Long-term climatic stability drives accumulation and maintenance of divergent freshwater fish lineages in a temperate biodiversity hotspot.

Anthropogenic climate change is forecast to drive regional climate disruption and instability across the globe. These impacts are likely to be exacerbated within biodiversity hotspots, both due to the greater potential for species loss but also to the possibility that endemic lineages might not have experienced significant climatic variation in the past, limiting their evolutionary potential to respond to rapid climate change. We assessed the role of climatic stability on the accumulation and persistence of lineages in an obligate freshwater fish group endemic to the southwest Western Australia (SWWA) biodiversity hotspot. Using 19,426 genomic (ddRAD-seq) markers and species distribution modelling, we explored the phylogeographic history of western (Nannoperca vittata) and little (Nannoperca pygmaea) pygmy perches, assessing population divergence and phylogenetic relationships, delimiting species and estimating changes in species distributions from the Pliocene to 2100. We identified two deep phylogroups comprising three divergent clusters, which showed no historical connectivity since the Pliocene. We conservatively suggest these represent three isolated species with additional intraspecific structure within one widespread species. All lineages showed long-term patterns of isolation and persistence owing to climatic stability but with significant range contractions likely under future climate change. Our results highlighted the role of climatic stability in allowing the persistence of isolated lineages in the SWWA. This biodiversity hotspot is under compounding threat from ongoing climate change and habitat modification, which may further threaten previously undetected cryptic diversity across the region.

Phylogenomics, Lineage Diversification Rates, and the Evolution of Diadromy in Clupeiformes (Anchovies, Herrings, Sardines, and Relatives).

Migration independently evolved numerous times in animals, with a myriad of ecological and evolutionary implications. In fishes, perhaps the most extreme form of migration is diadromy, the migration between marine and freshwater environments. Key and longstanding questions are: how many times has diadromy evolved in fishes, how frequently do diadromous clades give rise to non-diadromous species, and does diadromy influence lineage diversification rates? Many diadromous fishes have large geographic ranges with constituent populations that use isolated freshwater habitats. This may limit gene flow among some populations, increasing the likelihood of speciation in diadromous lineages relative to non-diadromous lineages. Alternatively, diadromy may reduce lineage diversification rates if migration is associated with enhanced dispersal capacity that facilitates gene flow within and between populations. Clupeiformes (herrings, sardines, shads and anchovies) is a model clade for testing hypotheses about the evolution of diadromy because it includes an exceptionally high proportion of diadromous species and several independent evolutionary origins of diadromy. However, relationships among major clupeiform lineages remain unresolved and existing phylogenies sparsely sampled diadromous species, limiting the resolution of phylogenetically-informed statistical analyses. We assembled a phylogenomic dataset and used multi-species coalescent and concatenation-based approaches to generate the most comprehensive, highly-resolved clupeiform phylogeny to date, clarifying associations among several major clades and identifying recalcitrant relationships needing further examination. We determined that variation in rates of sequence evolution (heterotachy) and base-composition (non-stationarity) had little impact on our results. Using this phylogeny, we characterized evolutionary patterns of diadromy and tested for differences in lineage diversification rates between diadromous, marine, and freshwater lineages. We identified thirteen transitions to diadromy, all during the Cenozoic Era (ten origins of anadromy, two origins of catadromy, and one origin of amphidromy), and seven losses of diadromy. Two diadromous lineages rapidly generated non-diadromous species, demonstrating that diadromy is not an evolutionary dead-end. We discovered considerably faster transition rates out of diadromy than to diadromy. The largest lineage diversification rate increase in Clupeiformes was associated with a transition to diadromy, but we uncovered little statistical support for categorically faster lineage diversification rates in diadromous versus non-diadromous fishes. We propose that diadromy may increase the potential for accelerated lineage diversification, particularly in species that migrate long distances. However, this potential may only be realized in certain biogeographic contexts, such as when diadromy allows access to ecosystems in which there is limited competition from incumbent species.

Museum genomics reveals the hybrid origin of an extinct crater lake endemic.

