Lei Yang

For over a hundred years, the “river sharks” of the genus Glyphis
were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast
Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.

Recent surveys of the shark and ray catches of artisanal fishers in the Western Province of
Papua New Guinea (PNG) resulted in the rediscovery of the threatened river sharks, Glyphis
garricki and Glyphis glyphis. These represent the first records of both species in PNG
since the 1960s and 1970s and highlight the lack of studies of shark biodiversity in PNG.
Two individuals of G. garricki and three individuals of G. glyphis were recorded from coastal
marine waters of the Daru region of PNG in October and November 2014. The two G. garricki
specimens were small individuals estimated to be 100–105 cm and ~113 cm total
length (TL). The three G. glyphis specimens were all mature, one a pregnant female and
two adult males. These are the first adults of G. glyphis recorded to date providing a more
accurate maximum size for this species, i.e. ~260 cm TL. A single pup which was released
from the pregnant female G. glyphis, was estimated to be ~65 cm TL. Anecdotal information
from the fishers of pregnant females of G. glyphis containing 6 or 7 pups provides the first
estimate of litter size for this species. The jaws of the pregnant female G. glyphis were
retained and a detailed description of the dentition is provided, since adult dentition has not
been previously documented for this species. Genetic analyses confirmed the two species
cluster well within samples from these species collected in northern Australia.

Chondrichthyans are a class of fishes threatened with habitat destruction and fishing pressures. The current study presents the complete mitochondrial genome sequence (17,688 bp) of the vulnerable Hortle’s whipray, Himantura hortlei. The mitochondrial genome arrangement is consistent with that seen in most vertebrates, containing 13 protein-coding, 22 tRNA, 2 rRNA genes, and 1 control region. Phylogenetic analyses were performed based on mitogenome and ND2 sequences. Under our taxon sampling scheme, Himantura hortlei was found to be most closely related to H. fai.

Cyprininae is the largest subfamily (>1300 species) of the family Cyprinidae and contains more polyploid species (􏰀400) than any other group of fishes. We examined the phylogenetic relationships of the Cyprininae based on extensive taxon, geographical, and genomic sampling of the taxa, using both mitochondrial and nuclear genes to address the phylogenetic challenges posed by polyploidy. Four datasets were analyzed in this study: two mitochondrial gene datasets (465 and 791 taxa, 5604 bp), a mitogenome dataset (85 taxa, 14,771 bp), and a cloned nuclear RAG1 dataset (97 taxa, 1497 bp). Based on resulting trees, the subfamily Cyprininae was subdivided into 11 tribes: Probarbini (new; Probarbus + Catlocarpio), Labeonini Bleeker, 1859 (Labeo & allies), Torini Karaman, 1971 (Tor, Labeobarbus & allies), Smiliogastrini Bleeker, 1863 (Puntius, Enteromius & allies), Poropuntiini (Poropuntius & allies), Cyprinini Rafinesque, 1815 (Cyprinus & allies), Acrossocheilini (new; Acrossocheilus & allies), Spinibarbini (new; Spinibarbus), Schizothoracini McClelland, 1842 (Schizothorax & allies), Schizopygopsini Mirza, 1991 (Schizopygopsis & allies), and Barbini Bleeker, 1859 (Barbus & allies). Phylogenetic relationships within each tribe were dis- cussed. Two or three distinct RAG1 lineages were identified for each of the following tribes Torini, Cyprinini, Spinibarbini, and Barbini, indicating their hybrid origin. The hexaploid African Labeobarbus & allies and Western Asian Capoeta are likely derived from two independent hybridization events between their respective maternal tetraploid ancestors and Cyprinion.

Squatinid sharks are among the most threatened of cartilaginous fishes. Here we describe the complete mitochondrial genome sequence (16,690bp) of the Endangered Taiwanese angelshark, Squatina formosa. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement.

We determined the complete mitochondrial genome sequence (16,760bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

We determined the complete mitochondrial genome sequence from the holotype of the southern Mandarin dogfish Cirrhigaleus australis. It has a length of 16,543 bp and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The gene composition and genome organization is similar to most other vertebrates. Data obtained in this study will be important for resolving possible taxonomic issues related to C. australis and will contribute to the reconstruction of phylogenetic relationships among chondrichthyan species.

