Molecular phylogenetics of slit‐faced bats (Chiroptera: Nycteridae) reveal deeply divergent African lineages

The bat family Nycteridae contains only the genus Nycteris, which comprises 13 currently recognized species from Africa and the Arabian Peninsula, one species from Madagascar, and two species restricted to Malaysia and Indonesia in South‐East Asia. We investigated genetic variation, clade membership, and phylogenetic relationships in Nycteridae with broad sampling across Africa for most clades. We sequenced mitochondrial cytochrome b (cytb) and four independent nuclear introns (2,166 bp) from 253 individuals. Although our samples did not include all recognized species, we recovered at least 16 deeply divergent monophyletic lineages using independent mitochondrial and multilocus nuclear datasets in both gene tree and species tree analyses. Mean pairwise uncorrected genetic distances among species‐ranked Nycteris clades (17% for cytb and 4% for concatenated introns) suggest high levels of phylogenetic diversity in Nycteridae. We found a large number of designated clades whose members are distributed wholly or partly in East Africa (10 of 16 clades), indicating that Nycteris diversity has been historically underestimated and raising the possibility that additional unsampled and/or undescribed Nycteris species occur in more poorly sampled Central and West Africa. Well‐resolved mitochondrial, concatenated nuclear, and species trees strongly supported African ancestry for SE Asian species. Species tree analyses strongly support two deeply diverged subclades that have not previously been recognized, and these clades may warrant recognition as subgenera. Our analyses also strongly support four traditionally recognized species groups of Nycteris. Mitonuclear discordance regarding geographic population structure in Nycteris thebaica appears to result from male‐biased dispersal in this species. Our analyses, almost wholly based on museum voucher specimens, serve to identify species‐rank clades that can be tested with independent datasets, such as morphology, vocalizations, distributions, and ectoparasites. Our analyses highlight the need for a comprehensive revision of Nycteridae.     

Multilocus phylogenetics of a rapid radiation in the genus Thomomys (Rodentia: Geomyidae)

Species complexes undergoing rapid radiation present a challenge in molecular systematics because of the possibility that ancestral polymorphism is retained in component gene trees. Coalescent theory has demonstrated that gene trees often fail to match lineage trees when taxon divergence times are less than the ancestral effective population sizes. Suggestions to increase the number of loci and the number of individuals per taxon have been proposed; however, phylogenetic methods to adequately analyze these data in a coalescent framework are scarce. We compare two approaches to estimating lineage (species) trees using multiple individuals and multiple loci: the commonly used partitioned Bayesian analysis of concatenated sequences and a modification of a newly developed hierarchical Bayesian method (BEST) that simultaneously estimates gene trees and species trees from multilocus data. We test these approaches on a phylogeny of rapidly radiating species wherein divergence times are likely to be smaller than effective population sizes, and incomplete lineage sorting is known, in the rodent genus, Thomomys. We use seven independent noncoding nuclear sequence loci (total approximately 4300 bp) and between 1 and 12 individuals per taxon to construct a phylogenetic hypothesis for eight Thomomys species. The majority-rule consensus tree from the partitioned concatenated analysis included 14 strongly supported bipartitions, corroborating monophyletic species status of five of the eight named species. The BEST tree strongly supported only the split between the two subgenera and showed very low support for any other clade. Comparison of both lineage trees to individual gene trees revealed that the concatenation method appears to ignore conflicting signals among gene trees, whereas the BEST tree considers conflicting signals and downweights support for those nodes. Bayes factor analysis of posterior tree distributions from both analyses strongly favor the model underlying the BEST analysis. This comparison underscores the risks of overreliance on results from concatenation, and ignoring the properties of coalescence, especially in cases of recent, rapid radiations.     

