Steven J.B. Cooper

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.

Genetic evidence for a family structure in stable social aggregations of the Australian lizard Egernia stokesii

In this study we used data from six unlinked microsatellite loci to examine stable aggregations of Egernia stokesii, from a population in the southern Flinders Ranges of South Australia. We show that these aggregations are comprised of breeding partners, their offspring from two or more cohorts, and related adults, providing the first genetic evidence of a family structure in any lizard species. Despite this high level of relatedness within aggregations, most breeding pairs were unrelated and partners were less closely related to each other than they were to other potential within‐group partners. Where individuals dispersed, both sexes usually moved to social groups close to their natal group. Although both sexes showed natal philopatry, there was some evidence that females in groups were more related than males in groups. These data suggest that an active choice of unrelated partners and male‐biased dispersal may be the mechanisms used by E. stokesii to avoid inbreeding within groups.

Subterranean archipelago in the Australian arid zone: mitochondrial DNA phylogeography of amphipods from central Western Australia

In 1998, a unique subterranean ecosystem was discovered in numerous isolated calcrete (carbonate) aquifers in the arid Yilgarn region of Western Australia. Previous morphological and genetic analyses of a subterranean water beetle fauna suggest that calcrete aquifers are equivalent to closed island habitats that have been isolated for millions of years. We tested this hypothesis further by phylogeographic analyses of subterranean amphipods (Crangonyctoidea: Paramelitidae and Hyalidae) using mitochondrial DNA sequence data derived from the cytochrome oxidase I gene. Phylogenetic analyses and population genetic analyses (samova) provided strong evidence for the existence of at least 16 crangonyctoid and six hyalid divergent mitochondrial lineages, each restricted in their distribution to a single calcrete aquifer, in support of the ‘subterranean island (archipelago) hypothesis’ and extending its scope to include entirely water respiring invertebrates. Sequence divergence estimates between proximate calcrete populations suggest that calcretes have been isolated at least since the Pliocene, coinciding with a major aridity phase that led to the intermittent drying of surface water. The distribution of calcretes along palaeodrainage channels and on either side of drainage divides, have had less influence on the overall phylogeographic structure of populations, with evidence that ancestral crangonyctoid and hyalid species moved between catchments multiple times prior to their isolation within calcretes. At least two potential modes of evolution may account for the diversity of subterranean amphipod populations: dispersal/vicariance of stygobitic species or colonization of calcretes by surface species and independent evolution of stygobitic characteristics.

Social monogamy and extra-pair fertilization in an Australian lizard, Tiliqua rugosa

Abstract This study investigates social monogamy in the Australian sleepy lizard, Tiliqua rugosa. At a 70-ha site near Mount Mary, South Australia, we radio tracked 55 adult female and 39 adult male lizards during their spring activity periods. Each lizard was observed in 1–5 years. Females were observed with a single male partner on an average of 10.8 days per year, although in 17.3% of cases, females were observed on 2 or fewer days with a male. The most intense pairing period each year was 15 September–15 November when females were with male partners on an average of 36% of observation days. Partnerships lasted an average of 43.3 days each year. After mating in early November, the pairs separated. Observations of females pairing with other males were rare. Most males (82%) were also consistently monogamous, although 7 were observed pairing with 2 females within one season. To investigate paternity, we allowed 21 gravid females to give birth to 42 offspring in the laboratory. We determined genotypes at five polymorphic microsatellite DNA loci for the females, their male partners and their offspring. Four litters (19%) and 6 of the offspring from those litters (14.3%) showed evidence of extra-pair fertilization (EPF). Although the sample sizes are small, females of polygynous males were more likely to experience EPF.