The complete mitochondrial genome of the blacknose shark Carcharhinus acronotus has been determined in this work. It has a length of 16,719 bp and consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The gene composition and genome organization was similar to other vertebrates. This study represents part of an ongoing effort to obtain mitochondrial genome sequences for chondrichthyan species in order to better estimate their phylogenetic relationships.

Evolutionary radiations, especially adaptive radiations, have been widely studied but mainly for recent events such as in cichlid fish or Anolis lizards. Here, we investigate the radiation of the subfamily Cyprininae, which includes more than 1300 species and is estimated to have originated from Southeast Asia around 55 Ma. In order to decipher a potential adaptive radi- ation, within a solid phylogenetic framework, we investigated the trophic apparatus, and especially the pharyngeal dentition, as teeth have proved to be important markers of ecological specialization. We compared two tribes within Cyprininae, Poropuntiini and Labeonini, displaying divergent dental patterns, as well as other characters related to their trophic apparatus. Our results suggest that the anatomy of the trophic apparatus and diet are clearly correlated and this explains the difference in dental patterns observed between these two tribes. Our results illustrate the diversity of mechanisms that account for species diversity in this very diverse clade: diversification of dental characters from an ancestral pattern on the one hand, conservation of a basal synapomorphy leading to ecological specialization on the other hand. By integrating morphological, ecological and phylogenetic analyses, it becomes possible to investigate ancient radiation events that have shaped the present diversity of species.

The cyprinid subfamily Oxygastrinae is composed of a diverse group of fishes that has been taxonomically and phyloge- netically problematic. Their great variation in appearance, life histories, and trophic diversity resulted in uncertainty re- garding their relationships, which led to their historical classification across many disparate subfamilies. The phylogenetic relationships of Oxygastrinae are resolved based on sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. A combined data matrix consisting of 4114 bp for 144 taxa was compiled and analyzed using maximum likelihood and parsimony optimality criteria. The subfamily Oxygastrinae is recovered as a monophyletic group that includes Ancherythroculter, Aphyocypris, Candidia, Chanodichthys, Ctenopharyngodon, Culter, Distoechodon, Elopichthys, Hainania, Hemiculter, Hemiculterella, Hemigrammocypris, Hypophthalmichthys, Ischikauia, Macrochirichthys, Megalobrama, Metzia, Mylopharyngodon, Nicholsicypris, Nipponocypris, Ochetobius, Opsariichthys, Oxygaster, Parabramis, Parachela, Paralaubuca, Pararasbora, Parazacco, Plagiognathops, Pseudobrama, Pseudohe- miculter, Pseudolaubuca, Sinibrama, Squaliobarbus, Toxabramis, Xenocyprioides, Xenocypris, Yaoshanicus, and Zacco. Of these genera, the following were found to be monophyletic: Aphyocypris, Distoechodon, Hypophthalmichthys, Nip- ponocypris, Opsariichthys, Parachela, Paralaubuca, Plagiognathops, Xenocyprioides, and Xenocypris. The following genera were not monophyletic: Metzia, Hemiculter, Toxabramis, Ancherythroculter, Chanodichthys, Culter, Megalobra- ma. The remainder are either monotypic or were represented by only a single species. Four genera not examined in this study are provisionally classified in Oxygastrinae: Anabarilius, Longiculter, Pogobrama, and Rasborichthys.

The cyprinid tribe Labeonini (sensu Rainboth, 1991) is a large group of freshwater fishes containing around 40 genera and 400 species. They are characterized by an amazing diversity of modifications to their lips and associated structures. In this study, a total of 34 genera and 142 species of putative mem- bers of this tribe, which represent most of the generic diversity and more than one third of the species diversity of the group, were sampled and sequenced for four nuclear genes and five mitochondrial genes (totaling 9465 bp). Phylogenetic relationships and subdivision of this tribe were investigated and the placement and status of most genera are discussed. Partitioned maximum likelihood analyses were per- formed based on the nuclear dataset, mitochondrial dataset, combined dataset, and the dataset for each nuclear gene. Inclusion of the genera Paracrossochilus, Barbichthys, Thynnichthys, and Linichthys in the Labeonini was either confirmed or proposed for the first time. None of the genera Labeo, Garra, Bangana, Cirrhinus, and Crossocheilus are monophyletic. Taxonomic revisions of some genera were made: the gen- eric names Gymnostomus Heckel, 1843, Ageneiogarra Garman, 1912 and Gonorhynchus McClelland, 1839 were revalidated; Akrokolioplax Zhang and Kottelat, 2006 becomes a junior synonym of Gonorhynchus; the species Osteochilus nashii was found to be a member of the barbin genus Osteochilichthys. Five histor- ical hypotheses on the classification of the Labeonini were tested and rejected. We proposed to subdivide the tribe, which is strongly supported as monophyletic, into four subtribes: Labeoina, Garraina, Osteochi- lina, and Semilabeoina. The taxa included in each subtribe were listed and those taxa that need taxo- nomic revision were discussed.