Phylogeography of Rhinichthys cataractae (Teleostei: Cyprinidae): pre‐glacial colonization across the Continental Divide and Pleistocene diversification within the Rio Grande drainage

The longnose dace, Rhinichthys cataractae, is a primary freshwater fish inhabiting riffle habitats in small headwater rivers and streams across the North American continent, including drainages east and west of the Continental Divide. The mitochondrially encoded cytochrome b gene (1140 bp) and 2298–2346 bp of the nuclear‐encoded genes S7 and RAG1 were obtained from 87 individuals of R. cataractae (collected from 17 sites throughout its range) and from several close relatives. Phylogenetic analyses recovered a monophyletic R. cataractae species‐group that contained Rhinichthys evermanni, Rhinichthys sp. ‘Millicoma dace’, and a non‐exclusive R. cataractae. Within the R. cataractae species‐group, two well‐supported lineages were identified, including a western lineage (containing R. evermanni, R. sp. ‘Millicoma dace’ and individuals of R. cataractae from Pacific slope drainages) and an eastern lineage (containing individuals of R. cataractae from Arctic, Atlantic, and Gulf slope drainages). Within the eastern lineage of R. cataractae, two well‐supported groups were recovered: a south‐eastern group, containing individuals from the Atlantic slope, southern tributaries to the Mississippi River, and the Rio Grande drainage; and a north‐eastern group, containing individuals from the Arctic slope and northern tributaries to the Mississippi River. Estimates of the timing of divergence within the R. cataractae species‐group, combined with ancestral area‐reconstruction methods, indicate a separation between the eastern and western lineages during the Pliocene to early‐Pleistocene, with a direction of colonization from the west of the Continental Divide eastward. Within the southern portion of its range, R. cataractae likely entered the Rio Grande drainage during the Pleistocene via stream capture events between the Arkansas River (Mississippi River drainage) and headwaters of the Rio Grande. A close relationship between populations of R. cataractae in the Rio Grande drainage and the adjacent Canadian River (Mississippi River drainage) is consistent with hypothesized stream capture events between the Pecos (Rio Grande drainage) and Canadian rivers during the late‐Pleistocene. The population of R. cataractae in the lower Rio Grande may have become separated from other populations in the Rio Grande drainage (upper Rio Grande and Pecos River) and Canadian River during the late‐Pleistocene, well before initiation of recent and significant anthropogenic disturbance within the Rio Grande drainage. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 317–333.     

Phylogenies from genetic and morphological characters do not support a revision of Gigasporaceae (Glomeromycota) into four families and five genera

The family Gigasporaceae consisted of the two genera Gigaspora and Scutellospora when first erected. In a recent revision of this classification, Scutellospora was divided into three families and four genera based on two main lines of evidence: (1) phylogenetic patterns of coevolving small and large rRNA genes and (2) morphology of spore germination shields. The rRNA trees were assumed to accurately reflect species evolution, and shield characters were selected because they correlated with gene trees. These characters then were used selectively to support gene trees and validate the classification. To test this new classification, a phylogenetic tree was reconstructed from concatenated 25S rRNA and β-tubulin gene sequences using 35% of known species in Gigasporaceae. A tree also was reconstructed from 23 morphological characters represented in 71% of known species. Results from both datasets showed that the revised classification was untenable. The classification also failed to accurately represent sister group relationships amongst higher taxa. Only two clades were fully resolved and congruent among datasets: Gigaspora and Racocetra (a clade consisting of species with spores having one inner germinal wall). Other clades were unresolved, which was attributed in part to undersampling of species. Topology of the morphology-based phylogeny was incongruent with gene evolution. Five shield characters were reduced to three, of which two were phylogenetically uninformative because they were homoplastic. Therefore, most taxa erected in the new classification are rejected. The classification is revised to restore the family Gigasporaceae, within which are the three genera Gigaspora, Racocetra, and Scutellospora. This classification does not reflect strict topology of either gene or morphological evolution. Further revisions must await sampling of additional characters and taxa to better ascertain congruence between datasets and infer a more accurate phylogeny of this important group of fungi.     

Species tree estimation for a deep phylogenetic divergence in the New World monkeys (Primates: Platyrrhini)