Molecular Phylogenetics of Allodapine Bees, with Implications for the Evolution of Sociality and Progressive Rearing

Allodapine bees have long been regarded as providing useful material for examining the origins of social behavior. Previous researchers have assumed that sociality arose within the Allodapini and have linked the evolution of sociality to a transition from mass provisioning to progressive provisioning of brood. Early phylogenetic studies of allodapines were based on morphological and life-history data, but critical aspects of these studies relied on small character sets, where the polarity and coding of characters is problematic. We used nucleotide sequence data from one nuclear and two mitochondrial gene fragments to examine phylogenetic structure among nine allodapine genera. Our data set comprised 1506 nucleotide positions, of which 402 were parsimony informative. Maximum parsimony, log determinant, and maximum likelihood analyses produced highly similar phylogenetic topologies, and all analyses indicated that the tropical African genus Macrogalea was the sister group to all other allodapines. This finding conflicts with that of previous studies, in which Compsomelissa + Halterapis formed the most basal group. Changing the basal node of the Allodapini has major consequences for understanding evolution in this tribe. Our results cast doubt on the previous hypotheses that progressive provisioning and castelike social behavior evolved among lineages leading to the extant allodapine taxa. Instead, our results suggest that mass provisioning in Halterapis is a derived feature and that social behavior is an ancestral trait for all allodapine lineages. The forms of social behavior present in extant allodapines are likely to have resulted from a long evolutionary history, which may help explain the complexity of social traits found in many allodapine bees.

Deep phylogeographic structuring of populations of the trapdoor spider Moggridgea tingle (Migidae) from southwestern Australia: evidence for long-term refugia within refugia

Southwestern Australia has been recognized as a biodiversity hot spot of global significance, and it is particularly well known for its considerable diversity of flowering plant species. Questions of interest are how this region became so diverse and whether its fauna show similar diverse patterns of speciation. Here, we carried out a phylogeographic study of trapdoor spiders (Migidae: Moggridgea), a presumed Gondwanan lineage found in wet forest localities across southwestern Australia. Phylogenetic, molecular clock and population genetic analyses of mitochondrial (mtDNA) COI gene and ITS rRNA (internal transcribed spacer) data revealed considerable phylogeographic structuring of Moggridgea populations, with evidence for long‐term (>3 million years) isolation of at least nine populations in different geographic locations, including upland regions of the Stirling and Porongurup Ranges. High levels of mtDNA divergence and no evidence of recent mitochondrial gene flow among valley populations of the Stirling Range suggest that individual valleys have acted as refugia for the spiders throughout the Pleistocene. Our findings support the hypothesis that climate change, particularly the aridification of Australia after the late Miocene, and the topography of the landscape, which allowed persistence of moist habitats, have been major drivers of speciation in southwestern Australia.

Subterranean archipelago: mitochondrial DNA phylogeography of stygobitic isopods (Oniscidea:Haloniscus ) from the Yilgarn region of Western Australia

The arid Yilgarn region of Western Australia contains numerous isolated calcrete aquifers, within which a diverse subterranean fauna has been discovered. Genetic and morphological studies of subterranean dytiscid beetles and amphipods have suggested that individual calcretes are equivalent to closed island habitats, which have been isolated for millions of years. Here we test this ‘subterranean island’ hypothesis further by phylogeographic analyses of subterranean oniscidean isopods (Haloniscus), using mitochondrial DNA (mtDNA) sequence data derived from the cytochrome c oxidase subunit I gene. Phylogenetic and population genetic analyses provided evidence for significant phylogeographic structuring of isopod populations, with evidence for at least 24 divergent mtDNA lineages, each restricted in their distribution to a single calcrete aquifer. The high level of divergence among calcrete populations (generally >25%) and several mtDNA lineages within calcretes (>16%) suggests that each lineage is likely to represent a distinct species. These analyses, together with comparative phylogeographic data from dytiscid beetles and amphipods, provide strong support for the ‘subterranean island’ hypothesis, applying to both air-breathing and fully aquatic arthropod groups. The finding of several epigean lineages that grouped with stygobitic Haloniscus populations, and the overall phylogeographic structure of populations, suggests that the majority of stygobitic species evolved within individual calcretes following independent colonisation by epigean ancestors.

High levels of genetic monogamy in the group-living Australian lizard Egernia stokesii.