The traditionally accepted relationships among basal jawed vertebrates have been challenged by some molecular phylogenetic analyses based on mitochondrial sequences. Those studies split extant gnathostomes into two monophyletic groups: tetrapods and piscine branch, including Chondrichthyes, Actinopterygii and sarcopterygian fishes. Lungfish and bichir are found in a basal position on the piscine branch. Based on transcriptomes of an armored bichir (Polypterus delhezi) and an African lungfish (Protopterus sp.) we generated, expressed sequences and whole genome sequences available from public databases, we obtained 111 genes to reconstruct the phylogenetic tree of basal jawed vertebrates and estimated their times of divergence. Our phylogenomic study supports the traditional relationship. We found that gnathostomes are divided into Chondrichthyes and the Osteichthyes, both with 100% support values (posterior probabilities and bootstrap values). Chimaeras were found to have a basal position among cartilaginous fishes with a 100% support value. Osteichthyes were divided into Actinopterygii and Sarcopterygii with 100% support value. Lungfish and tetrapods form a monophyletic group with 100% posterior probability. Bichir and two teleost species form a monophyletic group with 100% support value. The previous tree, based on mitochondrial data, was significantly rejected by an approximately unbiased test (AU test, p = 0). The time of divergence between lungfish and tetrapods was estimated to be 391.8 Ma and the divergence of bichir from pufferfish and medaka was estimated to be 330.6 Ma. These estimates closely match the fossil record. In conclusion, our phylogenomic study successfully resolved the relationship of basal jawed vertebrates based on transtriptomes, EST and whole genome sequences.

The barbin genera Discherodontus Rainboth 1989, Chagunius Smith 1938 and Hypselobarbus Bleeker 1860 are distrib- uted in Southeast and South Asia and are among the least studied taxa of the order Cypriniformes. Few morphological studies have been conducted on these genera and only a very limited number of morphological characters were employed to hypothesize or infer their monophyly, inter-relationships, and relationships with other barbins. The main aim of this study is to examine the monophyly of these three genera and propose hypothesis of relationship among these taxa and other barbins based on a molecular phylogeny of the subfamily Cyprininae. A total of 106 cypriniform species were sam- pled, including 64 species and 31 genera of barbins collected from Eurasia and Africa. Partitioned maximum likelihood analysis was performed using DNA sequences derived from five mitochondrial genes (5601 bp): cytochrome c oxidase subunit I (COI), cytochrome b (Cyt b), 16S ribosomal RNA (16S), NADH dehydrogenase subunits 4 (ND4) and subunits 5 (ND5). The resulting phylogeny demonstrates that, under current taxon sampling, Discherodontus, Chagunius, and Hypselobarbus are all monophyletic genera. Together they do not form a monophyletic group, as hypothesized in previous studies, but are instead part of three distinct and unrelated clades. Discherodontus constitutes the basal lineage of a clade formed by Southeast Asian barbins (e.g. Poropuntius, Hypsibarbus, Balantiocheilos); Chagunius is basal to a clade formed mainly by Puntius and allies (although this relationship was only weakly supported); Hypselobarbus and Barbus carnaticus formed a clade sister to a clade including Tor, Neolissochilus, Labeobarbus, and Varicorhinus. Homoplasy and shared plesiomorphy of some hypothesized important morphological characters employed in previous studies that led re- searchers to hypothesize earlier relationships are discussed.

