Michelle T. Guzik

Evolution in caves: Darwin's 'wrecks of ancient life' in the molecular era

Cave animals have historically attracted the attention of evolutionary biologists because of their bizarre ‘regressive’ characters and convergent evolution. However, understanding of their biogeographic and evolutionary history, including mechanisms of speciation, has remained elusive. In the last decade, molecular data have been obtained for subterranean taxa and their surface relatives, which have allowed some of the classical debates on the evolution of cave fauna to be revisited. Here, we review some of the major studies, focusing on the contribution of phylogeography in the following areas: biogeographic history and the relative roles of dispersal and vicariance, colonization history, cryptic species diversity and modes of speciation of cave animals. We further consider the limitations of current research and prospects for the future. Phylogeographic studies have confirmed that cave species are often cryptic, with highly restricted distributions, but have also shown that their divergence and potential speciation may occur despite the presence of gene flow from surface populations. Significantly, phylogeographic studies have provided evidence for speciation and adaptive evolution within the confines of cave environments, questioning the assumption that cave species evolved directly from surface ancestors. Recent technical developments involving ‘next generation’ DNA sequencing and theoretical developments in coalescent and population modelling are likely to revolutionize the field further, particularly in the study of speciation and the genetic basis of adaptation and convergent evolution within subterranean habitats. In summary, phylogeographic studies have provided an unprecedented insight into the evolution of these unique fauna, and the future of the field should be inspiring and data rich.

Phylogeography of the ancient Parabathynellidae (Crustacea : Bathynellacea) from the Yilgarn region of Western Australia

The crustacean order Bathynellacea is a primitive group of subterranean aquatic (stygobitic) invertebrates that typically inhabits freshwater interstitial spaces in alluvia. A striking diversity of species from the bathynellacean family Parabathynellidae have been found in the calcretes of the Yilgarn palaeodrainage system in Western Australia. Taxonomic studies show that most species are restricted in their distribution to a single calcrete, which is consistent with the findings of other phylogeographic studies of stygofauna. In this, the first molecular phylogenetic and phylogeographic study of interspecific relationships among parabathynellids, we aimed to explore the hypothesis that species are short-range endemics and restricted to single calcretes, and to investigate whether there were previously unidentified cryptic species. Analyses of sequence data based on a region of the mitochondrial (mt) DNA cytochrome c oxidase 1 gene showed the existence of divergent mtDNA lineages and species restricted in their distribution to a single calcrete, in support of the broader hypothesis that these calcretes are equivalent to closed island habitats comprising endemic taxa. Divergent mtDNA lineages were also observed to comprise four new and 12 recognised morphospecies. These results reflect the findings of previous studies of stygobitic arthropods (beetles, amphipods and isopods) from the Yilgarn region and reinforce the usefulness of using DNA-sequence data to investigate species boundaries and the presence of cryptic species.

Fine-scale comparative phylogeography of a sympatric sister species triplet of subterranean diving beetles from a single calcrete aquifer in Western Australia.

Calcrete aquifers in the arid Yilgarn region of central Western Australia are a biodiversity hotspot for stygofauna. A distinct pattern of interspecific size class variation among subterranean dytiscid beetle species has been observed in 29 of these aquifers where either two or three small, medium and/or large sympatric species are found that are in some cases sister species. We used a 3.5 km2 grid of bores to sample dytiscids on a fine‐scale and employed a comparative phylogeographical and population genetic approach to investigate the origins of a sympatric sister species triplet of diving beetles from a single aquifer. Mitochondrial DNA sequence data from the Cytochrome oxidase c subunit I gene revealed that all three species have high levels of haplotype diversity with ancient (∼1 million years ago) intra‐specific coalescence of haplotypes, but low levels of nucleotide diversity. Population analyses provide evidence for multiple expansion events within each species. There was spatial heterogeneity in the distribution of genetic variation and abundance both within and among the three taxa. Population analyses revealed significant fine‐scale differentiation with isolation by distance for Paroster macrosturtensis and P. mesosturtensis, but not the smallest species P. microsturtensis. Haplotype network analyses provided limited or no evidence for past population fragmentation within the large and small species, but substantial historical divergence was observed in P. mesosturtensis that was not spatially structured. A patchy population structure with contemporaneous and historical isolation by distance in the three species is likely to have been a significant isolating and diversifying force, preventing us from ruling out a potential role for allopatric divergence during speciation of this beetle sister triplet.

Extraordinary micro-endemism in Australian desert spring amphipods.

