Evolution underground: A molecular phylogenetic investigation of Australian burrowing freshwater crayfish (Decapoda: Parastacidae) with particular focus on Engaeus Erichson

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Abstract

Phylogenetic relationships and species boundaries of Australian burrowing freshwater crayfish belonging to the genera Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus are investigated using combined mitochondrial and nuclear DNA sequence data and Bayesian and Maximum Parsimony methods. Phylogenies are statistically compared to previously published hypotheses. Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus form a strongly supported monophyletic clade. This grouping is independently supported by morphology but unites geographically highly disjunct lineages. Our data show two cryptic species in Geocharax, one cryptic species in Gramastacus and two cryptic species within the highly divergent Engaeus lyelli lineage. Using a Bayesian relaxed molecular clock method, the 16S rDNA data show generic-level diversification coinciding with the transition from a wet to arid palaeoclimate near the mid Miocene.

Introduction

Freshwater crayfish are found on every continent except continental Africa, the Indian sub-continent and Antarctica (Crandall and Buhay, 2008). The consensus of geographical, morphological, molecular and palaeontological evidence strongly supports their monophyletic origin from marine ancestors (e.g. Crandall et al., 2000, Scholtz, 1993, Sinclair et al., 2004, Tsang et al., 2008) sometime around 280 million years ago (Ma) (Porter et al., 2005). Freshwater crayfish then dispersed throughout the Pangean supercontinent before it divided (∼185 Ma) into the Laurasian (northern hemisphere) and Gondwanan (southern hemisphere) continents. Reciprocal monophyly of the northern and southern hemisphere superfamilies—the Astacoidea Latreille, 1802 and the Parastacoidea Huxley, 1878, respectively—support this model (Crandall and Buhay, 2008, Porter et al., 2005, Sinclair et al., 2004).

The Parastacoidea contains one family, the Parastacidae Huxley, 1878, and has its centre of species diversity in southeastern Australia where eight of the 10 currently recognised extant Australian genera are found. Four additional genera are scattered about the southern hemisphere with two in southern South America, one in Madagascar and one in New Zealand. Crayfish body and trace fossils (burrows) confirm a Parastacid presence in southeastern Australia since at least 106–116 million years (m.y.) before the present (Martin et al., 2008). The distribution of extant Parastacidae remains consistent with ancient Gondwanan connections and fossils pre-date complete separation of Australia from Antarctica (Bedatou et al., 2008, Crandall and Buhay, 2008, Martin et al., 2008, Sampson et al., 1998, Veevers, 2006). Many forms of evidence corroborate southeastern Australia as an important area for freshwater crayfish evolution. Based on a phylogenetic assessment, Whiting et al. (2000) recommended southeastern Australia (including Tasmania) as a priority region for the conservation of freshwater crayfish.

Since the 19th century, various conflicting evolutionary scenarios for the Parastacidae have been assembled from morphological, geographical and/or palaeontological data (e.g. Huxley, 1880, Ortmann, 1902, Riek, 1959, Riek, 1969, Riek, 1972, Rode and Babcock, 2003). More recently, molecular data have been used, sometimes in combination with morphological and ecological data, to address questions of relationships between the Australian genera or between the Australian and New Zealand genera (e.g. Austin, 1995, Crandall et al., 1999, Crandall et al., 1995, Patak and Baldwin, 1984, Patak et al., 1989, Schultz et al., 2007). From these studies, it is evident that many aspects of the phylogeny of the Australian Parastacidae remain unclear. These uncertainties require resolution before a proper understanding of evolutionary diversification within the Parastacidae can be achieved.

A conspicuous feature of Australian Parastacidae is their ecological and morphological diversity. This principally relates to some genera having successfully radiated into semipermanent aquatic environments, evolving a largely underground lifestyle and an ability to construct large and often complex burrow systems (Horwitz, 1985, Horwitz and Richardson, 1986). Adaptation to burrowing has resulted in the evolution of unique or distinctive morphological features, such as the reduction of the size and width of the abdomen (Hobbs, 1974, Horwitz, 1988b, Horwitz, 1990a, Horwitz and Richardson, 1986, Suter, 1977a, Suter, 1977b) and vertically or sub-vertically inclined great chelae (the first pereopod) (Riek, 1969, Riek, 1972).

Based on orientation of the great chelae and burrowing habits, Riek (1972) denoted two divisions within Parastacidae: the moderate burrowers, which hold the great chelae and move the fingers in a horizontal or oblique plane, and the strong burrowers, which hold the great chelae and move the fingers in a vertical or sub-vertical plane. Within the Australian genera, Riek (1972) classified moderate burrowers as Astacopsis Huxley, 1878, Euastacus Clark, 1936, Cherax Erichson, 1846, Parastacoides (Erichson, 1846) (now Ombrastacoides Hansen and Richardson, 2006 and Spinastacoides Hansen and Richardson, 2006), Geocharax Clark, 1936 and Gramastacus Riek, 1972. He proposed that these genera were monophyletic and sister to the strong burrowers, Engaeus Erichson, 1846, Engaewa Riek, 1967 and Tenuibranchiurus Riek, 1951. However, Engaeus, Engaewa and Tenuibranchiurus, together with Geocharax and Gramastacus, possess a distinctive abdominal anterolateral flap not found in any other genus of freshwater crayfish, and it is postulated that this character is a synapomorphic trait (Horwitz, 1988b; but see also Horwitz, 1985; Horwitz, 1990a, Horwitz, 1990b; Horwitz and Adams, 2000). This hypothesis is inconsistent with that of Riek’s (1972). Of further note, Riek’s (1972) phylogenetic arrangement is not supported by mitochondrial 16S rDNA nucleotide data (Crandall et al., 1999), which show non-monophyly of Engaeus, Engaewa and Tenuibranchiurus. Horwitz and Adams (2000) argued that the results of Crandall et al. (1999) were inconclusively presented and that additional sequence data are needed to readdress the placement of Engaewa.

This paper seeks to resolve the phylogeny of this group of genera (Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus), which is required to comprehend more fully their ecological, morphological, genetic and geographical diversification. Such an objective necessitates a large nucleotide dataset from as near complete a taxon sample as possible.

The distributions of these genera give rise to some intriguing biogeographic questions. Engaewa has a very restricted distribution in the extreme southwest of Western Australia. Engaeus, Geocharax and Gramastacus occur in the extreme southeast, and Tenuibranchiurus occurs along the central-east coast of Australia. If they were indeed more closely related to one another than they are to other taxa, then assessments of divergence times between the genera Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus would enable a better understanding of the evolutionary context of their successful adaptive radiation.

Of the 10 extant Australian genera, comprising approximately 150 species (e.g. Austin and Ryan, 2002, Coughran, 2005a, Coughran, 2005b, Crandall et al., 1999, Hansen and Richardson, 2006, Horwitz, 1990a, Horwitz, 1994a, Horwitz and Adams, 2000, Morgan, 1997), Euastacus (∼49 species), Cherax (∼42 species) and Engaeus (∼35 species) hold the most species diversity, with Engaeus being comparatively the least studied using nucleotide data. Despite this, some key taxonomic studies of Engaeus have been performed, using electrophoretic, morphological and nucleotide data, which have revealed uncertainties in the relationships within and between species of Engaeus (Horwitz, 1990a, Horwitz et al., 1990, Schultz et al., 2007). For example, preliminary mitochondrial 16S rDNA data suggest that Engaeus lyelli (Clark, 1936) is highly divergent from the other Engaeus species and may in fact represent a new genus (Schultz et al., 2007). This suggestion is not, in itself, entirely new, as nearly every author who has dealt with the taxonomy of E. lyelli has disagreed with regard to its designation (Clark, 1936, Horwitz, 1990a, Kane, 1964, Riek, 1969). Such findings still await a full treatment of Engaeus and phylogenetic analysis with the addition of nuclear nucleotide data.

Engaeus has a relatively restricted distribution, occurring throughout the northern part of the island of Tasmania and the southeastern Australian mainland region. The geographical density of species in this genus is very high (Horwitz, 1990a), even in comparison to the more widespread and species-rich genera Cherax (e.g. see Riek, 1969) and Euastacus (e.g. see Morgan, 1997). The habitats of Engaeus species have endured geological shifts (Horwitz, 1988a, Lambeck and Chappell, 2001, Schultz et al., 2008) and contemporary anthropogenic impacts (e.g. see Ierodiaconou et al., 2005, Horwitz, 1990b, Horwitz, 1994b, Horwitz, 1995)—the latter providing conservation challenges.

From a conservational perspective, greater than 21% of known parastacid species (38 of 177 recognised species) are already listed as vulnerable, endangered or critically endangered and fourteen of these are Engaeus species (∼40% of the genus) (Department of Primary Industries and Water, 2002, Department of Sustainability and Environment, 2007, IUCN, 2008). Three of five Engaewa species (60% of the genus) are listed as vulnerable, endangered or critically endangered (Horwitz and Adams, 2000) and Gramastacus insolitus Riek, 1972 is listed as threatened (Department of Sustainability and Environment, 2007).

In this study, we build upon the phylogenetic information presented in molecular studies of Schultz et al. (2007) and Crandall et al. (1999), the allozyme electrophoretic study of Horwitz et al. (1990) and the morphological studies of Horwitz (1990a) and Riek (1972). We collect a substantial nucleotide-sequence dataset, comprising mitochondrial (ribosomal) and nuclear (protein-coding) markers, and use this to test phylogenetic hypotheses of Crandall et al., 1999, Horwitz, 1988b and Riek (1972) and to establish relationships among the Australian genera of burrowing crayfish: Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus. Additionally, we use mitochondrial nucleotide data and a near-complete taxon sampling of Engaeus to perform a phylogenetic-based analysis, inferring inter- and intra-specific taxonomic relationships in Engaeus. This study is the first to use nucleotide data from the glyceraldehyde-3-phosphate dehydrogenase (nuclear) gene to examine phylogenetic and taxonomic relationships within the southern hemisphere freshwater crayfish family, the Parastacidae.

In addition to providing a rich source of data for phylogenetic analysis, nucleotide data are useful for dating speciation and evolutionary events and radiations (e.g. Drummond et al., 2006). Porter et al. (2005) estimated divergence times within the Decapoda Latreille, 1802 using a combined geological, geographical, palaeontological and molecular dataset; however, the only samples of parastacid crayfish included in their study were single representatives of the genera Cherax and Astacopsis. Various other estimates of divergence times within Parastacidae have also been made but are limited to intra-generic estimates for Cherax, Euastacus or Geocharax (Gouws et al., 2006, Munasinghe et al., 2004, Ponniah and Hughes, 2004, Ponniah and Hughes, 2006, Schultz et al., 2007). Since no assessments of divergence times between the genera Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus have yet been made, we estimated divergence times among these genera in this study.

Section snippets

Sampling, laboratory procedures and data collection

Samples representing six genera of burrowing freshwater crayfish were obtained for this study: Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus, with Cherax included for comparative purposes. One species from each of the genera Euastacus and Paranephrops White, 1842 were included as outgroup taxa. The outgroup taxa were selected after a preliminary analysis during this study and from the results of Crandall et al. (1999). The genera Astacopsis, Ombrastacoides and Spinastacoides

Results

We obtained 59 new 16S rDNA sequences comprising 2 Cherax, 3 Engaewa, 50 Engaeus and 4 Tenuibranchiurus specimens; and 53 new GAPDH sequences comprising 16 Cherax, 15 Engaeus, 2 Engaewa, 1 Euastacus, 11 Geocharax, 4 Gramastacus, 1 Paranephrops and 4 Tenuibranchiurus specimens. Sixty-four previously published 16S rDNA GenBank sequences were also included in the analyses, comprising 14 Cherax, 34 Engaeus, 1 Euastacus, 10 Geocharax, 4 Gramastacus and 1 Paranephrops. NCBI GenBank accession numbers

Discussion

In this paper, we present the first comprehensive molecular phylogenetic and systematic treatment of a taxonomically challenging and conservationally important group of Australian burrowing freshwater crayfish using nucleotide sequence data from a combination of mitochondrial and nuclear genes. This study is the first to explore the use of the single-copy, nuclear, protein-coding GAPDH gene for a phylogenetic assessment of southern hemisphere freshwater crayfish (superfamily Parastacoidea).

Conclusions

The hypothesis that Engaeus, Engaewa, Geocharax, Gramastacus and Tenuibranchiurus form a monophyletic clade is supported by Bayesian analysis of combined mitochondrial 16S rDNA and nuclear GAPDH nucleotide data. An anterolateral flap on the second abdominal pleonite present in mature females of these genera independently supports their monophyletic relationship. Engaeus lyelli warrants recognition as a new genus (comprising at least two species) so the number of genera included in this

Acknowledgments

This work was supported by ARC Discovery grant number DP0557840 to A.M.M.R., C.M.A., P.H. and K.A.C., and partly funded by Charles Darwin University, Deakin University and Dragonfly Environmental P/L (http://www.dfe.net.au). For access to museum specimens, we thank Dr. Gary Poore and Dr. Joanne Taylor (Museum Victoria), Dr. Peter Davie (Queensland Museum), Dr. George Wilson, Dr. Stephen Keable and Dr. Shane Ahyong (Australian Museum). For their generation of some sequence data, we thank Dr.

References (134)

  • M.L. Porter et al.

    Model-based multi-locus estimation of decapod phylogeny and divergence times

    Mol. Phylogenet. Evol.

    (2005)
  • M.J.F. Pulquério et al.

    Dates from the molecular clock: how wrong can we be?

    Trends Ecol. Evol.

    (2007)
  • H. Akaike

    A new look at the statistical model identification

    IEEE Trans. Autom. Contr.

    (1974)
  • C.M. Austin

    The definition and phylogenetic position of the genus Cherax (Decapoda: Parastacidae)

    Freshw. Crayfish

    (1995)
  • C.M. Austin et al.

    Systematics of the freshwater crayfish genus Cherax Erichson (Decapoda: Parastacidae) in south-western Australia: electrophoretic, morphological and habitat variation

    Aust. J. Zool.

    (1996)
  • C.M. Austin et al.

    The taxonomy and phylogeny of the ‘Cherax destructor’ complex (Decapoda: Parastacidae) examined using mitochondrial 16S sequences

    Aust. J. Zool.

    (2003)
  • C.M. Austin et al.

    Allozyme evidence for a new species of freshwater crayfish of the genus Cherax Erichson (Decapoda: Parastacidae) from the south-west of Western Australia

    Invertebr. Syst.

    (2002)
  • D.A. Baum et al.

    Genealogical perspectives on the species problem

  • M.C. Brandley et al.

    Partitioned Bayesian analyses, partition choice, and the phylogenetic relationships of scincid lizards

    Syst. Biol.

    (2005)
  • C.P. Burridge et al.

    Stepping stone gene flow in an estuarine-dwelling sparid from south-east Australia

    J. Fish Biol.

    (2004)
  • J. Castresana

    Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis

    Mol. Biol. Evol.

    (2000)
  • E. Clark

    The freshwater and land crayfishes of Australia

    Mem. Nat. Mus. Vict.

    (1936)
  • E. Clark

    Tasmanian Parastacidae

    Pap. Proc. R. Soc. Tasman.

    (1939)
  • E. Clark

    New species of Australian freshwater and land crayfishes (Family Parastacidae)

    Mem. Nat. Mus. Vict.

    (1941)
  • J. Coughran

    Cherax leckii n. sp. (Decapoda: Parastacidae): a new crayfish from coastal, northeastern New South Wales

    J. Aust. N. Fishes Assoc.

    (2005)
  • J. Coughran

    New crayfishes (Decapoda: Parastacidae: Euastacus) from northeastern New South Wales

    Aust. Rec. Aust. Mus.

    (2005)
  • K. Crandall et al.

    Global diversity of crayfish (Astacidae, Cambaridae, and Parastacidae–Decapoda) in freshwater

    Hydrobiologia

    (2008)
  • K.A. Crandall et al.

    Phylogenetic relationships among the Australian and New Zealand genera of freshwater crayfishes (Decapoda: Parastacidae)

    Aust. J. Zool.

    (1999)
  • K.A. Crandall et al.

    Crayfish molecular systematics: using a combination of procedures to estimate phylogeny

    Syst. Biol.

    (1996)
  • K.A. Crandall et al.

    The monophyletic origin of freshwater crayfish estimated from nuclear and mitochondrial DNA sequences

    Proc. R. Soc. Lond. B

    (2000)
  • K.A. Crandall et al.

    A preliminary examination of the molecular phylogenetic relationships of some crayfish genera from Australia (Decapoda: Parastacidae)

    Freshw. Crayfish

    (1995)
  • Department of Primary Industries and Water, 2002. Threatened species protection act 1995—threatened species list...
  • Department of Sustainability and Environment, 2007. Action statement, Western Swamp Crayfish Gramastacus insolitus....
  • A.C. Driskell et al.

    Prospects for building the tree of life from large sequence databases

    Science

    (2004)
  • A.J. Drummond et al.

    Relaxed phylogenetics and dating with confidence

    PLoS Biol.

    (2006)
  • Drummond, A.J., Ho, S.Y.W., Rawlence, N., Rambaut, A., 2007. A rough guide to BEAST 1.4. Available from:...
  • A.J. Drummond et al.

    BEAST: Bayesian evolutionary analysis by sampling trees

    BMC Evol. Biol.

    (2007)
  • R.C. Edgar

    MUSCLE: multiple sequence alignment with high accuracy and high throughput

    Nucleic Acids Res.

    (2004)
  • W.F. Erichson

    Übersicht der Arten der Gattung Astacus

    Wiegmann’s Archiv für Naturgeschichte

    (1846)
  • J. Felsenstein

    Confidence limits on phylogenies: an approach using the Bootstrap

    Evolution

    (1985)
  • A.Y. Glikson

    Milestones in the evolution of the atmosphere with reference to climate change

    Aust. J. Earth Sci.

    (2008)
  • N. Goldman et al.

    Likelihood-based tests of topologies in phylogenetics

    Syst. Biol.

    (2000)
  • G. Gouws et al.

    Phylogeographic structure of a freshwater crayfish (Decapoda: Parastacidae: Cherax preissii) in south-western Australia

    Mar. Freshw. Res.

    (2006)
  • P. Hamr

    A revision of the Tasmanian freshwater crayfish genus Astacopsis Huxley (Decapoda: Parastacidae)

    Pap. Proc. R. Soc. Tasman.

    (1992)
  • B. Hansen et al.

    A revision of the Tasmanian endemic freshwater crayfish genus Parastacoides (Crustacea: Decapoda: Parastacidae)

    Invertebr. Syst.

    (2006)
  • D.M. Hillis et al.

    An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis

    Syst. Biol.

    (1993)
  • H.H.J. Hobbs

    On the distribution and phylogeny of the crayfish genus Cambarus

  • H.H.J. Hobbs

    Synopsis of the families and genera of crayfishes (Crustacea: Decapoda)

    Smithsonian Contrib. Zool.

    (1974)
  • P. Horwitz

    Aspects of the life history of the burrowing freshwater crayfish Engaeus leptorhyncus at Rattrays Marsh, north east Tasmania

    Tasman. Nat.

    (1985)
  • P. Horwitz

    Sea-level fluctuations and the distributions of some freshwater crayfishes of the genus Engaeus (Decapoda; Parastacidae) in the Bass Strait area

    Aust. J. Mar. Freshwat. Res.

    (1988)
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