The estimation of a robust phylogeny is a necessary first step in understanding the biological diversification of the platyrrhines. Although the most recent phylogenies are generally robust, they differ from one another in the relationship between Aotus and other genera as well as in the relationship between Pitheciidae and other families. Here, we used coding and non-coding sequences to infer the species tree and embedded gene trees of the platyrrhine genera using the Bayesian Markov chain Monte Carlo method for the multispecies coalescent (?BEAST) for the first time and to compared the results with those of a Bayesian concatenated phylogenetic analysis. Our species tree, based on all available sequences, shows a closer phylogenetic relationship between Atelidae and Cebidae and a closer relationship between Aotus and the Cebidae clade. The posterior probabilities are lower for these conflictive tree nodes compared to those in the concatenated analysis; this finding could be explained by some gene trees showing no concordant topologies between Aotus and the other genera. Moreover, the topology of our species tree also differs from the findings of previous molecular and morphological studies regarding the position of Aotus. The existence of discrepancies between morphological data, gene trees and the species tree is widely reported and can be related to processes such as incomplete lineage sorting or selection. Although these processes are common in species trees with low divergence, they can also occur in species trees with deep and rapid divergence. The sources of the inconsistency of morphological and molecular traits with the species tree could be a main focus of further research on platyrrhines.     

Mitochondrial DNA Variation, Phylogenetic Relationships, and Evolution of Four Mediterranean Genera of Soles (Soleidae, Pleuronectiformes)

To increase knowledge about the systematics and evolution of Mediterranean soles, we assessed mitochondrial DNA variation, molecular phylogeny, and evolution in eight species from the genera Solea, Microchirus, Monochirus, and Buglossidium by large ribosomal subunit (16S) and cytochrome b (cytb) sequence analysis. Relevant molecular features are the great variation of base composition among species at the third codon in cytb and the heterogeneity of the nucleotide substitution rate. Phylogenies recovered using 16S nucleotide and cytb amino acid sequences agree with those based on morphology in assessing monophyly of Solea species and ancestry of Buglossidium luteum, but they are against the intergeneric differentiation of Microchirus and Monochirus. Conversely, phylogenetic trees based on cytb nucleotide sequences yielded relationships among taxa regardless of their evolutionary histories. The incongruities between morphological and molecular issues suggest the need for reassessing the systematic value of some morphological characters. Approximate estimates of the divergence time of Mediterranean soleid lineages range from 40 to 13 Mya (Oligocene–Miocene), indicating an ancient origin for the group. Received August 31, 1999; accepted December 17, 1999.     

Molecular and morphometric data provide evidence of intraspecific variation in shape and pigmentation pattern in Otocinclus cocama (Siluriformes: Loricariidae) across major river drainages

Otocinclus cocama, a uniquely colored species of the loricariid catfish genus Otocinclus described solely from the type locality in the lower Ucayali River in northern Peru, is reported occurring in the Tigre River, a tributary to the Marañón River that drains a different section of the Andean Mountain range in the western Amazon. Both populations differ in the number of dark bars spanning the flanks of the body, and we investigated whether these morphotypes constitute distinct species. The body shapes of populations from the Tigre and Ucayali rivers were compared using geometric morphometrics. Although principal component analysis detected a broad overlap between populations, multivariate analysis of variance and linear driscriminat analysis revealed a subtle differentiation between the populations of the two hydrographic basins. Average body shape of the Ucayali River population tend to be slightly higher than that of the Tigre River, with the caudal peduncle stretched vertically in the Ucayali population. Multivariate regression of shape and centroid size revealed an allometric effect of 10.7% (p < 0.001), suggesting that the variation between Tigre and Ucayali populations was purely shape variation. Molecular data of coI, cytb, nd2, and 16S mitochondrial genes indicated a nucleotide diversity range from 0.001 to 0.003, and haplotypic diversity range from 0.600 ± 0.11 to 0.79 ± 0.07. The median-joining haplotype network for the concatenated matrix exhibited two divergent haplogroups related to the geographic area and separated by <10 mutational steps. The molecular species delimitation methods based on distance (automatic barcode gap discovery and assemble species by automatic partitioning) recovered two molecular lineages evolving independently, being one of the lineages formed by individuals from both populations. Tree-based methods (generalized mixed Yule coalescent and Bayesian implementation of the Poisson tree process) recovered similar topologies and supported single lineage recognition. Methods of molecular delimitation of species disclosed the high similarity between the two populations of Otocinclus cocama, further supported by the presence of old haplotypes common to both groups which could indicate that the populations still maintain gene flow. Although the morphological data reveal a subtle variation between both river basins, the molecular data suggest a weak population structuration based on hydrographic areas, but not different species lineages, therefore Otocinclus cocama is composed of a single lineage with two distinct morphotypes.     

