Long-term ecological research in freshwaters enabled by regional biodiversity collections, stable isotope analysis, and environmental informatics.

Biodiversity collections are experiencing a renaissance fueled by the intersection of informatics, emerging technologies, and the extended use and interpretation of specimens and archived databases. In this article, we explore the potential for transformative research in ecology integrating biodiversity collections, stable isotope analysis (SIA), and environmental informatics. Like genomic DNA, SIA provides a common currency interpreted in the context of biogeochemical principles. Integration of SIA data across collections allows for evaluation of long-term ecological change at local to continental scales. Challenges including the analysis of sparse samples, a lack of information about baseline isotopic composition, and the effects of preservation remain, but none of these challenges is insurmountable. The proposed research framework interfaces with existing databases and observatories to provide benchmarks for retrospective studies and ecological forecasting. Collections and SIA add historical context to fundamental questions in freshwater ecological research, reference points for ecosystem monitoring, and a means of quantitative assessment for ecosystem restoration.

Using low-coverage whole genome sequencing (genome skimming) to delineate three introgressed species of buffalofish (Ictiobus).

Consumption of buffalofish has been sporadically associated with Haff disease-like illnesses involving sudden onset muscle pain and weakness due to skeletal muscle rhabdomyolysis, but determination of precisely which species are associated with these illnesses has been impeded by a lack of species-specific DNA-based markers. Here, three closely related species of buffalofish native to the Mississippi River Basin (Ictiobus bubalus, Ictiobus cyprinellus and Ictiobus niger) that have previously proven genetically indistinguishable using both mitochondrial and nuclear single-locus sequencing were reliably discriminated using low-coverage whole genome sequencing ('genome skimming'). Using 44 specimens representing the three species collected from the mid/upper (Missouri) and lower (Louisiana) regions of the species' native ranges, the SISRS (Site Identification from Short Read Sequences) bioinformatics pipeline was adapted to (1) identify over 620Mbp of putatively homologous nuclear sequence data and (2) isolate over 140,000 single-nucleotide polymorphisms (SNPs) that supported accurate species delimitation, all without the use of a reference genome or annotation data. These sites were used to classify Ictiobus spp. samples with genome-skim data, along with a larger set (n = 67) where ultraconserved elements (UCEs) were sequenced. Analyses of whole mitochondrial data revealed more limited signal. Nearly all samples matched their purported species based on morphologic identification, but two Missouri samples morphologically identified as I. niger grouped with samples of I. bubalus, albeit with significant enrichment of I. niger SNPs. To our knowledge this is the first report of a DNA-based tool to reliably discriminate these three morphologically distinct species.

Haff disease associated with consumption of buffalofish (Ictiobus spp.) in the United States, 2010-2020, with confirmation of the causative species.

BACKGROUND: In the United States, buffalofish (Ictiobus spp.) are sporadically associated with sudden onset muscle pain and weakness due to rhabdomyolysis within 24 h of fish consumption (Haff disease). Previous genetic analyses of case-associated samples were unable to distinguish the three species of buffalofish that occur in the US, Ictiobus cyprinellus (bigmouth buffalo), Ictiobus bubalus (smallmouth buffalo), and Ictiobus niger (black buffalo). METHODS: Ten events were investigated between 2010 and 2020 and demographic and clinical information was collected for 24 individuals. Meal remnants were collected from 5 of 10 events with additional associated samples (n = 24) collected from another five of 10 events. Low-coverage whole-genome sequencing (genome skimming) was used to identify meal remnants. RESULTS: Patients (26-75 years of age) ranged from 1-4 per event, with 90% involving ≥2 individuals. Reported symptoms included muscle tenderness and weakness, nausea/vomiting, and brown/tea-colored urine. Median incubation period was 8 h. Ninety-six percent of cases were hospitalized with a median duration of four days. The most commonly reported laboratory finding was elevated creatine phosphokinase and liver transaminases. Treatment was supportive including intravenous fluids to prevent renal failure. Events occurred in California (1), Illinois (2), Louisiana (1), New York (1), Mississippi (1), Missouri (2), New Jersey (1), and Texas (1) with location of harvest, when known, being Illinois, Louisiana, Mississippi, Missouri, Texas, and Wisconsin. Meal remnants were identified as I. bubalus (n = 4) and I. niger (n = 1). Associated samples were identified as I. bubalus (n = 16), I. cyprinellus (n = 5), and I. niger (n = 3). DISCUSSION: Time course, presentation of illness, and clinical findings were all consistent with previous domestic cases of buffalofish-associated Haff disease. In contrast to previous reports that I. cyprinellus is the causative species in US cases, data indicate that all three buffalofish species are harvested but I. bubalus is most often associated with illness.

