Vol. 533: 237–244, 2015 MARINE ECOLOGY PROGRESS SERIES Published August 6 doi: 10.3354/meps11354 Mar Ecol Prog Ser OPENPEN ACCESSCCESS Whole mitogenome sequencing refines population structure of the Critically Endangered sawfish Pristis pristis Pierre Feutry1,7,*, Peter M. Kyne1, Richard D. Pillans2, Xiao Chen3, James R. Marthick4, David L. Morgan5, Peter M. Grewe6 1Research Institute for the Environment and Livelihoods, Charles Darwin University, Ellengowan Drive, Darwin 0909, Northern Territory, Australia 2CSIRO Oceans and Atmosphere Flagship, 41 Boggo Road, Dutton Park 4102, Queensland, Australia 3Guangxi Mangrove Research Center, Guangxi Academy of Sciences, Beihai 536000, PR China 4Menzies Institute for Medical Research, University of Tasmania, 17 Liverpool Street, Hobart 7000, Tasmania, Australia 5Freshwater Fish Group & Fish Health Unit, Centre for Fish & Fisheries Research, School of Veterinary & Life Sciences, Murdoch University, South Street, Murdoch 6150, Western Australia, Australia 6CSIRO Oceans and Atmosphere Flagship, Castray Esplanade, Hobart 7000, Tasmania, Australia 7Present address: CSIRO Oceans and Atmosphere Flagship, Castray Esplanade, Hobart 7000, Tasmania, Australia ABSTRACT: The largetooth sawfish Pristis pristis (Linnaeus, 1758) is a highly threatened euryha- line elasmobranch that in recent times has undergone a significant range contraction. It now only remains in a few areas, with northern Australia being the main stronghold. Previous work using a single mitochondrial gene approach suggested the existence of regional barriers to gene flow in northern Australia. In this study, whole mitochondrial sequences of 92 P. pristis from 7 river drainages across northern Australia were used to refine the population structure. This approach revealed barriers to gene flow at a scale as fine as between adjacent river drainages. Except for those flowing into the Gulf of Carpentaria, all river drainages appeared to host a genetically dis- tinct population. The apparent genetic homogeneity in the Gulf is probably due to freshwater con- nectivity between river drainages, either during the last glaciation event when the Gulf was a freshwater lake or through contemporary wet season flooding. These results suggest that each river drainage across the species’ range should be considered a discrete management unit unless there is evidence of freshwater connectivity. More broadly, the improved resolution of population structure obtained with whole mitogenome analysis compared to single mitochondrial gene approaches suggests that female reproductive philopatry may have been overlooked in previous studies of some elasmobranch species. KEY WORDS: Population genetics · Elasmobranch · Philopatry · Dispersal · Control region · D-loop INTRODUCTION and extirpation from large parts of its former distribu- tion (Kyne et al. 2013). Significant range contractions The largetooth (or freshwater) sawfish Pristis pristis have occurred in the tropical Eastern Atlantic, West- (Linnaeus, 1758) was once widespread, but is now ern Atlantic, Eastern Pacific and Indo-West Pacific listed as Critically Endangered on the IUCN Red List Oceans, and the species is now only found in a lim- of Threatened Species, due to population declines ited number of core population areas (Peverell 2005, © The authors 2015. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 238 Mar Ecol Prog Ser 533: 237–244, 2015 Morgan et al. 2011, Kyne et al. 2013, Dulvy et al. recent study, Phillips et al. (2011) provided the first 2014, Moore 2014). The distribution of P. pristis over- population genetic analyses of each of the Indo-West laps with areas of intense human activity in coastal Pacific Pristis species and found evidence of female zones, and the species has suffered from both unreg- reproductive philopatry in P. pristis. They analysed a ulated and regulated fishing, and habitat modifica- portion of the mitochondrial control region (CR) or d- tion and loss (with threats persisting across its range) loop across 149 individuals from northern Australia (Peverell 2005, Morgan et al. 2011, Kyne et al. 2013). and demonstrated the existence of several barriers to P. pristis reaches at least 6.5 m total length (TL) gene flow at a broad scale. The P. pristis CR study (Compagno & Last 1999), has a late age at maturity revealed a low genetic diversity, with >80% of the (8−10 yr) (Thorson 1982, Peverell 2008), is estimated individuals harbouring 1 of the 2 main haplotypes to live to 44 yr (Tanaka 1991) and have 1−13 young (Phillips et al. 2011). every 1 or 2 yr (Thorson 1976, Peverell 2008). These The power to detect population structure directly life history traits