Higher‐level phylogenetic affinities of the Neotropical genus Mastigodryas Amaral, 1934 (Serpentes: Colubridae), species‐group definition and description of a new genus for Mastigodryas bifossatus

Most Neotropical colubrid snakes belong to a single, well‐supported lineage. Relationships between the major constituents of this clade remain. Here, we explore the phylogenetic relationships of Mastigodryas and its affinities to other Neotropical colubrid genera by combining DNA and morphological data. Analyses demonstrate that the concatenation of multiple individuals into a single terminal can mask the detection of new taxa. Further, non‐random missing data and/or taxa in some empirical datasets can bias species tree analyses more than concatenation approaches. Our results place Mastigodryas in a strongly supported clade that includes Drymarchon, Rhinobothryum, Drymoluber, Simophis and Leptodrymus. Mastigodryas bifossatus is more closely related to species of Drymoluber and Simophis than to its congeners. Thus, we erect a new genus to accommodate it and recover a monophyletic Mastigodryas. We highlight the importance of the use of morphological characters to diagnose suprageneric clades by showing that some key external and hemipenial characteristics are phylogenetically informative.     

Phylogenetic analysis of RAD‐seq data: examining the influence of gene genealogy conflict on analysis of concatenated data

One of the major issues in phylogenetic analysis is that gene genealogies from different gene regions may not reflect the true species tree or history of speciation. This has led to considerable debate about whether concatenation of loci is the best approach for phylogenetic analysis. The application of Next‐generation sequencing techniques such as RAD‐seq generates thousands of relatively short sequence reads from across the genomes of the sampled taxa. These data sets are typically concatenated for phylogenetic analysis leading to data sets that contain millions of base pairs per taxon. The influence of gene region conflict among so many loci in determining the phylogenetic relationships among taxa is unclear. We simulated RAD‐seq data by sampling 100 and 500 base pairs from alignments of over 6000 coding regions that each produce one of three highly supported alternative phylogenies of seven species of Drosophila. We conducted phylogenetic analyses on different sets of these regions to vary the sampling of loci with alternative gene trees to examine the effect on detecting the species tree. Irrespective of sequence length sampled per region and which subset of regions was used, phylogenetic analyses of the concatenated data always recovered the species tree. The results suggest that concatenated alignments of Next‐generation data that consist of many short sequences are robust to gene tree/species tree conflict when the goal is to determine the phylogenetic relationships among taxa.     

Multi‐locus phylogenetics,lineage sorting,and reticulation in Pinus subsection Australes

Premise of the Study

Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long‐lived, wind‐pollinated plants with large population sizes and weak reproductive barriers.

Methods

We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low‐copy‐number nuclear genes and high‐copy‐number plastomes (Hyb‐Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation.

Key Results

Concatenation‐ and coalescent‐based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed‐cone pines) and Oocarpae (the egg‐cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non‐monophyly of the Attenuatae in the plastid tree was equivocal.

Conclusions

Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow.     

Embracing discordance: Phylogenomic analyses provide evidence for allopolyploidy leading to cryptic diversity in a Mediterranean Campanula (Campanulaceae) clade

The Mediterranean Basin harbors a remarkable amount of biodiversity, a high proportion of which is endemic to this region. Here, we present an in‐depth study of an angiosperm species complex, in which cryptic taxonomic diversity has been hypothesized. Specifically, we focus on four currently recognized species in the Roucela complex, a well‐supported clade in the Campanulaceae/Campanuloideae: Campanula creutzburgii, C. drabifolia, C. erinus, and C. simulans. This study takes a phylogenomic approach, utilizing near‐complete plastomes and 130 nuclear loci, to uncover cryptic diversity and test hypotheses regarding hybridization and polyploidy within this clade. Genome size estimates recovered tetraploid and octoploid lineages within the currently recognized, widespread species C. erinus, showing an east‐west geographic pattern. Though genomic data clearly differentiate these two cytotypes, we failed to discern morphological differences. The formation of a cryptic octoploid lineage, distributed across the eastern Mediterranean, is hypothesized to be the result of an allopolyploid event in which one parental morphology is retained. The tetraploid C. erinus and C. creutzburgii (also a tetraploid) are implicated as parental lineages. Our results highlight the utility of target‐enrichment approaches for obtaining genomic datasets for thorough assessments of species diversity and the importance of carefully considering gene‐tree discordance within such datasets.     