Tube-like structures within the ovarian follicle of Hydrolagus colliei indicate the early origins of follicle cell processes.

Follicle cell processes (FCP) are actin-based, tube-like structures that connect the developing oocyte to the follicle cells throughout oogenesis. They were first described in Selachians (sharks) where their suggested roles were facilitating the transport of metabolites to the developing oocyte and providing structural support to the large egg cells of sharks, an early stage in the evolution of viviparity. Subsequent studies found that FCP are absent in Rajiformes (skates), suggesting that FCP may have been novel structures specific to the sharks. Here, FCP in Hydrolagus colliei, a Chimaeriform, were described. The FCP of H. colliei differ morphologically from those previously described in sharks, but as they also contain actin, they presumably play similar roles provisioning the developing oocyte and providing structural support. The presence of FCP in the order Chimaeriformes suggests that their origin predates the split of the elasmobranchs and the holocephalans.

A new Western North Atlantic Ocean kitefin shark (Squaliformes: Dalatiidae) from the Gulf of Mexico.

A new species of kitefin shark (Squaliformes; Dalatiidae) is described from the Gulf of Mexico (Western North Atlantic Ocean) based on five diagnostic features not seen on the only other known Mollisquama specimen, the holotype of Mollisquama parini Dolganov which was captured in the Eastern South Pacific Ocean. The new species, Mollisquama mississippiensis sp. nov., is distinguished from its congener by a putative pit organ located ventrally just posterior of the lower jaw margin center, photophores irregularly distributed along many areas of the body, 16 distinct ventral-abdominal photophore aggregations, and two differences associated with the dentition. Other potential distinguishing features are 10 fewer vertebrae than Mollisquama parini and six morphometric proportional differences that exceeded +/- 20% from the holotype.

A new species of cyprinoid fish from the Tana River, Kenya (Actinopterygii: Danionidae).

Sampling of streams in the middle reaches of the Tana River Basin in Meru National Park, Kenya, from 2010 to 2012 for an NSF-funded International Research Experiences for Students (IRES) project, resulted in the capture of a number of specimens of what were first thought to be Neobola fluviatilis.  On closer examination the specimens were determined to represent a distinct species, endemic to the Tana River basin, which is herein formally described. The new species is readily diagnosed from N. fluviatilis by higher counts of lateral line, pre-dorsal, and caudal peduncle circumferential scales, higher numbers of pectoral rays, lower numbers of anal fin rays, and a shorter anal-fin base length.

Integrative taxonomy of the red-finned barb, Enteromius apleurogramma (Cyprininae: Smiliogastrini) from Kenya, supports recognition of E. amboseli as a valid species.

Research on freshwater ecosystems in East Africa is providing a better understanding of the biodiversity in the region. Recent studies of the Kenyan barbs (Cyprininae: Smiliogastrini) revealed diversity within several nominal species from the region. In this study, we examine the molecular and morphological variation in the red-finned barb (Enteromius apleurogramma). The results of this study support the recognition of E. amboseli as a valid species that is endemic to the middle Athi River drainage in southern Kenya. Enteromius amboseli is geographically isolated and distinguished from E. apleurogramma in having longer posterior barbels, a shorter dorsal fin, and generally fewer scales in the longitudinal series. Genetic divergence between E. apleurogramma populations in Lake Victoria and Lake Kanyaboli, provide novel estimates for rates of molecular evolution in the group. Additionally, the biogeography of these barbs and the conservation status of E. amboseli are discussed.

Cranial morphology in Mollisquama sp. (Squaliformes; Dalatiidae) and patterns of cranial evolution in dalatiid sharks.

Dalatiid sharks are members of a family of predominantly small, midwater meso- and bathypelagic chondrichthyans. The family is notable for both its number of monotypic genera and high morphological disparity. Three of the seven dalatiid genera are known only from holotype specimens (Mollisquama parini) or from only a handful of specimens (Euprotomicroides zantedeschia, Heteroscymnoides marleyi), with the only detailed anatomical work consistent across all taxa being studies of dentition. Here, we present detailed anatomical description of the second-ever specimen of Mollisquama (Mollisquama sp.) covering chondrocranial, jaw, dental, and muscular anatomy, derived from a phase-contrast synchrotron microtomographic scan. Mollisquama sp. is unique among dalatiids in possessing a deep carinal process, extending ventrally from the bar between the subethmoid region and basal angle in squaloid sharks, containing a large fenestra infiltrated by the suborbitalis muscle. Mollisquama sp. also exhibits additional possibly diagnostic features, including a planar configuration of the labial cartilages and the absence of labial folds; a pad-like orbital process on the palatoquadrate; and the origination of the suborbitalis muscle solely on the carina, rather than the intraorbital wall. Character optimization of anatomical data onto a phylogeny of dalatiid sharks suggests Mollisquama sp. to be among the most specialized in the family, expanding the existing dalatiid morphospace. However, the functional significance of such transformations remains unclear. Synchrotron-derived data, which do not require chemical pretreatment of specimens, may elucidate soft-tissue functional correlates in future studies of undersampled taxa, such as dalatiids.

Multi-locus phylogeny reveals instances of mitochondrial introgression and unrecognized diversity in Kenyan barbs (Cyprininae: Smiliogastrini).

The phylogenetics and taxonomic status of small African barbs (Cyprininae: Smiliogastrini) remains unresolved despite the recent decision to elevate the genus name Enteromius for the group. The main barrier to understanding the origin of African small barbs and evolutionary relationships within the group is the poor resolution of phylogenies published to date. These phylogenies usually rely on mitochondrial markers and have limited taxon sampling. Here we investigate the phylogenetic relationships of small barbs of Kenya utilizing cytochrome b, Growth Hormone (GH) intron 2, and RAG1 markers from multiple populations of many species in the region. This multi-locus study produced well-supported phylogenies and revealed additional issues that complicate understanding the relationships among East African barbs. We observed widespread mtDNA introgression within the Kenyan barbs, highlighting the need to include nuclear markers in phylogenetic studies of the group. The GH intron 2 resolved heterospecific individuals and aided in inferring the species level phylogeny. The study reveals unrecognized diversity within the group, including within species reported to occur throughout East Africa, and it provides the groundwork for future taxonomic work in the region and across Africa.

High levels of endemism in suckermouth catfishes (Mochokidae: Chiloglanis) from the Upper Guinean forests of West Africa.

Freshwater systems are under threat globally, yet the biodiversity in many areas is still unknown. This is especially true for the aquatic biodiversity of the Upper Guinean forests of Guinea, Sierra Leone, Liberia, and Côte d'Ivoire. Access to this area is largely restricted, though recent surveys by the authors are allowing us to reassess the area's diversity. This area has vast mineral reserves and hydroelectric potential. As the area emerges from civil strife and the recent public health crisis, policy makers and resource managers require accurate accounts of biodiversity to evaluate future development projects. Here we look at the diversity of the suckermouth catfishes (Chiloglanis) populations from the area; inferred from mitochondrial (cyt b) and nuclear (Growth Hormone intron) markers. The phylogenies revealed additional lineages, independent of the currently recognized taxa, suggesting the presence of ten new candidate species. These new taxa are largely endemic and allopatrically distributed in rivers of the Upper Guinean forests. Our results suggest that the aquatic diversity within the Upper Guinean forests is currently underestimated. This study provides the foundation for elucidating the historical biogeography of the region and highlights the endemism within rivers in the Upper Guinean forests and surrounding areas.

Two new species of African suckermouth catfishes, genus Chiloglanis (Siluriformes: Mochokidae), from Kenya with remarks on other taxa from the area.

Recent expeditions in Kenya and examination of existing collections confirmed the presence of two undescribed Chiloglanis species and revealed previously unknown diversity within the Athi River system. The two new species are easily distinguished from described congeners in the area by external morphology, allopatric distributions, and genetic markers. Chiloglanis kerioensis sp. nov., is restricted to the Kerio River system and is the only known suckermouth catfish from the Lake Turkana basin. Chiloglanis devosi sp. nov., is known only from the type locality, the Northern Ewaso Nyiro (Ng'iro) below Chanler's Falls. In addition to these two new species, this study confirmed the presence of an undescribed Chiloglanis sp. occurring sympatrically with Chiloglanis brevibarbis in the Tsavo River. A dichotomous key for identifying all described Chiloglanis species found within Kenya is presented along with comments.

Nomenclatural changes should not be based on equivocally supported phylogenies: Reply to Yang et al. 2015.

