complete mitochondrial genome and assembled DNA barcoding analysis of Lutjanus fulgens (Valenciennes, 1830) and its comparison with other Lutjanus species

Lutjanus fulgens (Valenciennes, 1830) is a teleost species classified under the family Lutjanidae which is a native of the Eastern Atlantic Ocean. Though highly commercialized due to its abundance and good taste, the production output has declined in recent years. This is an indication of the need for effective management and conservation measures. However, accurate species identification will ensure strategic management and conservation measure. DNA‐based species identification has proven its reliability in this regard via precise species identification. Several researchers have confirmed the accuracy of DNAbarcode as a species identification tool as well as species phylogeny analysis based on both the complete mitogenome and COI gene. Currently, nine specimens of L. fulgens were sampled from Ghana and subjected to DNA‐based analysis, namely, complete mitochondrial DNAand COI gene (DNA barcoding) analyses. The mitogenomic result revealed that L. fulgens is made up of a 16,500 base pairs (bp) mtDNA which consists of 22 transfer RNAs, 13 protein‐coding genes, and two ribosomal RNAs (GenBank Accession Number: MN398650). Furthermore, a sequence polymorphism analysis of the COIgene (MN986442–MN986450) detected two haplotypes. These haplotypes were both collected from the same fish landing site which suggests a possible cryptic linage diversity in the L. fulgens population at Vodza. According to the phylogeny examination, a close taxonomic relationship exists between L. fulgens and Lutjanus buccanella caused by a recent evolution termed as sympatric speciation. This study serves as a novel study for this species, building the foundation for future molecular‐based study for this species and as a DNA barcode reference data.     

Introgression and selection shaped the evolutionary history of sympatric sister‐species of coral reef fishes (genus: Haemulon)

Closely related marine species with large overlapping ranges provide opportunities to study mechanisms of speciation, particularly when there is evidence of gene flow between such lineages. Here, we focus on a case of hybridization between the sympatric sister‐species Haemulon maculicauda and H. flaviguttatum, using Sanger sequencing of mitochondrial and nuclear loci, as well as 2422 single nucleotide polymorphisms (SNPs) obtained via restriction site‐associated DNA sequencing (RADSeq). Mitochondrial markers revealed a shared haplotype for COI and low divergence for CytB and CR between the sister‐species. On the other hand, complete lineage sorting was observed at the nuclear loci and most of the SNPs. Under neutral expectations, the smaller effective population size of mtDNA should lead to fixation of mutations faster than nDNA. Thus, these results suggest that hybridization in the recent past (0.174–0.263 Ma) led to introgression of the mtDNA, with little effect on the nuclear genome. Analyses of the SNP data revealed 28 loci potentially under divergent selection between the two species. The combination of mtDNA introgression and limited nuclear DNA introgression provides a mechanism for the evolution of independent lineages despite recurrent hybridization events. This study adds to the growing body of research that exemplifies how genetic divergence can be maintained in the presence of gene flow between closely related species.     

Slow Mitochondrial COI Sequence Evolution at the Base of the Metazoan Tree and Its Implications for DNA Barcoding

The evolution rates of mtDNA in early metazoans hold important implications for DNA barcoding. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and Scyphozoa) using a data set of 619 sequences from 224 species. We found variation within and between species to be much lower in Porifera and Anthozoa compared to Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans. Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution could have originated independently in Medusozoa and Bilateria or been acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding. Caution is also advised for Porifera and Hydrozoa because of relatively low identification success rates as even threshold divergence that maximizes the “barcoding gap” does not improve identification success. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Old and new DNA unweave the phylogenetic position of the eastern Atlantic gall crab Detocarcinus balssi (Monod, 1956) (Decapoda: Cryptochiridae)

Phylogeny reconstructions based on mtDNA and nDNA have become the standard in studies on relationships between taxa. Difficulties in obtaining material, for example because of small endemic distributions, often lead to gaps in datasets. Collections in natural history museums can provide us with material to fill these gaps, but extracting DNA from historical specimens can be challenging. We used a PCR protocol for small amounts of sample material and high PCR yield on eggs of specimens of the coral‐dwelling gall crab family Cryptochiridae collected in 1984, including material from the eastern Atlantic species Detocarcinus balssi. We obtained DNA sequences from seven older museum specimens using newly developed primers, which we combined with COI sequences from recently collected material. Results show that D. balssi is closest to the Indo‐Pacific species Utinomiella dimorpha and not closely related to one of the other three Atlantic Cryptochiridae species. The remaining newly acquired DNA sequences from museum material cluster with the respective sequences from recently collected specimens.     