Increasing pressure for water in the Australian arid zone is placing enormous stress on the diverse endemic communities inhabiting desert springs. Detailed information about the evolutionary processes occurring within and between individual endemic species will help to develop effective and biologically relevant management strategies this fragile ecosystem. To help determine conservation priorities, we documented the genetic structure of the endemic freshwater amphipod populations in springs fed by the Great Artesian Basin in central Australia. Phylogenetic and phylogeographic history and genetic diversity measures were examined using nuclear and mitochondrial DNA from approximately 500 chiltoniid amphipods across an entire group of springs. Pronounced genetic diversity was identified, demonstrating that levels of endemism have been grossly underestimated in these amphipods. Using the GMYC model, 13 genetically divergent lineages were recognized as Evolutionarily Significant Units (ESUs), all of which could be considered as separate species. The results show that due to the highly fragmented ecosystem, these taxa have highly restricted distributions. Many of the identified ESUs are endemic to a very small number of already degraded springs, with the rarest existing in single springs. Despite their extraordinarily small ranges, most ESUs showed relative demographic stability and high levels of genetic diversity, and genetic diversity was not directly linked to habitat extent. The relatively robust genetic health of ESUs does not preclude them from endangerment, as their limited distributions ensure they will be highly vulnerable to future water extraction.

The ink sac clouds octopod evolutionary history

Difficulties in elucidating the evolutionary history of the octopods have arisen from problems in identifying informative morphological characters. Recent classifications have divided the largest group, the incirrate octopods, into five groups. These include the pelagic superfamily Argonautoidea and three gelatinous pelagic families (Vitreledonellidae, Bolitaenidae, Amphitretidae). All benthic incirrate octopods have been accommodated in the family Octopodidae, itself divided into four subfamilies, Octopodinae, Eledoninae, Bathypolypodinae and Graneledoninae, which are defined by the presence or absence of an ink sac, and uniserial or biserial sucker arrangements on the arms. We used relaxed clock models in a Bayesian framework and maximum likelihood methods to analyse three nuclear and four mitochondrial genes of representatives from each of the previous subfamilies. Strong evidence indicates that the family Octopodidae is paraphyletic and contains the gelatinous pelagic families. The subfamilies of Octopodidae recognised in earlier works do not reflect evolutionary history. The following clades were supported in all analyses: (1) Eledone/Aphrodoctopus, (2) Callistoctopus/Grimpella/Macroctopus/Scaeurgus, (3) Abdopus/Ameloctopus/Amphioctopus/Cistopus/Hapalochlaena/Octopus, (4) Enteroctopus/Muusoctopus/Vulcanoctopus, (5) Vitreledonella/Japetella, (6) Southern Ocean endemic and deep-sea taxa with uniserial suckers. These clades form the basis for a suite of taxa assigned family taxonomic rank: Amphitretidae, Bathypolypodidae, Eledonidae, Enteroctopodidae, Megaleledonidae and Octopodidae sensu nov. They are placed within the superfamily Octopodoidea.

Evidence for population fragmentation within a subterranean aquatic habitat in the Western Australian desert

The evolution of subterranean animals following multiple colonisation events from the surface has been well documented, but few studies have investigated the potential for species diversification within cavernicolous habitats. Isolated calcrete (carbonate) aquifers in central Western Australia have been shown to contain diverse assemblages of aquatic subterranean invertebrate species (stygofauna) and to offer a unique model system for exploring the mechanisms of speciation in subterranean ecosystems. In this paper, we investigated the hypothesis that microallopatric speciation processes (fragmentation and isolation by distance (IBD)) occur within calcretes using a comparative phylogeographic study of three stygobiontic diving beetle species, one amphipod species and a lineage of isopods. Specimens were sequenced for the mitochondrial cytochrome c oxidase 1 gene from three main sites: Quandong Well, Shady Well (SW) and Mt. Windarra (MW), spanning a 15 km region of the Laverton Downs Calcrete. Phylogenetic and haplotype network analyses revealed that each species possessed a single divergent clade of haplotypes that were present only at the southern MW site, despite the existence of other haplotypes at MW that were shared with SW. IBD between MW and SW was evident, but the common phylogeographic pattern most likely resulted from fragmentation, possibly by a salt lake adjacent to MW. These findings suggest that microallopatric speciation within calcretes may be a significant diversifying force, although the proportion of stygofauna species that may have resulted from in situ speciation in this system remains to be determined.

Desert Springs: Deep Phylogeographic Structure in an Ancient Endemic Crustacean (Phreatomerus latipes)

Desert mound springs of the Great Artesian Basin in central Australia maintain an endemic fauna that have historically been considered ubiquitous throughout all of the springs. Recent studies, however, have shown that several endemic invertebrate species are genetically highly structured and contain previously unrecognised species, suggesting that individuals may be geographically ‘stranded in desert islands’. Here we further tested the generality of this hypothesis by conducting genetic analyses of the obligate aquatic phreatoicid isopod Phreatomerus latipes. Phylogenetic and phylogeographic relationships amongst P. latipes individuals were examined using a multilocus approach comprising allozymes and mtDNA sequence data. From the Lake Eyre region in South Australia we collected data for 476 individuals from 69 springs for the mtDNA gene COI; in addition, allozyme electrophoresis was conducted on 331 individuals from 19 sites for 25 putative loci. Phylogenetic and population genetic analyses showed three major clades in both allozyme and mtDNA data, with a further nine mtDNA sub-clades, largely supported by the allozymes. Generally, each of these sub-clades was concordant with a traditional geographic grouping known as spring complexes. We observed a coalescent time between ∼2–15 million years ago for haplotypes within each of the nine mtDNA sub-clades, whilst an older total time to coalescence (>15 mya) was observed for the three major clades. Overall we observed that multiple layers of phylogeographic history are exemplified by Phreatomerus, suggesting that major climate events and their impact on the landscape have shaped the observed high levels of diversity and endemism. Our results show that this genus reflects a diverse fauna that existed during the early Miocene and appears to have been regionally restricted. Subsequent aridification events have led to substantial contraction of the original habitat, possibly over repeated Pleistocene ice age cycles, with P. latipes populations becoming restricted in the distribution to desert springs.