Crater lake fishes are common evolutionary model systems, with recent studies suggesting a key role for gene flow in promoting rapid adaptation and speciation. However, the study of these young lakes can be complicated by human-mediated extinctions. Museum genomics approaches integrating genetic data from recently extinct species are therefore critical to understanding the complex evolutionary histories of these fragile systems. Here, we examine the evolutionary history of an extinct Southern Hemisphere crater lake endemic, the rainbowfish Melanotaenia eachamensis. We undertook comprehensive sampling of extant rainbowfish populations of the Atherton Tablelands of Australia alongside historical museum material to understand the evolutionary origins of the extinct crater lake population and the dynamics of gene flow across the ecoregion. The extinct crater lake species is genetically distinct from all other nearby populations due to historic introgression between two proximate riverine lineages, similar to other prominent crater lake speciation systems, but this historic gene flow has not been sufficient to induce a species flock. Our results suggest that museum genomics approaches can be successfully combined with extant sampling to unravel complex speciation dynamics involving recently extinct species.

Phylogeography of a widespread Australian freshwater fish, western carp gudgeon (Eleotridae: Hypseleotris klunzingeri): Cryptic species, hybrid zones, and strong intra-specific divergences.

Despite belonging to the most abundant and widespread genus of freshwater fishes in the region, the carp gudgeons of eastern Australia (genus Hypseleotris) have proved taxonomically and ecologically problematic to science since the 19th century. Several molecular studies and a recent taxonomic revision have now shed light on the complex biology and evolutionary history that underlies this group. These studies have demonstrated that carp gudgeons include a sexual/unisexual complex (five sexual species plus an assortment of hemiclonal lineages), many members of which also co-occur with an independent sexual relative, the western carp gudgeon (H. klunzingeri). Here, we fill yet another knowledge gap for this important group by presenting a detailed molecular phylogeographic assessment of the western carp gudgeon across its entire and extensive geographic range. We use a suite of nuclear genetic markers (SNPs and allozymes) plus a matrilineal genealogy (cytb) to demonstrate that H. klunzingeri s.l. also displays considerable taxonomic and phylogeographic complexity. All molecular datasets concur in recognizing the presence of multiple candidate species, two instances of historic between-species admixture, and the existence of a natural hybrid zone between two of the three candidate species found in the Murray-Darling Basin. We also discuss the major phylogeographic patterns evident within each taxon. Together, these analyses provide a robust molecular, taxonomic, and distributional framework to underpin future morphological and ecological investigations on this prominent member of regional freshwater ecosystems in eastern Australia.

Diversification of the sleepers (Gobiiformes: Gobioidei: Eleotridae) and evolution of the root gobioid families.

Eleotridae (sleepers) and five smaller families are the earliest diverging lineages within Gobioidei. Most inhabit freshwaters in and around the Indo-Pacific, but Eleotridae also includes species that have invaded the Neotropics as well as several inland radiations in the freshwaters of Australia, New Zealand, and New Guinea. Previous efforts to infer phylogeny of these families have been based on sets of mitochondrial or nuclear loci and have yielded uncertain resolution of clades within Eleotridae. We expand the taxon sampling of previous studies and use genomic data from nuclear ultraconserved elements (UCEs) to infer phylogeny, then calibrate the hypothesis with recently discovered fossils. Our hypothesis clarifies ambiguously resolved relationships, provides a timescale for divergences, and indicates the core crown Eleotridae diverged over a short period 24.3-26.3 Ma in the late Oligocene. Within Eleotridae, we evaluate diversification dynamics with BAMM and find evidence for an overall slowdown in diversification over the past 35 Ma, but with a sharp increase 3.5 Ma in the genus Mogurnda, a clade of brightly colored species found in the freshwaters of Australia and New Guinea.

Phylogenomics and biogeography of arid-adapted Chlamydogobius goby fishes.