limit productivity and restrict the depends on the variability of the genetic marker(s) ability of this species to recover from depletion analysed (Ryman et al. 2006), as the typical way to (Dulvy et al. 2014). test for population differentiation is to assay 1 or International trade in P. pristis is regulated through more loci and to test the null hypothesis of identical the species’ listing on the Convention on International allele frequencies. Thus, finer-scale resolution of Trade in Endangered Species (CITES) Appendix I. P. population structuring can be missed if an appropri- pristis is also protected in many countries (Dulvy et al. ate marker (i.e. one exhibiting enough variability) is 2014), including across its Australian range, where it not analysed (Feutry et al. 2014). Shamblin et al. is listed as Vulnerable in Australia’s Environment Pro- (2012) and Feutry et al. (2014) demonstrated that the tection and Biodiversity Conservation Act. Northern use of whole mitogenome sequencing can greatly in - Australia is a key remaining population centre of crease the resolution of matrilineal genetic structure global significance for P. pristis (Peverell 2005, Mor- patterns compared to single gene approaches, espe- gan et al. 2011, Kyne et al. 2013, Dulvy et al. 2014). cially in groups where mitochondrial gene evolution- Much of northern Australia remains remote and ary rates are low, such as elasmobranchs (Martin et sparsely inhabited, although pressure is mounting to al. 1992). develop the region (The Parliament of the Common- In the present study, we apply whole mitogenome wealth of Australia 2014), which has the potential to sequencing to investigate patterns of matrilineal degrade natural systems, including key P. pristis habi- gene flow in P. pristis amongst northern Australian tats (Chin et al. 2012). rivers. Phillips et al. (2011) noted limitations in both P. pristis is a euryhaline elasmobranch, with life- sample size and the number of locations sampled, cycle stages that encompass freshwater, estuarine which hindered the examination of fine-scale pat- and marine waters; juveniles are born in estuaries terns of variation (e.g. that study had only 8 individu- and move into river systems, including upstream als sampled from the Northern Territory [NT]). The reaches and floodplain waterholes, where they are main objective is to further refine the population believed to spend some 4−5 yr before moving back to boundaries discovered by Phillips et al. (2011) for this the sea (Thorburn et al. 2007, Peverell 2008, Whitty Critically Endangered species, which is crucial infor- et al. 2009, Kyne et al. 2013). Adults are elusive and mation for conservation and recovery planning. their rarity has rendered them difficult to sample for scientific research. However, samples of juveniles are more easily collected in river drainages, and MATERIALS AND METHODS genetic analyses from different river drainages (nurs- eries) can inform population structure and manage- Tissue samples from 92 juvenile Pristis pristis were ment strategies if admixture is limited. collected between 2009 and 2013 from 7 major river Mitochondrial DNA (mtDNA) markers are mater- drainages across their northern Australian range, nally inherited and thus reflect female behaviour, from the Kimberley region of Western Australia (WA) which is particularly useful to detect female repro- to the eastern Gulf of Carpentaria in Queensland ductive philopatry, as is often exhibited by elasmo- (QLD). From west to east, samples were obtained branch species (Hueter et al. 2005). Different phylo- from the Fitzroy River (n = 20) in WA; the Daly River geographic signals can be observed using nuclear (n = 21), Adelaide River (n = 13) and South Alligator DNA instead of mtDNA if males display a reproduc- River (n = 5) in the NT; and the Norman River (n = tive behaviour which is in contrast to females. In a 11), Gilbert River (n = 11) and Mitchell River (n = 11) Feutry et al.: Mitogenomics of largetooth sawfish 239 in QLD (Fig. 1). DNA was extracted from fin clips or ming were discarded as well as sequences with >15 biopsies using DNeasy Blood & Tissue kits (Qiagen). low-quality bases. Remaining reads for each individ- The P. pristis mitogenomes were amplified in 2 ual were mapped onto a reference sequence (P. Feu- overlapping fragments using 2 new primer pairs: try et al. unpubl. data) using the custom sensitivity PM6405F: 5’-TAG ACG TAG ACA CTC GAG CC-3’ tool with the following parameters: Maximum gaps and PM15026R: 5’-TGA GTA GGG AGG TTA GGA per read = 10; Word length = 24; Ignore word re- CAA G-3’; and PM14570F: 5’-TGG CTG ACT CAT peated more than 8 times; Maximum mismatches