What lies beneath: Molecular phylogenetics and ancestral state reconstruction of the ancient subterranean Australian Parabathynellidae (Syncarida, Crustacea)

The crustacean family Parabathynellidae is an ancient and significant faunal component of subterranean ecosystems. Molecular data were generated in order to examine phylogenetic relationships amongst Australian genera and assess the species diversity of this group within Australia. We also used the resultant phylogenetic framework, in combination with an ancestral state reconstruction (ASR) analysis, to explore the evolution of two key morphological characters (number of segments of the first and second antennae), previously used to define genera, and assess the oligomerization principle (i.e. serial appendage reduction over time), which is commonly invoked in crustacean systematics. The ASR approach also allowed an assessment of whether there has been convergent evolution of appendage numbers during the evolution of Australian parabathynellids. Sequence data from the mtDNA COI and nDNA 18S rRNA genes were obtained from 32 parabathynellid species (100% of described genera and ~25% of described species) from key groundwater regions across Australia. Phylogenetic analyses revealed that species of each known genus, defined by traditional morphological methods, were monophyletic, suggesting that the commonly used generic characters are robust for defining distinct evolutionary lineages. Additionally, ancestral state reconstruction analysis provided evidence for multiple cases of convergent evolution for the two morphological characters evaluated, suggesting that caution needs to be shown when using these characters for elucidating phylogenetic relationships, particularly when there are few morphological characters available for reconstructing relationships. The ancestral state analysis contradicted the conventional view of parabathynellid evolution, which assumes that more simplified taxa (i.e. those with fewer-segmented appendages and setae) are derived and more complex taxa are primitive.

Molecular phylogenetic analysis of Western Australian troglobitic chthoniid pseudoscorpions (Pseudoscorpiones : Chthoniidae) points to multiple independent subterranean clades

Abstract. The Yilgarn and Pilbara regions of Western Australia are considered biodiversity hotspots for subterranean invertebrates. While the relatively well studied (aquatic) stygofauna are typically constrained to geographically isolated habitats (‘subterranean islands’) and have likely originated from multiple independent epigean ancestors, the troglofauna found in cavernicolous calcretes and fractured rock remains largely unstudied. Here we focus on the pseudoscorpion genera Tyrannochthonius Chamberlin, 1929 and Lagynochthonius Beier, 1951, as common components of the troglofauna, to determine whether they also display highly restricted distributional patterns, and have independent origins. Bayesian and maximum likelihood analyses of sequence data from the mtDNA cytochrome c oxidase I (COI) and the small subunit 18S nuclear genes for subterranean and epigean species from both genera reveal divergent mtDNA lineages that are restricted to single aquifers and/or geographic locations. This strong geographic structuring of troglobitic pseudoscorpions is indicative of short-range endemism and supports the ‘subterranean island’ hypothesis. Further, independent sister relationships between subterranean and epigean taxa indicate multiple invasions into subterranean habitats, likely driven by post-Miocene aridification, consistent with that predicted for the stygofauna. The phylogeny also reveals that Tyrannochthonius + Lagynochthonius is monophyletic but that Lagynochthonius is polyphyletic and nested inside Tyrannochthonius. The results of this study point to common processes that have shaped the diversity and uniqueness of both stygofaunal and troglofaunal communities in Western Australia.

Desert spring refugia: museums of diversity or evolutionary cradles?

Refugia play a critical role in preserving species unable to move or adapt to cope with environmental change. The role of refugia as ‘museums of diversity’ means these environments have a high conservation priority. Less well known, however, is the role that isolated and fragmented refugia can play in the generation of new diversity. Here, we examined the diversification and evolutionary history of a community of endemic invertebrates that inhabit Australian desert spring refugia. We compared the phylogenies of seven endemic groups (Haloniscus and Phreatomerus isopods, chiltoniid amphipods, Ngarawa ostracods, Trochidrobia and Fonscochlea snails and Gymnochthebius beetles) from these springs and examine the rates and timing of diversification and reconstructed the phylogeographic history of each taxon. Despite major life‐history differences among these taxa, they demonstrate remarkable similarities in their evolutionary histories. All groups have multiple lineages that extend back to a time before the formation of present‐day deserts, and significant geographic‐based diversification since adapting to a refugial habitat. The results provide further evidence that refugia act as museums of biodiversity, preserving lineages that would have otherwise gone extinct. However, we also observed that isolation in refugia corresponds with significant diversification, leading to a recently evolved, novel endemic fauna, supporting the idea that fragmented refugia provide ideal conditions for the generation of future biodiversity hotspots.