JOURNAL OF CRUSTACEAN BIOLOGY, 23(1): 169–177, 2003 MOLECULAR TAXONOMY AND PHYLOGENETICS OF SOME SPECIES OF AUSTRALIAN PALAEMONID SHRIMPS Nicholas P. Murphy and Christopher M. Austin School of Ecology and Environment, Deakin University, P.O. Box 423, Warrnambool 3280, Australia (e-mail: [email protected]) ABSTRACT The evolutionary history and classification of the palaemonid shrimps has been the subject of constant speculation and debate. At present, all major systematic treatments have been based on morphological characteristics. To help resolve the phylogenetic relationships, and thus enable the creation of a classification system that reflects evolutionary history, a region of the 16S mitochondrial rRNA gene was sequenced for a number of Australian Palaemonidae. The resulting phylogenetic analyses indicated the presence of major anomalies in the current classification of Australian Palaemonidae. Significantly, three species belonging to three separate genera, Macrobrachium intermedium, Palaemon serenus, and Palaemonetes australis, are closely related, with genetic differences more characteristic with that of congeneric species. The results also demonstrate non-monophyly in Australian palaemonids with respect to both Palaemonetes and Macrobrachium. The Palaemonidae family of shrimps is a the Palaemonidae, both within and between very successful group of decapods, inhabiting genera. The only phylogenetic study to focus on marine, estuarine, and freshwater environments the palaemonids as a whole was by Pereira throughout the world. The Palaemonidae are (1997), who performed a cladistic analysis of currently divided into four subfamilies, the morphological characteristics. The majority of Palaemoninae, Pontoniinae, Euryrhynchinae, other studies have focused on the genus Macro- and Typhlocaridinae, with the first containing brachium, the largest within the subfamily perhaps the most familiar palaemonid genera: Palaemoninae (containing approximately 65% Macrobrachium, Palaemon, and Palaemonetes. of the species within this subfamily) (Hedge- Whilst it is generally considered that the fam- cock et al., 1979; Lindenfelser, 1984; Mashiko ily Palaemonidae represents a natural group and Namuchi, 1993; Short, 2000; Murphy and (Pereira, 1997), the relationships within the fam- Austin, unpublished). Of these, Short (2000) pre- ily are far from clear. sents the most comprehensive phylogenetic study There have been many acknowledged prob- using morphological, biological, and ecological lems associated with the classification of the characters to assess the evolutionary relation- Palaemonidae. These occur at the specific ships of 30 Australian and non-Australian species (Lindenfelser, 1984; Short, 2000), generic (Hol- of Macrobrachium and nine species of related thuis, 1952; Fincham, 1987; Short, 2000), and genera of the Palaemoninae. Both Short (2000) family (Boulton and Knott, 1984; Pereira, 1997) and Pereira (1997) found evidence for anomalies levels. The problems have been attributed to dif- within the current taxonomic classification of ficulties in the interpretation of the significance the palaemonids. Pereira (1997) concluded that of morphological characteristics used to classify although the family Palaemonidae represents palaemonid shrimp and have hindered the de- a monophyletic group, several major lineages velopment of stable classification systems. within this family are, in fact paraphyletic. Short Holthuis(1950,1952)establishedthemostwidely (2000) provided further evidence that the current used classification of the Palaemonidae based on classification does not accurately reflect evolu- a number of morphological characteristics. How- tionary history, by identifying polyphyletic ever, this scheme has been criticized for not relationships within the genus Macrobrachium. accurately reflecting evolutionary relationships Until recently, all major systematic treat- within the group (Chace, 1972; Pereira, 1997). ments of palaemonids have been based on Recently, attempts have been made to de- morphological characteristics alone (Holthuis, velop hypotheses for the evolutionary history of 1950; Riek, 1951; Holthuis, 1952; Bray, 1976; 169 170 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 23, NO. 1, 2003 Choy, 1984; Fincham, 1987; Chace and Bruce, long debated, with a particular focus upon the 1993; Pereira, 1997), with the exception of representatives of the genus Palaemonetes Short (2000), who included some biological and in southwest Australia. Palaemonetes is repre- ecological characters. A few allozyme-based sented by two species in Australia, the largely studies have been undertaken (Trudaeu, 1978; estuarine Palaemonetes atrinubes and the Boulton and Knott, 1984; Lindenfelser, 1984; freshwater species Palaemonetes australis. Chow