The progressive aridification of the Australian continent from âˆ¼ 20 million years ago posed severe challenges for the persistence of its resident biota. A key question involves the role of refugial habitats - specifically, their ability to mediate the effects of habitat loss and fragmentation, and their potential to shape opportunities for allopatric speciation. With freshwater species, for example, the patchiness, or absence, of water will constrain distributions. However, aridity may not necessarily isolate populations if disjunct refugia experience frequent hydrological connections. To investigate this potential dichotomy, we explored the evolutionary history of the Chlamydogobius gobies (Gobiiformes: Gobiidae), an arid-adapted genus of six small, benthic fish species that exploit all types of waterbodies (i.e. desert springs, waterholes and bore-fed wetlands, coastal estuarine creeks and mangroves) across parts of central and northern Australia. We used Anchored Phylogenomics to generate a highly resolved phylogeny of the group from sequence data for 260 nuclear loci. Buttressed by companion allozyme and mtDNA datasets, our molecular findings infer the diversification of Chlamydogobius in arid Australia, and provide a phylogenetic structure that cannot be simply explained by invoking allopatric speciation events reflecting current geographic proximity. Our findings are generally consistent with the existing morphological delimitation of species, with one exception: at the shallowest nodes of phylogenetic reconstruction, the molecular data do not fully support the current dichotomous delineation of C. japalpa from C. eremius in Kati Thanda-Lake Eyre-associated waterbodies. Together these findings illustrate the ability of structural (hydrological) connections to generate patterns of connectivity and isolation for an ecologically moderate disperser in response to ongoing habitat aridification. Finally, we explore the implications of these results for the immediate management of threatened (C. gloveri) and critically endangered (C. micropterus, C. squamigenus) congeners.

Phylogeny, diversification, and biogeography of a hemiclonal hybrid system of native Australian freshwater fishes (Gobiiformes: Gobioidei: Eleotridae: Hypseleotris).

BACKGROUND: Carp gudgeons (genus Hypseleotris) are a prominent part of the Australian freshwater fish fauna, with species distributed around the western, northern, and eastern reaches of the continent. We infer a calibrated phylogeny of the genus based on nuclear ultraconserved element (UCE) sequences and using Bayesian estimation of divergence times, and use this phylogeny to investigate geographic patterns of diversification with GeoSSE. The southeastern species have hybridized to form hemiclonal lineages, and we also resolve relationships of hemiclones and compare their phylogenetic placement in the UCE phylogeny with a hypothesis based on complete mitochondrial genomes. We then use phased SNPs extracted from the UCE sequences for population structure analysis among the southeastern species and hemiclones. RESULTS: Hypseleotris cyprinoides, a widespread euryhaline species known from throughout the Indo-Pacific, is resolved outside the remainder of the species. Two Australian radiations comprise the bulk of Hypseleotris, one primarily in the northwestern coastal rivers and a second inhabiting the southeastern region including the Murray-Darling, Bulloo-Bancannia and Lake Eyre basins, plus coastal rivers east of the Great Dividing Range. Our phylogenetic results reveal cytonuclear discordance between the UCE and mitochondrial hypotheses, place hemiclone hybrids among their parental taxa, and indicate that the genus Kimberleyeleotris is nested within the northwestern Hypseleotris radiation along with three undescribed species. We infer a crown age for Hypseleotris of 17.3 Ma, date the radiation of Australian species at roughly 10.1 Ma, and recover the crown ages of the northwestern (excluding H. compressa) and southeastern radiations at 5.9 and 7.2 Ma, respectively. Range-dependent diversification analyses using GeoSSE indicate that speciation and extinction rates have been steady between the northwestern and southeastern Australian radiations and between smaller radiations of species in the Kimberley region and the Arnhem Plateau. Analysis of phased SNPs confirms inheritance patterns and reveals high levels of heterozygosity among the hemiclones. CONCLUSIONS: The northwestern species have restricted ranges and likely speciated in allopatry, while the southeastern species are known from much larger areas, consistent with peripatric speciation or allopatric speciation followed by secondary contact. Species in the northwestern Kimberley region differ in shape from those in the southeast, with the Kimberley species notably more elongate and slender than the stocky southeastern species, likely due to the different topographies and flow regimes of the rivers they inhabit.

Variation in intraspecific demography drives localised concordance but species-wide discordance in response to past climatic change.