The Australian lizard Egernia stokesii lives in spatially and temporally stable groups of up to 17 individuals. We have recently shown that these groups are comprised of breeding partners, their offspring and, in some cases, highly related adults, providing the first genetic evidence of a family structure in any lizard species. Here we investigated the mating system of E. stokesii using data from up to eight polymorphic microsatellite loci and tested the hypothesis that breeding partners are monogamous both within and between mating seasons. Among 16 laboratory‐born litters from field collected gravid females from two sites in South Australia, 75% had a single male parent and no male contributed to more than one litter, indicating a high level of genetic monogamy within a season. Additional analyses of field caught individuals, captured between 1994 and 1998, enabled assignment of parentage for 70 juveniles and subadults. These data showed that most young (88.6%) had both parents from within the same group and that high proportions of males (88.9%) and females (63.6%) have multiple cohorts of offspring only with the same partner. Our results suggest that monogamy both within and between seasons is a common mating strategy of E. stokesii and that breeding partners maintain stable associations together and with multiple cohorts of their offspring over periods of up to at least 5 years.

Assessment of prey overlap between a native (Polistes humilis) and an introduced (Vespula germanica) social wasp using morphology and phylogenetic analyses of 16S rDNA

In newly invaded communities, interspecific competition is thought to play an important role in determining the success of the invader and its impact on the native community. In southern Australia, the native Polistes humilis was the predominant social wasp prior to the arrival of the exotic Vespula germanica (Hymenoptera: Vespidae). Both species forage for similar resources (water, pulp, carbohydrate and protein prey), and concerns have arisen about potential competition between them. The aim of this study was to identify the protein foods that these wasps feed on. As many prey items are masticated by these wasps to the degree that they cannot be identified using conventional means, morphological identification was complemented by sequencing fragments of the mitochondrial 16S rRNA gene. GenBank searches using blast and phylogenetic analyses were used to identify prey items to at least order level. The results were used to construct complete prey inventories for the two species. These indicate that while P. humilis is restricted to feeding on lepidopteran larvae, V. germanica collects a variety of prey of invertebrate and vertebrate origin. Calculated values of prey overlap between the two species are used to discuss the implications of V. germanica impacting on P. humilis. Results obtained are compared to those gained by solely ‘conventional’ methods, and the advantages of using DNA‐based taxonomy in ecological studies are emphasized.

Molecular phylogenetics of the exoneurine allodapine bees reveal an ancient and puzzling dispersal from Africa to Australia

Previous phylogenetic studies of the bee tribe Allodapini suggested a puzzling biogeographic problem: one of the key basal divergences involved separation of the southern African and southern Australian clades at a very early stage in allodapine evolution, but no taxa occur in the Palaearctic or Asian regions that might suggest a Laurasian dispersal route. However, these studies lacked sufficient sequence data and appropriate maximum likelihood partition models to provide reliable phylogenetic estimates and enable alternative biogeographic hypotheses to be distinguished. Using Bayesian and penalized likelihood approaches and an expanded sequence and taxon set we examine phylogenetic relationships between the Australian, African, and Malagasy groups and estimate divergence times for key nodes. We show that divergence of the three basal Australian clades (known as the exoneurines) occurred at least 25 Mya following a single colonization event, and that this group diverged from the African + Madagascan clade at least 30 Mya, but actual divergence dates are likely to be much older than these very conservative limits. The bifurcation order of the exoneurine clades was not resolved and analyses could not rule out the existence of a hard polytomy, suggesting rapid radiation after colonization of Australia. Their divergence involved major transitions in life history traits and these placed constraints on the kinds of social organization that subsequently evolved in each lineage. Early divergence between the African, Malagasy, and Australian clades presents a major puzzle for historical biogeography: node ages are too recent for Gondwanan vicariance hypotheses, but too early for Laurasian dispersal scenarios. We suggest a scenario involving island hopping across the Indian Ocean via a series of now largely submerged elements of the Kergulen Plateau and Broken Ridge provinces, both of which are known to have had subaerial formations during the Cenozoic. [Bayesian; biogeography; dispersal; Gondwana; Kerguelen Plateau; penalized likelihood.].