The members of the cyprinid subfamily Gobioninae, commonly called gudgeons, form one of the most well-established assemblages in the family Cyprinidae. The subfamily is a species-rich group of fishes, these fishes display diverse life histories, appearances, and behavior. The phylogenetic relationships of Gobioninae are examined using sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. This investigation produced a data matrix of 4114 bp for 162 taxa that was analyzed using parsimony, maximum likelihood, and Bayesian inference methods. The phylogenies our analyses recovered corroborate recent studies on the group. The subfamily Gobion- inae is monophyletic and composed of three major lineages. We find evidence for a Hemibarbus–Squalidus group, and the tribes Gobionini and Sarcocheilichthyini, with the Hemibarbus–Squalidus group sister to a clade of Gobionini–Sarcocheilichthyini. The Hemibarbus–Squalidus group includes those two genera; the tribe Sarcocheilichthyini includes Coreius, Coreoleuciscus, Gnathopogon, Gobiocypris, Ladislavia, Paracant- hobrama, Pseudorasbora, Pseudopungtungia, Pungtungia, Rhinogobio, and Sarcocheilichthys; the tribe Gobio- nini includes Abbottina, Biwia, Gobio, Gobiobotia, Huigobio, Microphysogobio, Platysmacheilus, Pseudogobio, Romanogobio, Saurogobio, and Xenophysogobio. The monotypic Acanthogobio is placed into the synonymy of Gobio. We tentatively assign Belligobio to the Hemibarbus–Squalidus group and Mesogobio to Gobionini; Paraleucogobio and Parasqualidus remain incertae sedis. Based on the topologies presented, the evolution of swim bladder specializations, a distinctive feature among cyprinids, has occurred more than once within the subfamily.

Carps (e.g. Koi) of the genus Cyprinus and Crucian carps (e.g. Goldfish) of the genus Car- assius are among the most popular freshwater fishes around the world. However, their phy- logenetic positions within the subfamily Cyprininae, relationships with their allies (e.g. Procypris, Carassioides), and the monophyly of the group formed by them and their allies, which is referred as the tribe Cyprinini sensu stricto, are far from clear. Historically, the Cyprinini was defined by different people according to whether a cyprinine fish possessed a spinous anal-fin ray (or anal spine), the spine was serrated or not, and occasionally, the number of branched dorsal-fin rays. Some definitions were established without providing any diagnostic characters. In this study, we investigated the monophyly of the tribe Cyprinini sensu stricto, based on four different historical definitions, and explored the phy- logenetic relationships of these members in the subfamily Cyprininae. Using five mito- chondrial genes as markers, both maximum-likelihood and Bayesian trees were constructed using the optimal partitioning strategy. Both analyses successfully resolved a monophyletic Cyprininae and recovered seven major clades from this subfamily. The diagnosis limiting the tribe Cyprinini sensu stricto to four genera, Cyprinus, Carassius, Carassioides and Procypris, received most support. We propose that only those cyprinines that possess a serrated anal spine and have no <10 branched dorsal-fin rays should be considered members of this tribe. Cyprinini is sister to the Sinocyclocheilus clade, a group traditionally considered a bar- bin, and together they form the ‘Cyprinini-Sinocyclocheilus’ clade. Procypris forms the basal clade of the Cyprinini, whereas species of Carassius and Carassioides locate at the top.

The Labeonini (sensu Rainboth, 1991) is a tribe of the subfamily Cyprininae, the largest subfamily of Cyp- riniformes. With around 400 species in 34 genera, this tribe is widely distributed in the freshwaters of tropical Africa and Asia. Most species are adapted to fast-flowing streams and rivers, and exhibit unique morphological modifications associated with their lips and other structures around the mouth. The monophyly of this tribe has been tested and generally accepted in previous morphological and molecular studies. The major objectives of this study were to reconstruct the phylogenetic relationships within the tribe Labeonini, test its monophyly and explore the taxonomic subdivisions, intrarelationships and bio- geography of the group. The value of the morphological characters associated with the lips and other associated structures in the taxonomic classification of labeonins was also discussed. Nucleotide sequences (3867 bp) of four unlinked nuclear loci were obtained from 51 species in 18 Labeonini genera from throughout the range of the tribe. Maximum parsimony, partitioned maximum likelihood and par- titioned Bayesian analyses were used for phylogenetic inference from combined and separate gene data sets. Based on our results, the monophyly of Labeonini was well supported. Two major clades could be recovered within the tribe. Three subclades could further be recognized from the first clade. These clades/subclades are not consistent with groupings of any of previous workers using either morphological or molecular characters for phylogenetic inference. Only five currently recognized genera in this analysis are monophyletic. The similarity between some lips and associated structures (e.g. suctorial discs) of labeonins may due to convergence or parallelism instead of common ancestry. Labeonins of Southeast Asia, India and China are closely related to each other; the multiple clades of African taxa do not form a single monophyletic group, indicating multiple, independent dispersal events of labeonins into Africa from Asia.