BACKGROUND: Understanding how species biology may facilitate resilience to climate change remains a critical factor in detecting and protecting species at risk of extinction. Many studies have focused on the role of particular ecological traits in driving species responses, but less so on demographic history and levels of standing genetic variation. Additionally, spatial variation in the interaction of demographic and adaptive factors may further complicate prediction of species responses to environmental change. We used environmental and genomic datasets to reconstruct the phylogeographic histories of two ecologically similar and largely co-distributed freshwater fishes, the southern (Nannoperca australis) and Yarra (N. obscura) pygmy perches, to assess the degree of concordance in their responses to Plio-Pleistocene climatic changes. We described contemporary genetic diversity, phylogenetic histories, demographic histories, and historical species distributions across both species, and statistically evaluated the degree of concordance in co-occurring populations. RESULTS: Marked differences in contemporary genetic diversity, historical distribution changes and historical migration were observed across the species, with a distinct lack of genetic diversity and historical range expansion suggested for N. obscura. Although several co-occurring populations within a shared climatic refugium demonstrated concordant demographic histories, idiosyncratic population size changes were found at the range edges of the more spatially restricted species. Discordant responses between species were associated with low standing genetic variation in peripheral populations. This might have hindered adaptive potential, as documented in recent demographic declines and population extinctions for the two species. CONCLUSION: Our results highlight both the role of spatial scale in the degree of concordance in species responses to climate change, and the importance of standing genetic variation in facilitating range shifts. Even when ecological traits are similar between species, long-term genetic diversity and historical population demography may lead to discordant responses to ongoing and future climate change.

Longitudinal monitoring of neutral and adaptive genomic diversity in a reintroduction.

Restoration programs in the form of ex-situ breeding combined with reintroductions are becoming critical to counteract demographic declines and species losses. Such programs are increasingly using genetic management to improve conservation outcomes. However, the lack of long-term monitoring of genetic indicators following reintroduction prevents assessments of the trajectory and persistence of reintroduced populations. We carried out an extensive monitoring program in the wild for a threatened small-bodied fish (southern pygmy perch, Nannoperca australis) to assess the long-term genomic effects of its captive breeding and reintroduction. The species was rescued prior to its extirpation from the terminal lakes of Australia's Murray-Darling Basin, and then used for genetically informed captive breeding and reintroductions. Subsequent annual or biannual monitoring of abundance, fitness, and occupancy over a period of 11 years, combined with postreintroduction genetic sampling, revealed survival and recruitment of reintroduced fish. Genomic analyses based on data from the original wild rescued, captive born, and reintroduced cohorts revealed low inbreeding and strong maintenance of neutral and candidate adaptive genomic diversity across multiple generations. An increasing trend in the effective population size of the reintroduced population was consistent with field monitoring data in demonstrating successful re-establishment of the species. This provides a rare empirical example that the adaptive potential of a locally extinct population can be maintained during genetically informed ex-situ conservation breeding and reintroduction into the wild. Strategies to improve biodiversity restoration via ex-situ conservation should include genetic-based captive breeding and longitudinal monitoring of standing genomic variation in reintroduced populations.

Monitoreo Longitudinal de la Diversidad Genómica Neutral y Adaptativa en una Reintroducción Marshall et al. 21-643 Resumen Los programas de restauración a manera de reproducción ex situ combinada con reintroducciones se están volviendo críticos para contrarrestar las declinaciones demográficas y la pérdida de especies. Dichos programas usan cada vez más la gestión genética para mejorar los resultados de conservación. Sin embargo, la falta de monitoreo a largo plazo de los indicadores genéticos posteriores a la reintroducción evita que se realicen evaluaciones de la trayectoria y la persistencia de las poblaciones reintroducidas. Se rescató un pez de talla pequeña (percha pigmea del sur [Nannoperca australis]) previo a su extirpación de los lagos terminales de la Cuenca Murray-Darling en Australia para después reproducirlo en cautiverio con información genética y reintroducirlo. Realizamos monitoreos anuales o bianuales de la abundancia, aptitud y ocupación en vida silvestre durante once años, además de un muestreo genético posterior a la reintroducción. Analizamos los datos genómicos de los grupos originales rescatados, los nacidos en cautiverio y los reintroducidos. Nuestro objetivo era evaluar los efectos genómicos a largo plazo de la reproducción en cautiverio y la reintroducción de esta especie. Esto reveló baja endogamia y el sólido mantenimiento de la diversidad genómica neutral y adaptativa durante varias generaciones. Encontramos una coherencia entre la tendencia creciente en el tamaño de la población efectiva de la población reintroducida y los datos de campo que demostraron el restablecimiento exitoso de la especie. Nuestro estudio proporciona un raro ejemplo empírico de cómo el potencial adaptativo de una población localmente extinta puede mantenerse durante la reproducción de conservación ex situ genéticamente informada y su reintroducción. Las estrategias para mejorar la restauración de la biodiversidad por medio de la conservación ex situ deberían incluir la reproducción en cautiverio basada en la genética y el monitoreo longitudinal de la variación genómica actual de las poblaciones reintroducidas.