Phylogenies produced by Yang et al. 2015 provide reasonably well-supported hypotheses of relationships among 11 proposed tribes of cyprinine fishes and present an interesting hypothesis about the origin of a number of polyploid cyprinine lineages. However, support for relationships within some of the tribes is equivocal. Herein we address the treatment of African diploid and tetraploid cyprinine genera within tribe Smiliogastrini. More specifically, we reject the revalidation of Enteromius based on the evidence presented and discuss the ramifications of the proposed revalidation.

First record of Mollisquama sp. (Chondrichthyes: Squaliformes: Dalatiidae) from the Gulf of Mexico, with a morphological comparison to the holotype description of Mollisquama parini Dolganov.

The description of the pocket shark genus Mollisquama (M. parini Dolganov, 1984) is based on a single known specimen collected from the Nazca Ridge of the southeast Pacific Ocean. A second Mollisquama specimen has been captured in the central Gulf of Mexico establishing a considerable range extension and a parturition locality because the specimen has a healed vitelline scar. Both the holotype of M. parini and the Gulf of Mexico specimen possess the remarkable pocket gland with its large slit-like external opening located just above the pectoral fin. Features found on the Gulf of Mexico specimen that were not noted in the description of M. parini include a series of ventral abdominal photophore agglomerations and a modified dermal denticle surrounded by a radiating arrangement of denticles just posterior to the mouth. Based on a morphometric and meristic comparison of the Gulf of Mexico specimen with information in the description of M. parini, the Gulf of Mexico specimen is identified as Mollisquama sp. due to differences in tooth morphology and vertebral counts. Phylogenetic analysis of NADH2 gene sequences places Mollisquama sister to Dalatias plus Isistius within the family Dalatiidae.

Phylogeny of suckermouth catfishes (Mochokidae: Chiloglanis) from Kenya: the utility of Growth Hormone introns in species level phylogenies.

African suckermouth catfishes (Mochokidae: Chiloglanis) occur in freshwater throughout tropical Africa. Specimens from all major drainages across Kenya were collected over three field seasons. Here we present a phylogeny inferred from both mitochondrial cytochrome b (cyt b) and introns of the nuclear Growth Hormone gene (GH). The phylogeny inferred from introns is largely congruent with the results from an analysis of cyt b. The length and variability of GH introns make them ideal species level nuclear markers without the problem of introgression commonly encountered with mitochondrial genes. This analysis confirmed the presence of two previously known undescribed Chiloglanis species and also suggests the presence of previously unknown diversity within the Athi River system. The resulting phylogeny also indicates the presence of two separate lineages within C. brevibarbis. The historical biogeography of Chiloglanis within Kenya is discussed. The utility of GH intron for species level phylogenies of Siluriformes is compared to that in other groups.

Limits and phylogenetic relationships of East Asian fishes in the subfamily Oxygastrinae (Teleostei: Cypriniformes: Cyprinidae).

The cyprinid subfamily Oxygastrinae is composed of a diverse group of fishes that has been taxonomically and phylogenetically problematic. Their great variation in appearance, life histories, and trophic diversity resulted in uncertainty regarding 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, Pseudohemiculter, Pseudolaubuca, Sinibrama, Squaliobarbus, Toxabramis, Xenocyprioides, Xenocypris, Yaoshanicus, and Zacco. Of these genera, the following were found to be monophyletic: Aphyocypris, Distoechodon, Hypophthalmichthys, Nipponocypris, Opsariichthys, Parachela, Paralaubuca, Plagiognathops, Xenocyprioides, and Xenocypris. The following genera were not monophyletic: Metzia, Hemiculter, Toxabramis, Ancherythroculter, Chanodichthys, Culter, Megalobrama. 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.

A different perspective on the phylogenetic relationships of the Moxostomatini (Cypriniformes: Catostomidae) based on cytochrome-b and Growth Hormone intron sequences.

We have examined phylogenetic relationships of suckers of tribe Moxostomatini (Cypriniformes, Catostomidae) using cytochrome-b and Growth Hormone gene intron sequences. Phylogenies were significantly different from recent estimates of relationships based primarily on morphology (Smith, 1992) and cytochrome-b sequences (Harris et al., 2002). Overall, there was little support for many basal nodes in the phylogeny, however it was clear that Scartomyzon and Moxostoma were not monophyletic, despite morphological evidence provided Robins and Raney (1956, 1957), Jenkins (1970), and Smith (1992). Growth Hormone sequences provided good support for a monophyletic Western Scartomyzon lineage and thus suggested a single ancestral invasion of Scartomyzon-like fishes into drainages of Texas and Mexico. Phylogenetic relationships of Western Scartomyzon are structured geographically and do not conform well to current taxonomy.