Polymorphic population genetic markers for the Australian wood cockroach Panesthia australis

Primers for five polymorphic nuclear DNA (nDNA) markers and one mitochondrial DNA (mtDNA) gene (COI) were developed for the Australian wood cockroach Panesthia australis using a range of techniques. Eight mitochondrial haplotypes and four to 36 alleles per nuclear locus were detected in 744 cockroaches. Observed heterozygosity ranged from 0.117 to 0.816 in a sample of 30 animals from one population. The markers will be useful for population biology and for the measurement of the effects of habitat fragmentation on this ecologically important forest‐dependent species.     

Long‐term panmixia in a cosmopolitan Indo‐Pacific coral reef fish and a nebulous genetic boundary with its broadly sympatric sister species

Phylogeographical studies have shown that some shallow‐water marine organisms, such as certain coral reef fishes, lack spatial population structure at oceanic scales, despite vast distances of pelagic habitat between reefs and other dispersal barriers. However, whether these dispersive widespread taxa constitute long‐term panmictic populations across their species ranges remains unknown. Conventional phylogeographical inferences frequently fail to distinguish between long‐term panmixia and metapopulations connected by gene flow. Moreover, marine organisms have notoriously large effective population sizes that confound population structure detection. Therefore, at what spatial scale marine populations experience independent evolutionary trajectories and ultimately species divergence is still unclear. Here, we present a phylogeographical study of a cosmopolitan Indo‐Pacific coral reef fish Naso hexacanthus and its sister species Naso caesius, using two mtDNA and two nDNA markers. The purpose of this study was two‐fold: first, to test for broad‐scale panmixia in N. hexacanthus by fitting the data to various phylogeographical models within a Bayesian statistical framework, and second, to explore patterns of genetic divergence between the two broadly sympatric species. We report that N. hexacanthus shows little population structure across the Indo‐Pacific and a range‐wide, long‐term panmictic population model best fit the data. Hence, this species presently comprises a single evolutionary unit across much of the tropical Indian and Pacific Oceans. Naso hexacanthus and N. caesius were not reciprocally monophyletic in the mtDNA markers but showed varying degrees of population level divergence in the two nuclear introns. Overall, patterns are consistent with secondary introgression following a period of isolation, which may be attributed to oceanographic conditions of the mid to late Pleistocene, when these two species appear to have diverged.     

Effects of salinity on the survival and aggression of the invasive Rio Grande cichlid (Herichthys cyanoguttatus)

The spread of non-native Rio Grande cichlids (Herichthys cyanoguttatus) in southeast Louisiana includes brackish habitats. We studied the effects of three different salinity levels on the biology of juvenile H. cyanoguttatus for 13 months to determine the potential of this species to spread through local estuaries. The highest salinity tolerated was 30.0 psu, and these fish did not survive acclimation to the 32 psu treatment. Fish in brackish conditions grew slower than fish in freshwater conditions, indicating a potential long-term detriment to juvenile fish living in brackish conditions. Aggression levels were notably higher for fish in brackish (15 psu) vs. freshwater conditions. This persisted through the entire experiment even after acclimation back to freshwater. This study indicates that higher salinity habitats in Louisiana can be tolerated by this species. It also raises a question about the effect of higher salinities on aggressive behavior.     