The roles of aridification and sea level changes in the diversification and persistence of freshwater fish lineages.

While the influence of Pleistocene climatic changes on divergence and speciation has been well-documented across the globe, complex spatial interactions between hydrology and eustatics over longer timeframes may also determine species evolutionary trajectories. Within the Australian continent, glacial cycles were not associated with changes in ice cover and instead largely resulted in fluctuations from moist to arid conditions across the landscape. We investigated the role of hydrological and coastal topographic changes brought about by Plio-Pleistocene climatic changes on the biogeographic history of a small Australian freshwater fish, the southern pygmy perch Nannoperca australis. Using 7958 ddRAD-seq (double digest restriction-site associated DNA) loci and 45,104 filtered SNPs, we combined phylogenetic, coalescent and species distribution analyses to assess the various roles of aridification, sea level and tectonics and associated biogeographic changes across southeast Australia. Sea-level changes since the Pliocene and reduction or disappearance of large waterbodies throughout the Pleistocene were determining factors in strong divergence across the clade, including the initial formation and maintenance of a cryptic species, N. 'flindersi'. Isolated climatic refugia and fragmentation due to lack of connected waterways maintained the identity and divergence of inter- and intraspecific lineages. Our historical findings suggest that predicted increases in aridification and sea level due to anthropogenic climate change might result in markedly different demographic impacts, both spatially and across different landscape types.

A review of the Glossogobius giuris complex in Australia, with wider discussion on nomenclature and possible synonymies.

Two species that had previously been referred to as Glossogobius giuris from Australia are redescribed and tentatively treated as Glossogobius giuris and Glossogobius laticeps. The two species are largely allopatric in Australia with G. laticeps found on the east coast of Queensland and some rivers of the Northern Territory, while G. giuris is distributed from the Northern Territory to rivers of the west coast of Western Australia. The two species are known to be genetically distinct and with some differences in colour patterns. Morphological features separating the species are minor and there is considerable overlap in many of the characters separating them. High variation in the Hammersley Ranges and Kimberley region of Western Australia further complicates separation of the species. The status of the Glossogobius giuris group from India is discussed as a key area to resolving an overall "giuris" species complex. Information is provided on the current identity of a number of species that had previously been considered synonyms of Glossogobius giuris. Extant types and difficulties assigning the types of known species are also discussed. A key is provided to aid identification of Australian species of Glossogobius.

A revision of the gobiid fish genus Pseudogobius/ (Teleostei, Gobiidae, Tridentigerinae), with description of seven new species from Australia and South-east Asia.

The tridentigerine genus Pseudogobius is widespread, from temperate to tropical areas of the Indo-west Pacific. A morphological review of the genus was carried out, with the initial focus on South-east Asian and Australian species. There are about 21 nominal species in the genus; however, the type specimens for some nominal species are missing. Our work recognises 15 valid species so far, including seven new species, which are described herein: P. aquilonius n. sp., P. cinctus n. sp., P. eos n. sp., P. hoesei n. sp., P. jeffi n. sp., P. rhizophora n. sp. and P. verticalis n. sp. We also conclude that P. poicilosoma (Bleeker, 1849) is the senior synonym of P. javanicus (Bleeker, 1856). Pseudogobius poicilosoma, P. gastrospilos (Bleeker, 1853) and P. javanicus are accepted here as conspecific, although the types are in poor condition (note that all three names were given to specimens collected within close proximity to each other in coastal Java, Indonesia). A key to valid species is presented. The revision benefited from an adaptive feedback loop with companion genetic analyses, with the most comprehensive data available for the Australian region. Broader preliminary genetic data suggest the potential for recognition of additional cryptic species, with the current study providing a platform for future systematic work. Pseudogobius are a prominent part of estuarine biodiversity in the Indo-west Pacific, and this study helps to highlight the need to better understand the taxonomy and conservation requirements of cryptobenthic fishes.