Mitochondrial phylogeny and biogeography of the genus Gobio (Teleostei: Cyprinidae) in Turkey

Species of the cyprinid genus Gobio Cuvier, 1816 are widely distributed in freshwater lakes and rivers in Turkey, which is a hotspot for freshwater fish diversity and endemism. The mitochondrial 16S, coxI and cytb genes were sequenced for 217 individuals representing 15 species of Gobio from Turkey. A total of 23 haplotypes were identified for each mitochondrial gene. The genetic distance matrices show that Turkish Gobio species are clustered into three groups (northwestern, central and northeastern Anatolia). Phylogenetic trees constructed with combined dataset by using maximum parsimony, maximum likelihood and Bayesian inference methods revealed that the Turkish Gobio species belongs to three well-supported groups in accordance with their geographic distribution: Group I comprises species found in Central Anatolia, Group II species ranging from eastern Thrace to western Anatolia (northwestern Group) and Group III contains only one species from Western Transcaucasia (northeastern Group). The estimated divergence times between the three Gobio groups, calculated using a conventional 1% rate of mutation for a fish mitochondrial cytb gene per million years, coincide with the late Miocene period in which the tectonic uplift of Anatolia and global climate fluctuations occurred. The relatively low genetic distance between Gobio species in the Turkish Lake District indicate that they are not good species.     

Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae)

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18 mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate.     

A comprehensive molecular phylogeny of tiger beetles (Coleoptera,Carabidae, Cicindelinae)

Tiger beetles are a remarkable group that captivates amateur entomologists, taxonomists and evolutionary biologists alike. This diverse clade of beetles comprises about 2300 currently described species found across the globe. Despite the charisma and scientific interest of this lineage, remarkably few studies have examined its phylogenetic relationships with large taxon sampling. Prior phylogenetic studies have focused on relationships within cicindeline tribes or genera, and none of the studies have included sufficient taxon sampling to conclusively examine broad species patterns across the entire subfamily. Studies that have attempted to reconstruct higher‐level relationships of Cicindelinae have yielded conflicting results. Here, we present the first taxonomically comprehensive molecular phylogeny of Cicindelinae to date, with the goal of creating a framework for future studies focusing on this important insect lineage. We utilized all available published molecular data, generating a final concatenated dataset including 328 cicindeline species, with molecular data sampled from six protein‐coding gene fragments and three ribosomal gene fragments. Our maximum‐likelihood phylogenetic inferences recover Cicindelinae as sister to the wrinkled bark beetles of the subfamily Rhysodinae. This new phylogenetic hypothesis for Cicindelinae contradicts our current understanding of tiger beetle phylogenetic relationships, with several tribes, subtribes and genera being inferred as paraphyletic. Most notably, the tribe Manticorini is recovered nested within Platychilini including the genera Amblycheila Say, Omus Eschscholtz, Picnochile Motschulsky and Platychile Macleay. The tribe Megacephalini is recovered as paraphyletic due to the placement of the monophyletic subtribe Oxycheilina as sister to Cicindelini, whereas the monophyletic Megacephalina is inferred as sister to Oxycheilina, Cicindelini and Collyridini. The tribe Collyridini is paraphyletic with the subtribes Collyridina and Tricondylina in one clade, and Ctenostomina in a second one. The tribe Cicindelini is recovered as monophyletic although several genera are inferred as para‐ or polyphyletic. Our results provide a novel phylogenetic framework to revise the classification of tiger beetles and to encourage the generation of focused molecular datasets that will permit investigation of the evolutionary history of this lineage through space and time.     

TESTING MITOCHONDRIAL CAPTURE AND DEEP COALESCENCE IN AMAZONIAN CICHLID FISHES (CICHLIDAE: CICHLA)

Hybridization and introgression have important consequences in evolution, such as increasing the genetic diversity and adaptive potential of a species. One of their most conspicuous footprints is discordance among gene trees or between genes and phenotypes. However, most studies that report introgression fail to disprove the null hypothesis that genetic incongruence may result from stochastic sorting of ancestral allelic polymorphisms. In the case of ancient introgression, these two processes may be especially difficult to distinguish topologically, but they make different predictions about the patterns of coalescence among loci. Here we apply three methods, molecular dating, multispecies coalescent models, and gene tree simulation under coalescence, to compare these two hypotheses that explain the polyphyletic mtDNA of the butterfly peacock bass, Cichla orinocensis. In comparison with a species tree based on 20 unlinked nuclear loci, we determined that mtDNA divergences were too recent to be explained by ancestral polymorphism. Similarly, coalescent species tree branches were significantly shorter when putative introgressed mtDNA was incorporated, and simulations showed the mtDNA topology to be unlikely under lineage sorting only. We conclude that introgression approximately 1.5 million years ago resulted in capture by C. orinocensis of an mtDNA lineage ancestral to the modern subspecies C. oc. monoculus.     