Predator Generalization Decreases the Effect of Introduced Predators in the San Marcos Salamander,Eurycea nana

The introduction of novel predators into an environment can have detrimental consequences on prey species, especially if these species lack the ability to recognize these predators. One such species that may be negatively affected by introduced predators is the federally threatened San Marcos salamander (Eurycea nana). Previous research found that predator‐naïve (captive‐hatched) salamanders showed decreased activity in response to the chemical cues of both a native fish predator (Micropterus salmoides) and an introduced fish predator (Lepomis auritus), but not to a non‐predatory fish (Gambusia geiseri). We tested the hypothesis that E. nana recognized the introduced Lepomis (and other non‐native Lepomis) because they share chemical cues with other native congeneric Lepomis predators in the San Marcos River. We examined the antipredator response of predator‐naïve E. nana to chemical cues from (1) a sympatric native sunfish (Lepomis cyanellus; Perciformes: Centrarchidae); (2) a sympatric introduced sunfish (L. auritus); (3) an allopatric sunfish (Lepomis gibbosus); (4) a sympatric non‐native, non‐centrarchid cichlid (Herichthys cyanoguttatum; Perciformes: Cichlidae); and (5) a blank water control to determine whether individuals make generalizations about novel predators within a genus and across a family. Exposure to chemical cues from all fish predator treatments caused a reduction in salamander activity (antipredator response). Additionally, there were no differences in the antipredator responses to each predatory fish treatment. The similar responses to all sunfish treatments indicate that E. nana shows predator generalization in response to novel predators that are similar to recognized predators. Additionally, the antipredator response to H. cyanoguttatum indicates that predator generalization can occur among perciform families.     

Molecular patterns of differentiation in canyon treefrogs (Hyla arenicolor): evidence for introgressive hybridization with the Arizona treefrog (H. wrightorum) and correlations with advertisement call differences

Detection of genetic and behavioural diversity within morphologically similar species has led to the discovery of cryptic species complexes. We tested the hypothesis that US populations of the canyon treefrog (Hyla arenicolor) may consist of cryptic species by examining mate‐attraction signals among three divergent clades defined by mtDNA. Using a multi‐locus approach, we re‐analysed phylogenetic relationships among the three clades and a closely related, but morphologically and behaviourally dissimilar species, the Arizona treefrog (H. wrightorum). We found evidence for introgression of H. wrightorum’s mitochondrial genome into H. arenicolor. Additionally, the two‐clade topology based on nuclear data is more congruent with patterns of call variation than the three‐clade topology from the mitochondrial dataset. The magnitude of the call divergence is probably insufficient to promote isolation of the nuclear DNA‐defined clades should they become sympatric, but further divergence in call properties significant in species identification could promote speciation in the future.     

PERCHED AT THE MITO‐NUCLEAR CROSSROADS: DIVERGENT MITOCHONDRIAL LINEAGES CORRELATE WITH ENVIRONMENT IN THE FACE OF ONGOING NUCLEAR GENE FLOW IN AN AUSTRALIAN BIRD

Relationships among multilocus genetic variation, geography, and environment can reveal how evolutionary processes affect genomes. We examined the evolution of an Australian bird, the eastern yellow robin Eopsaltria australis, using mitochondrial (mtDNA) and nuclear (nDNA) genetic markers, and bioclimatic variables. In southeastern Australia, two divergent mtDNA lineages occur east and west of the Great Dividing Range, perpendicular to latitudinal nDNA structure. We evaluated alternative scenarios to explain this striking discordance in landscape genetic patterning. Stochastic mtDNA lineage sorting can be rejected because the mtDNA lineages are essentially distinct geographically for > 1500 km. Vicariance is unlikely: the Great Dividing Range is neither a current barrier nor was it at the Last Glacial Maximum according to species distribution modeling; nuclear gene flow inferred from coalescent analysis affirms this. Female philopatry contradicts known female‐biased dispersal. Contrasting mtDNA and nDNA demographies indicate their evolutionary histories are decoupled. Distance‐based redundancy analysis, in which environmental temperatures explain mtDNA variance above that explained by geographic position and isolation‐by‐distance, favors a nonneutral explanation for mitochondrial phylogeographic patterning. Thus, observed mito‐nuclear discordance accords with environmental selection on a female‐linked trait, such as mtDNA, mtDNA–nDNA interactions or genes on W‐chromosome, driving mitochondrial divergence in the presence of nuclear gene flow.