Surprising Pseudogobius: Molecular systematics of benthic gobies reveals new insights into estuarine biodiversity (Teleostei: Gobiiformes).

Snubnose gobies (genus Pseudogobius: Gobionellinae) are ubiquitous to, and important components of, estuarine ecosystems of the Indo-west Pacific. These small benthic fishes occur in freshwater, brackish and marine habitats such as mangroves, sheltered tide pools and lowland streams, and represent a model group for understanding the biodiversity and biogeography of estuarine fauna. To develop the species-level framework required for a concurrent morphological taxonomic appraisal, we undertook thorough sampling around the extensive Australian coastline, referenced to international locations, as part of a molecular systematic review using both nuclear and mitochondrial markers. The results indicate that while there are currently eight recognised species, the true diversity is close to double this, with a hotspot of endemism located in Australia. Complicated patterns were observed in southern Australia owing to two differing zones of introgression/admixture. Key drivers of diversity in the group appear to include plate tectonics, latitude, and historic barriers under glacial maxima, where an interplay between ready dispersal and habitat specialisation has led to regional panmixia but frequent geographic compartmentalisation within past and present landscapes. The findings have significant implications for biodiversity conservation, coastal and estuarine development, the basic foundations of field ecology, and for applied use such as in biomonitoring.

Unravelling the taxonomy and identification of a problematic group of benthic fishes from tropical rivers (Gobiidae: Glossogobius).

Flathead gobies (genus Glossogobius) include c. 40 small- to medium-sized benthic fishes found primarily in freshwater habitats across the Indo-Pacific, having biodiversity value as well as cultural and economic value as food fishes, especially in developing countries. To help resolve considerable confusion regarding the identification of some of the larger-growing Glossogobius species, a systematic framework was established using nuclear genetic markers, mitochondrial DNA barcoding and phenotypic evidence for a geographically widespread collection of individuals from the waterways of tropical northern Australia. Species boundaries and distribution patterns were discordant with those previously reported, most notably for the tank goby Glossogobius giuris, which included a cryptic species. Genetic divergence was matched with accompanying unique visual characters that aid field identification. Additional taxonomic complexity was also evident, by comparison with DNA barcodes from international locations, suggesting that the specific names applicable for two of the candidate species in Australia remain unresolved due to confusion surrounding type specimens. Although flathead gobies are assumed to be widespread and common, this study demonstrates that unrealised taxonomic and ecological complexity is evident, and this will influence assessments of tropical biodiversity and species conservation. This study supports the need for taxonomic studies of freshwater fishes to underpin management in areas subject to significant environmental change.

Comparison of genetic structure in co-occurring freshwater eleotrids (Actinopterygii: Philypnodon) reveals cryptic species, likely translocation and regional conservation hotspots.

Freshwater systems are naturally fragmented and heterogeneous habitats that promote genetic sub-division and speciation for aquatic biota. Here we provide a novel nuclear genetic perspective (49 allozyme loci) complimented with updated mitochondrial data for the eleotrid genus Philypnodon to investigate broad genetic sub-structure across south-eastern Australia as a foundation for management and conservation. The genus is nominally comprised of two small benthic fishes with contrasting physical and ecological traits, namely the Flathead Gudgeon P. grandiceps and the Dwarf Flathead Gudgeon P. macrostomus. Extensive sample coverage included 99 sites across 5 major drainage divisions and 48 river basins. Nuclear markers revealed strong, geographically-based divergence and sub-structure, contrasting with shallower but largely congruent patterns for mtDNA. The results flag that each nominal species represents a hyper-cryptic species complex, including both broadly distributed and narrow-range taxa, with complicated biogeographic patterns. Predictions on dispersal and genetic structure based on ecological traits were only partially supported and varied by region, with the potential signature of human-assisted translocation evident in several catchments. Further intensive sampling in an important area of high genetic diversity, coastal south-east Queensland, is recommended to better resolve species boundaries and conservation units. The findings provide new insights on regional ecology and biogeography, demonstrating that even supposedly common species can, in reality, have complex conservation and management needs.