Phylogeny of the gudgeons (Teleostei: Cyprinidae: Gobioninae).

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 Gobioninae 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, Paracanthobrama, Pseudorasbora, Pseudopungtungia, Pungtungia, Rhinogobio, and Sarcocheilichthys; the tribe Gobionini 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.

Morphological variation of the palatal organ and chewing pad of catostomidae (teleostei: cypriniformes).

We studied the morphology and shape variation of the palatal organ and chewing pad of sucker fishes, family Catostomidae. The palatal organ is a muscularized structure that forms a large mass on the roof of the posterior part of the buccopharyngeal cavity in cypriniform fishes. It functions in coordination with the branchial arches to separate food items from inorganic debris during feeding. The palatal organ exhibits considerable variability in morphology among catostomids. It is shorter, narrower, and thinner in species of the subfamily Cycleptinae (e.g., Cycleptus elongatus) than in other catostomid subfamilies. The thickest and widest palatal organ is seen in species of the subfamily Ictiobinae (e.g., Ictiobus cyprinellus). The shape and size of the palatal organ generally varies between these extremes in species of subfamily Catostominae (e.g., Catostomus and Moxostoma species). Principal components analysis and analysis of variance has differentiated means of various palatal organ measurements for each monophyletic subfamily and tribe of Catostomidae with statistical significance. These results corroborate previously established typological classification of catostomids based on pharyngeal tooth count, pharyngeal tooth morphology, and diet. A keratinized chewing pad forms on the posterior surface of the palatal organ in catostomids or on a skeletal process in cyprinids and serves as an occlusion surface for pharyngeal teeth. The chewing pad is lunate in catostomids and generally ovoid in cyprinids. It is absent from the species of loaches (e.g., botiids, cobitids, and nemacheilids) and gyrinocheilids examined. A synonymy of terms used in the past to describe the palatal organ and chewing pad of Cypriniformes is provided.

Phylogenetic relationships of the North American cyprinid subgenus Hydrophlox.

Notropis is one of the largest genera of North American fishes and is composed of a number of morphologically diagnosed subgroups; however, the validity of many has not been tested in a phylogenetic framework. One such subgroup is the subgenus Hydrophlox, which is composed of brilliantly colored species that engage in the symbiotic reproductive behavior of nest association. Although they have long been recognized as a cohesive group due to their nuptial coloration and fin tuberculation, very little is known about the relationships of species within Hydrophlox. We tested the monophyly of Hydrophlox using a mitochondrial marker (ND2) and two nuclear markers (ITS1 and RH), with Maximum Parsimony and Bayesian inference approaches. A well supported clade of "core"Hydrophlox was recovered and is composed of five taxa: Notropis chiliticus, Notropis rubricroceus, Notropis lutipinnis, Notropis chlorocephalus, and Notropis chrosomus. Hydrophlox s.l. is paraphyletic with respect to three taxa: Notropis baileyi, Notropis leuciodus and Notropis nubilus. While there was some discordance among the individual marker topologies, a combined evidence analysis recovered a topology that incorporated elements from all single-gene trees. Our analyses suggest that Hydrophlox is composed of five nominal species and additional undescribed diversity exists within this clade.

Evolutionary divergence of duplicate copies of the growth hormone gene in suckers (Actinopterygii: catostomidae).

Catostomid fishes (suckers) have duplicate copies of the growth hormone gene and other nuclear genes, due to a genome duplication event early in the group's history. Yet, paralogs of GH in suckers are more than 90% conserved in nucleotide (nt) and amino acid (aa) sequence. Within paralogs across species, variation in nt and aa sequence averages 3.33% and 4.46% for GHI, and 3.22% and 2.43% for GHII, respectively. Selection tests suggest that the two GH paralogs are under strong purifying selection. Consensus trees from phylogenetic analysis of GH coding region data for 23 species of suckers, other cypriniform fishes and outgroups resolved cypriniform relationships and relationships among GHI sequences of suckers more or less consistently with analyses based on other molecular data. However, the analysis failed to resolve all sucker GHI and GHII sequences as monophyletic sister groups. This unexpected topology did not differ significantly from topologies constrained to make all GH sequences monophyletic. We attribute this result either to limitations in our GHII data set or convergent adaptive changes in GHII of tribe Catostomini.