The order Cypriniformes is the most diverse clade of freshwater fishes and is natively distributed on all continents except South America, Australia, and Antarctica. Despite the diversity of the group and the fundamental importance of these species in both ecosystems and human culture, relatively little has been known about their relationships relative to their diversity. In recent years, with an international effort investigating the systematics of the group, more information as to their genealogical relationships has emerged and species discovery and their descriptions have increased. One of the more interesting aspects of this group has been a traditional lack of understanding of the relationships of the families, subfamilies, and other formally or informally identified groups. Historical studies have largely focused on smaller groups of species or genera. Because of the diversity of this group and previously published whole mito- chondrial genome evidence for relationships of major clades in the order, this clade serves as an excellent group to investigate the congruence between relationships reconstructed for major clades with whole mitogenome data and those inferred from a series of nuclear gene sequences. As descent has resulted in only one tree of life, do the phylogenetic relationships of these major clades converge on similar topol- ogies using the large number of available characters through this suite of nuclear genes and previously published mitochondrial genomes? In this study we examine the phylogenetic relationships of major clades of Cypriniformes using previously published mitogenomes and four putative single-copy nuclear genes of the same or closely related species. Combined nuclear gene sequences yielded 3810 bp, approx- imately 26% of the bp found in a single mitogenome; however homoplasy in the nuclear genes was mea- surably less than that observed in mitochondrial sequences. Relationships of taxa and major clades derived from analyses of nuclear and mitochondrial sequences were nearly identical and both received high support values. While some differences of individual gene trees did exist for species, it is predicted that these differences will be minimized with increased taxon sampling in future analyses.

The phylogenetic relationships of species are fundamental to any biological investigation, including all evolutionary studies. Accurate inferences of sister group relationships provide the researcher with an historical framework within which the attributes or geographic origin of species (or supraspecific groups) evolved. Taken out of this phylogenetic context, interpretations of evolutionary processes or origins, geographic distributions, or speciation rates and mechanisms, are subject to nothing less than a biological experiment without controls. Cypriniformes is the most diverse clade of freshwater fishes with estimates of diversity of nearly 3,500 species. These fishes display an amazing array of morphological, ecological, behavioral, and geographic diversity and offer a tremendous opportunity to enhance our understanding of the biotic and abiotic factors associated with diversification and adaptation to environments. Given the nearly global distribution of these fishes, they serve as an important model group for a plethora of biological investigations, including indicator species for future cli- matic changes. The occurrence of the zebrafish, Danio rerio, in this order makes this clade a critical component in understanding and predicting the relationship between mutagenesis and phenotypic expressions in vertebrates, including humans. With the tremendous diversity in Cypriniformes, our understanding of their phylogenetic relationships has not proceeded at an acceptable rate, despite a plethora of morphological and more recent mo- lecular studies. Most studies are pre-Hennigian in origin or include relatively small numbers of taxa. Given that analyses of small numbers of taxa for molecular characters can be compromised by peculiarities of long-branch attraction and nodal-density effect, it is critical that significant progress in our understanding of the relationships of these important fishes occurs with increasing sampling of species to mitigate these potential problems. The recent Cypriniformes Tree of Life initiative is an effort to achieve this goal with morphological and molecular (mitochondrial and nuclear) data. In this early synthesis of our understanding of the phylogenetic relationships of these fishes, all types of data have contributed historically to improving our understanding, but not all analyses are complementary in taxon sampling, thus precluding direct understanding of the impact of taxon sampling on achieving accurate phylogenetic inferences. However, recent molecular studies do provide some insight and in some instances taxon sampling can be implicated as a variable that can influence sister group relationships. Other instances may also exist but without inclusion of more taxa for both mitochondrial and nuclear genes, one cannot distinguish between inferences being dictated by taxon sampling or the origins of the molecular data.