Two new species of dwarf rainbowfishes (Atheriniformes: Melanotaeniidae) from northern Australia and southern New Guinea.

The 'maccullochi species group' of rainbowfishes are small and distinctly patterned freshwater fishes of streams and swamps, comprising around eight species. The species from which the group bears its name, Melanotaenia maccullochi Ogilby, 1915, has been thought to comprise three forms occurring in distinct geographic areas, and recent mitochondrial genetic data provides matching patterns of likely inter-specific divergence. Here we undertake a detailed investigation of the taxonomic status of M. maccullochi using a combined lines of evidence approach incorporating multiple nuclear genetic markers (55 allozyme loci), mitochondrial DNA sequence data (1141 bp cytochrome b) and morphology (examination of a suite of 38 morphometric and meristic characters). As all three datasets provide support for a three-way split, we accordingly describe two new species and redescribe M. maccullochi sensu stricto. McCulloch's Rainbowfish M. maccullochi, a species with brown body stripes and red fins occurs in northeast Queensland and is redescribed based on 338 specimens, 13.1-53.0 mm SL. This species was one of the first rainbowfishes to become known in the aquarium hobby. A second form with darker stripes on a contrasting light white-grey body and with distinct sub-marginal black bands in the dorsal and anal fins, distributed across northern and eastern Cape York Peninsula, Torres Strait and southern central New Guinea, is described as Sahul Rainbowfish M. sahulensis sp. nov. based on 267 specimens, 13.4-48.4 mm SL. A diminutive and well geographically isolated form occurring below the escarpment of the Tabletop Range in Litchfield National Park, Northern Territory possessing a more prominent and purplish mid-lateral stripe, is described as the Little Rainbowfish Melanotaenia wilsoni sp. nov. based on 50 specimens, 19.3-33.3 mm SL. A combination of morphological characters is useful for separating the respective taxa with M. wilsoni sp. nov. the most distinctive, typically having fewer vertebrae, lateral scales, cheek scales, procurrent caudal rays and anal rays and proportionally a shorter maxilla and snout than either of the other two species. Useful characters for further separating M. sahulensis sp. nov. from M. maccullochi include slightly higher counts of vertebrae, lateral scales and anal rays and proportionally greater body depth, body width and pre-dorsal distance. Information on the known distribution, habitats and conservation status of the three species is summarised, with the Northern Territory species being a narrow-range endemic with specific environmental requirements.

Cryptic biodiversity in the freshwater fishes of the Kimberley endemism hotspot, northwestern Australia.

The prevalence of unrecognised cryptic species impairs biodiversity estimates, clouds biological research and hinders conservation planning. As the rate of cryptic species detection increases globally, research is needed to determine how frequent cryptic species are, whether they are more common in given management regions, and whether these patterns are consistent across taxonomic groups. The Kimberley region in remote northwestern Australia harbours some of the most speciose, and morphologically and functionally diverse, endemic animal and plant communities on the continent. The rugged and changeable landscape also appears to contain a large proportion of cryptic terrestrial species, raising the question of whether similar patterns are also found among aquatic taxa, which have yet to be studied using integrative systematic approaches. If true, then the actual levels of aquatic biodiversity are yet to be fully realised. Here we conducted a molecular assessment of where species boundaries may exist in the Kimberley regions' most speciose freshwater fish family, the Terapontidae (grunters), with a combined morphological assessment of the regions' most speciose terapontid genus, Syncomistes. Assessment of nuclear markers (54 allozyme loci), sequence data (mitochondrial cytochrome b (cytb); nuclear recombination activation gene one (RAG1)) and 31 meristic and 36 morphometric characters provides evidence for 13 new candidate species across three different genera. Many of these candidate species are narrow range endemics. Our findings raise several questions about the evolutionary origin of the Kimberley's endemic fish fauna and highlight the likelihood that freshwater fish species diversity in the Kimberley is severely under-represented by current systematic frameworks, with significant implications for ecological research, conservation and management.

Revision of the Australian Wet Tropics endemic rainbowfish genus Cairnsichthys (Atheriniformes: Melanotaeniidae), with description of a new species.

The freshwater melanotaeniid genus Cairnsichthys is endemic to a relatively small area of specialised habitat within the Wet Tropics bioregion of north-eastern Queensland, Australia. It was previously considered as monotypic, including only a single species, C. rhombosomoides (Nichols Raven, 1928). The recent discovery of an apparently-isolated population in the Daintree rainforest, approximately 120 km north of the known range extent, prompted a detailed investigation of its taxonomic status using a combined lines of evidence approach. We provide compelling evidence from multiple nuclear genetic markers (52 allozyme loci), mitochondrial DNA sequence data (1141 bp cytochrome b) and morphology (examination of a suite of 38 morphometric and meristic characters) that supports north-south splitting of C. rhombosomoides. Accordingly, we describe the northern population as a distinct species, C. bitaeniatus sp. nov., on the basis of 25 specimens, 34.7-65.6 mm SL. The new species differs morphologically primarily by having a more slender and narrow shape, featuring a flatter, straighter predorsal profile and shorter second dorsal fin base; possession of slightly smaller scales, reflected in higher counts of lateral scales and predorsal scales; typically more vertebrae; and colour differences including a more robust, short black stripe across the upper operculum, a pronounced yellow patch on the anteroventral body and usually a more conspicuous second dark stripe on the lower body, with adult males generally having yellowish compared to reddish fins. We also provide a generic diagnosis for Cairnsichthys and a redescription of C. rhombosomoides. Information on the known distribution, habitats and conservation status of species in the genus is summarised, the new species being of particular concern as a narrow range endemic with specific environmental requirements.

Phylogenetic analysis of trophic niche evolution reveals a latitudinal herbivory gradient in Clupeoidei (herrings, anchovies, and allies).

Biotic and abiotic forces govern the evolution of trophic niches, which profoundly impact ecological and evolutionary processes and aspects of species biology. Herbivory is a particularly interesting trophic niche because there are theorized trade-offs associated with diets comprised of low quality food that might prevent the evolution of herbivory in certain environments. Herbivory has also been identified as a potential evolutionary "dead-end" that hinders subsequent trophic diversification. For this study we investigated trophic niche evolution in Clupeoidei (anchovies, sardines, herrings, and their relatives) and tested the hypotheses that herbivory is negatively correlated with salinity and latitude using a novel, time-calibrated molecular phylogeny, trophic guilds delimited using diet data and cluster analysis, and standard and phylogenetically-informed statistical methods. We identified eight clupeoid trophic guilds: molluscivore, terrestrial invertivore, phytoplanktivore, macroalgivore, detritivore, piscivore, crustacivore, and zooplanktivore. Standard statistical methods found a significant negative correlation between latitude and the proportion of herbivorous clupeoids (herbivorous clupeoid species/total clupeoid species), but no significant difference in the proportion of herbivorous clupeoids between freshwater and marine environments. Phylogenetic least squares regression did not identify significant negative correlations between latitude and herbivory or salinity and herbivory. In clupeoids there were five evolutionary transitions from non-herbivore to herbivore guilds and no transitions from herbivore to non-herbivore guilds. There were no transitions to zooplanktivore, the most common guild, but it gave rise to all trophic guilds, except algivore, at least once. Transitions to herbivory comprised a significantly greater proportion of diet transitions in tropical and subtropical (<35°) relative to temperate areas (>35°). Our findings suggest cold temperatures may constrain the evolution of herbivory and that herbivory might act as an evolutionary "dead-end" that hinders subsequent trophic diversification, while zooplanktivory acts as an evolutionary "cradle" that facilitates trophic diversification.

Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly.

Background: Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. Results: We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. Conclusions: We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.