Crayfish can be used as model organisms in phylogeographic and divergence time studies if reliable calibrations are available. This study presents a comprehensive investigation into the phylogeography of the European stone crayfish ( Austropotamobius torrentium ) and includes samples from previously unstudied sites. Two mitochondrial markers were used to reveal evolutionary relationships among haplogroups throughout the species’ distributional range and to estimate the divergence time by employing both substitution rates and geological calibration methods. Our haplotype network reconstruction and phylogenetic analyses revealed the existence of a previously unknown haplogroup distributed in Romania's Apuseni Mountains. This haplogroup is closely related to others that are endemic in the Dinarides, despite their vast geographical separation (~600 km). The separation is best explained by the well‐dated tectonic displacement of the Tisza–Dacia microplate, which started in the Miocene (~16 Ma) and possibly carried part of the A. torrentium population to the current location of the Apuseni Mountains. This population may thus have been isolated from the Dinarides for a period of ca. 11 m.y. by marine and lacustrine phases of the Pannonian Basin. The inclusion of this geological event as a calibration point in divergence time analyses challenges currently accepted crayfish evolutionary time frames for the region, constraining the evolution of this area's crayfish to a much earlier date. We discuss why molecular clock calibrations previously employed to date European crayfish species divergences should therefore be reconsidered.

Abstract:Anchialine caves contain haline bodies of water with underground connections to the ocean and limited exposure to open air. Despite being found on islands and peninsular coastlines around the world, the isolation of anchialine systems has facilitated the evolution of high levels of endemism among their inhabitants. The unique characteristics of anchialine caves and of their predominantly crustacean biodiversity nominate them as particularly interesting study subjects for evolutionary biology. However, there is presently a distinct scarcity of modern molecular methods being employed in the study of anchialine cave ecosystems. The use of current and emerging molecular techniques, e.g., next-generation sequencing (NGS), bestows an exceptional opportunity to answer a variety of long-standing questions pertaining to the realms of speciation, biogeography, population genetics, and evolution, as well as the emergence of extraordinary morphological and physiological adaptations to these unique environments. The integration of NGS methodologies with traditional taxonomic and ecological methods will help elucidate the unique characteristics and evolutionary history of anchialine cave fauna, and thus the significance of their conservation in face of current and future anthropogenic threats. Here we review previous contributions to our understanding of anchialine biodiversity and evolution, and discuss the potential of "speleogenomic" methods for future research in these threatened systems.

19A major goal of evolutionary biology is to understand the role of adaptive processes on 20 sensory systems. Visual capabilities are strongly influenced by environmental and 21 ecological conditions, and the evolutionary advantages of vision are manifest by its 22 29 developed a modified version of the Phylogenetically Informed Annotation tool to filter 30 and identify visual genes from transcriptomes in a scalable and efficient manner. 31 Additionally, reference genomes of these species were used to validate our pipeline 32 while characterizing the genomic architecture of the opsin genes. Next-generation 33 sequencing and phylogenetics provide future venues for the study of sensory systems, 34 adaptation, and evolution in model and non-model organisms. 35 36

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Animals that inhabit subterranean environments often undergo various distinct phenotypic modifications (referred to as "troglomorphy") as they transition to life in perpetual darkness. However, the molecular basis behind troglomorphy remains poorly understood, particularly in regards to the mechanisms involved in the reduction and/or loss of traits at the transcriptomic level. In this study, we investigate the transcriptional basis behind vision loss in populations of cave-dwelling crustaceans. We employ phylogenetic and transcriptomic methods on surface and cave-adapted populations of an emerging model species for biospeleology, the isopod Asellus aquaticus (Linnaeus, 1758), and the amphipod Niphargus hrabei S. Karaman, 1932. These two species show contrasting directionality in the surface-cave transition, which positions them as ideal study subjects. Asellus aquaticus is common in surface waters and is only occasionally found in caves, where its populations present different degrees of eye reduction and pigmentation. On the other hand, the eyeless N. hrabei has successfully colonized surface environments despite belonging to an almost exclusively cave-dwelling genus. By sequencing and assembling robust de novo transcriptomes we characterized differences in visual genes and pathways among surface and cave populations of the aforementioned species. Our results indicate that despite having reduced eyes, recent cave colonizer A. aquaticus is still capable of expressing functional visual opsins and major components of the phototransduction pathway within the cave. Niphargus hrabei, a species with an ancient cave origin, shows no clear indication of being capable of sight. However, the expression of putative functional visual opsins and other phototransduction genes was maintained, which suggests that this eyeless species might be capable of extraocular photoreception. With the present study, we aim to bring forth the Molnár János Cave system as a promising research avenue to improve our understanding of patterns of reduction and loss of vision in caves and other aphotic environments.