Comparative Phylogeography of Mississippi Embayment Fishes

The Mississippi Embayment is a prominent physiographic feature of eastern North America consisting of primarily lowland aquatic habitats and a fish fauna that is largely distinct from nearby highland regions. Numerous studies have demonstrated that both pre-Pleistocene and Pleistocene events have had a strong influence on the distributions and relationships of highland fishes in eastern North America. However, the extent to which these same events affected Embayment distributed taxa remains largely unexplored. The purpose of this study was to investigate the relative roles of pre-Pleistocene and Pleistocene events in shaping phylogeographic relationships of four stream dwelling fishes in the Mississippi Embayment. Molecular genetic analyses of the mitochondrial gene cytochrome b were performed for three ictalurid catfish species (Noturus miurus, n = 67; Noturus hildebrandi, n = 93, and Noturus phaeus, n = 44) and one minnow species (Cyprinella camura, n = 78), all distributed in tributary streams of the Mississippi Embayment. Phylogenetic relationships and divergence times among haplotypes for each species were estimated using maximum likelihood and Bayesian methods. Phylogenetic analyses recovered 6 major haplotype clades within N. miurus, 5 within N. hildbrandi, 8 within N. phaeus, and 8 within C. camura. All three Noturus species show a high degree of isolation by drainage, which is less evident in C. camura. A clade of haplotypes from tributaries in the southern portion of the Mississippi Embayment was consistently recovered in all four species. Divergence times among clades spanned the Pleistocene, Pliocene, and Miocene. Novel relationships presented here for C. camura and N. phaeus suggest the potential for cryptic species. Pre-Pleistocene and Pleistocene era sea level fluctuations coincide with some divergence events, but no single event explains any common divergence across all taxa. Like their highland relatives, a combination of both pre-Pleistocene and Pleistocene era events have driven divergences among Embayment lineages.     

Genetic Variations in Two Seahorse Species (Hippocampus mohnikei and Hippocampus trimaculatus): Evidence for Middle Pleistocene Population Expansion

Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b (cytb) and control region (CR) were obtained from 50 Hippocampus mohnikei and 92 H. trimaculatus from four zoogeographical zones. A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments.     

Picking holes in traditional species delimitations: an integrative taxonomic reassessment of the Parmotrema perforatum group (Parmeliaceae,Ascomycota)

Accurate species delimitation is important as species are a fundamental unit in ecological, evolutionary and conservation biology research. In lichenized fungi, species delimitation has been difficult due to a lack of taxonomically important characteristics and due to the limits of traditional, morphology‐based species concepts. In this study we reassess the current taxonomy of the Parmotrema perforatum group, which recognizes six closely related species divided into three species pairs, each pair comprising one apotheciate (sexual) and one sorediate (asexual) species. Each pair is further characterized by a distinct combination of secondary metabolites. It was hypothesized that the three apotheciate species are reproductively isolated sibling species and that each sorediate species evolved once from the chemically identical apotheciate species. In this study, species boundaries were re‐examined using an integrative approach incorporating morphological, chemical and molecular sequence data to delimit species boundaries. Phylogenetic trees were inferred from a seven‐locus DNA sequence dataset using concatenated gene tree and coalescent‐based species‐tree inference methods. Furthermore, we employed a multi‐species coalescent method to validate candidate species. Micromorphological measurements of conidia were found to be congruent with phylogenetic clusters. Each approach that we applied to the P. perforatum group consistently recovered four of the currently circumscribed species (P. perforatum, P. hypotropum, P. subrigidum and P. louisianae), whereas P. preperforatum and P. hypoleucinum were consistently combined and are thus interpreted as conspecific.