and Fujio, 1985a, b), but no phylogenetic (Bishop, 1967) suggested that Palaemonetes studies utilizing DNA-based data have been originated in Australia from larvae carried published. This is surprising, given the world- across the Indian Ocean from Africa. It has wide distribution of the family and the fact that been said that the palaemonids, in general, several species, including the giant freshwater appear to be in transition from marine to prawn Macrobrachium rosenbergii, are impor- freshwater environments (Hedgepeth, 1957). tant commercially, particularly in developing However Boulton and Knott (1984) suggest, countries. In contrast, other important decapod based on allozyme data, that the likely ancestor crustacean groups, such as penaeid prawns, of Palaemonetes australis is a M. intermedium- freshwater crayfish, and marine lobsters, have like form. been much more extensively studied using To further explore the taxonomic, phyloge- molecular genetic techniques (Palumbi and netic, and biogeographic questions pertaining to Benzie, 1991; Bouchon et al., 1994; Harding Australian palaemonids, sequencing of the 16S et al., 1997; Ovendon et al., 1997; Grandjean mtDNA gene region was undertaken. The 16S et al., 1998; Sarver et al., 1998; Crandall et al., mtDNA gene region has been found to be 1999). The use of DNA sequence data has the extremely useful for studying taxonomic ques- great advantage that it allows the creation of tions and phylogenetic relationships within a phylogeny that enables the testing of mor- a number of decapod crustacean groups phologically based systematic hypotheses and (Bucklin et al., 1995; Crandall and Fitzpatrick, for the independent assessment of morpholog- 1996; Ovendon et al., 1997; Kitaura et al., ical evolution. 1998; Tam and Kornfield, 1998; Crandall et al., In Australia, palaemonids are widespread, 1999). The 16S rRNA gene has both fast- and with representatives inhabiting marine, estua- slow-evolving regions and therefore can provide rine, and freshwater environments. In prelimi- useful information across a broad taxonomic nary work on the phylogenetics of Australian spectrum from the population to the family Macrobrachium sp. using 16S mitochondrial level. DNA (mtDNA) sequences, Murphy and Austin The object of this paper is to essentially (2002) showed evidence for the non-monophyly follow up Boulton and Knott’s (1984) allo- within Australian Macrobrachium. Murphy and zyme study of Palaemonidae in the Swan River Austin (2002) found that Macrobrachium Estuary, Western Australia, using powerful intermedium did not share a close evolution- DNA-based analyses. In addition to the samples ary relationship with other Australian Macro- examined by Boulton and Knott, specimens of brachium and instead was found to share Palaemon serenus and M. intermedium from a closer affinity with Palaemon serenus. eastern Australia and M. australiense and Boulton and Knott (1984), in a study of M. rosenbergii are included for comparative palaemonid species in the Swan River Estuary, purposes. Western Australia, also found inconsistencies between the current morphologically based MATERIALS AND METHODS classification system and genetic relation- ships between the species studied using allo- Taxa zyme data. Boulton and Knott (1984) showed The four Australian palaemonid species studied by M. intermedium to be closely allied to an- Boulton and Knott (1984), Macrobrachium intermedium (Stimpson, 1860); Palaemon serenus (Milne-Edwards, other species, Palaemonetes australis, suggest- 1837); Palaemonetes australis (Bray, 1976); and Palae- ing that they may be congeners. Short (2000) monetes atrinubes (Dakin, 1915) from the Swan River and Pereira (1997) also found evidence that estuary, Western Australia, were included in this study. M. intermedium is polyphyletic with respect to Also included were M. intermedium and P. serenus from . Warrnambool, Victoria, to allow for intraspecific compar- Macrobrachium isons, and M. australiense (Ortman, 1891) from inland The biogeographic relationships and the Australia and M. rosenbergii (De Man, 1879) from Papua origin of Australian palaemonids have been New Guinea to allow for species-level comparisons with MURPHY AND AUSTIN: MOLECULAR PHYLOGENY OF AUSTRALIAN PALAEMONIDAE 171 Macrobrachium. Macrobrachium intermedium is found were purified using a QIAGEN QIAquick PCR Purification in estuarine and some marine waters around the south- Kit, with final elution volumes of 50 ll per individual. The ern Australian coastline from Perth, W.A., to the central DNA concentrations were approximated against a Promega Queensland coast; P. serenus, a marine species, has a similar DNA/Hae 111 marker on a 2% agarose/TAE gel containing distribution. The two Palaemonetes species are found only ethidium bromide