BackgroundThe underlying mechanisms and processes that prompt the colonisation of extreme environments, such as caves, constitute major research themes of evolutionary biology and biospeleology. The special adaptations required to survive in subterranean environments (low food availability, hypoxic waters, permanent darkness), and the geographical isolation of caves, nominate cave biodiversity as ideal subjects to answer long-standing questions concerning the interplay amongst adaptation, biogeography, and evolution. The present project aims to examine the phylogeographic patterns exhibited by two sympatric species of surface and cave-dwelling peracarid crustaceans (Asellus aquaticus and Niphargus hrabei), and in doing so elucidate the possible roles of isolation and exaptation in the colonisation and successful adaptation to the cave environment.ResultsSpecimens of both species were sampled from freshwater hypogean (cave) and epigean (surface) habitats in Hungary, and additional data from neighbouring countries were sourced from Genbank. Sequencing of mitochondrial and nuclear loci revealed, through haplotype network reconstruction (TCS) and phylogenetic inference, the genetic structure, phylogeographic patterns, and divergence-time estimates of A. aquaticus and N. hrabei surface and cave populations. Contrasting phylogeographic patterns were found between species, with A. aquaticus showing strong genetic differentiation between cave and surface populations and N. hrabei lacking any evidence of genetic structure mediated by the cave environment. Furthermore, N. hrabei populations show very low levels of genetic differentiation throughout their range, which suggests the possibility of recent expansion events over the last few thousand years.ConclusionsIsolation by cave environment, rather than distance, is likely to drive the genetic structuring observed between immediately adjacent cave and surface populations of A. aquaticus, a predominantly surface species with only moderate exaptations to subterranean life. For N. hrabei, in which populations exhibit a fully ‘cave-adapted’ (troglomorphic) phenotype, the lack of genetic structure suggests that subterranean environments do not pose a dispersal barrier for this surface-cave species.Electronic supplementary materialThe online version of this article (10.1186/s12862-017-1094-9) contains supplementary material, which is available to authorized users.

High-quality RNA is an important precursor for high-throughput RNA sequencing (RNAseq) and subsequent analyses. However, the primary metric used to assess RNA quality, the RNA Integrity Number (RIN), was developed based on model bacterial and vertebrate organisms. Though the phenomenon is not widely recognized, invertebrate 28S ribosomal RNA (rRNA) is highly prone to a form of denaturation known as gap deletion, in which the subunit collapses into two smaller fragments. In many nonmodel invertebrates, this collapse of the 28S subunit appears as a single band similar in size to the 18S rRNA subunit. This phenomenon is hypothesized to be commonplace among arthropods and is often misinterpreted as a “degraded” rRNA profile. The limited characterization of gap deletion in arthropods, a highly diverse group, as well as other nonmodel invertebrates, often biases RNA quality assessments. To test whether the collapse of 28S is a general pattern or a methodological artifact, we sampled more than half of the major lineages within Arthropoda. We found that the 28S collapse is present in ∼90% of the species sampled. Nevertheless, RNA profiles exhibit considerable diversity with a range of banding patterns. High-throughput RNAseq and subsequent assembly of high-quality transcriptomes from select arthropod species exhibiting collapsed 28S subunits further illustrates the limitations of current RIN proxies in accurately characterizing RNA quality in nonmodel organisms. Furthermore, we show that this form of 28S denaturation, which is often mistaken for true “degradation,” can occur at relatively low temperatures.

Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans.