Fairy Shrimps in Distress

Hydrobiologia (2013) 700:313–327 DOI 10.1007/s10750-012-1240-8

PRIMARY RESEARCH PAPER

Fairy shrimps in distress: a molecular taxonomic review of the diverse fairy shrimp genus Branchinella (Anostraca: Thamnocephalidae) in Australia in the light of ongoing environmental change

Tom Pinceel • Bram Vanschoenwinkel • Aline Waterkeyn • Maarten P. M. Vanhove • Adrian Pinder • Brian V. Timms • Luc Brendonck

Received: 17 January 2012 / Revised: 22 June 2012 / Accepted: 23 June 2012 / Published online: 8 July 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Australia, and especially South-Western gene of 31 presumed species, complemented with Australia, is a diversity hotspot for large branchiopod analysis of morphological structures holding taxo- crustaceans. A significant proportion of this diversity nomic information. Results revealed the presence of at is found in the anostracans (Crustacea, Anostraca) and least three new cryptic species. On the other hand, particularly in the diverse genus Branchinella with at some Branchinella lineages, surviving in environ- least 34 species. Members of this genus are found ments subjected to contrasting selection regimes, exclusively in temporary aquatic habitats which are appeared to be conspecific. This suggests substantial increasingly threatened by secondary salinization and physiological plasticity or important adaptive varia- other anthropogenic pressures. The development of tion present in some species, potentially enabling them adequate conservation strategies is therefore consid- to better cope with environmental change, such as ered a priority. To define conservation units, however, secondary salinization. Overall, these results further thorough knowledge of the taxonomy and phyloge- illustrate the benefits of combining molecular markers netic position of extant lineages is essential. We and classic morphological taxonomy and phylogeny to reconstructed a large scale phylogeny of the Austra- assess biodiversity and define conservation units in lian Branchinella by analyzing the 16S mitochondrial cryptic groups.

Keywords Branchinella Molecular taxonomy Handling editor: Koen Martens Secondary salinization Conservation Australia

Electronic supplementary material The online version of this article (doi:10.1007/s10750-012-1240-8) contains supplementary material, which is available to authorized users.

T. Pinceel (&) B. Vanschoenwinkel A. Pinder A. Waterkeyn L. Brendonck Department of Environment and Conservation, Wildlife Laboratory of Aquatic Ecology and Evolutionary Biology, Place, Woodvale, WA 6026, Australia KULeuven, Charles Deberiotstraat 32, 3000 Leuven, Belgium B. V. Timms e-mail: [email protected] Australian Museum, 6-9 College St, Sydney 2000, Australia M. P. M. Vanhove Laboratory of Animal Diversity and Systematics, B. V. Timms KULeuven, Charles Deberiotstraat 32, 3000 Leuven, Australian Wetland and Rivers Centre, BEES, University Belgium of New South Wales, Kensington, NSW 2052, Australia 123 314 Hydrobiologia (2013) 700:313–327

Introduction aquatic habitats that periodically dry out (Fig. 1a). Some brine shrimp species, however, do occur in Australia, and South-Western Australia in particular, permanent salt lakes. With over 300 described is considered one of the world’s main diversity species worldwide, the anostracans are a diverse hotspots, not only for plants and terrestrial organ- group (Brendonck et al., 2008) which differs from isms (Myers et al., 2000) but also for freshwater other branchiopods by their predominantly pelagic invertebrates (Belk, 1998; Halse et al., 2000; Seger habitat use, relatively large size and lack of a & Shiel, 2003; Pinder et al., 2010). Over one protective carapace. In Australia, only five anostr- thousand freshwater invertebrate species have been acan genera occur that thrive in a wide variety of recorded in the Wheatbelt area in South-West habitats such as rock pools, clay pans and swamps Australia alone, of which about 20% are endemic (Timms, 2004, 2012). In contrast to the low generic (Pinder et al., 2004). A significant proportion of this diversity, Australia harbours at least 58 anostracan freshwater biodiversity is represented by primitive species, 40 of which occur in Western Australia. crustacean groups, such as the classes Ostracoda and This comprises about 15% of the world’s and 70% Branchiopoda (e.g. Halse, 2002; Halse & McRae, of the Australian anostracan diversity. Roughly 60% 2004; Pinder et al., 2004; Brendonck et al., 2008; (34 species) of the known Australian species belong Timms, 2008, 2012). Among branchiopod crusta- to Branchinella (Sayce, 1903; Timms, 2008, 2012) ceans, anostracans are generalist filter feeders typ- while the rest are mostly salt lake specialists and ical for the macrozooplankton community in fishless classified as Parartemia Daday, 1910.

Fig. 1 a Example of a large branchiopod crustacean assem- sampled Australian Branchinella populations. Localities from blage from a typical habitat: a clay pan in Western Australia. specimens obtained from Genbank could not be mapped as no b Illustration of the effects of secondary salinization of a lake exact coordinates were available. The Wheatbelt area in system in the West Australian Wheatbelt area. c Overview of the Western Australia, which is severely threatened by secondary salinity tolerance of Australian anostracan species as recon- salinization, is highlighted in white. (Photo 1a and 1b by Bram structed from literature and additional ﬁeld measurements (for Vanschoenwinkel) references, see text). d Geographical locations of the newly 123 Hydrobiologia (2013) 700:313–327 315

Many temporary aquatic habitats in Australia are genetic variation through the protection of Evolution- threatened by ongoing anthropogenic impact includ- ary Significant Units (ESUs). ing unsustainable agriculture, mining, river regulation, Our goal is to re-evaluate the phylogenetic rela- groundwater resource development and climate tionships in the diverse anostracan genus Branchinella change. One of the greatest threats, especially in the in Australia using primarily 16S mtDNA data and to Western Australian Wheatbelt area, is human induced assess the usefulness of these molecular data to define (secondary) salinization and altered hydrology appropriate conservation units by uncovering cryptic (McFarlane et al., 2004; Natural Resource Manage- genetic diversity. In order to achieve these goals we ment Ministerial Council, 2010) (Fig. 1b, d). Over the construct a molecular phylogeny based on a data set past 180 years, at least 18.3 million ha of South- which includes about 85% of the Branchinella species Western Australia was cleared of its natural deep- currently known to science as well as a number of rooted vegetation (mostly Eucalyptus and Acacia spp.) recently discovered lineages. Furthermore, based on to make way for shallow-rooted annual crops (Clarke this phylogeny, genetic distances among lineages as et al., 2002; Jardine et al., 2011). As a result, well as morphological and ecological information, we groundwater levels rose, carrying salts from deeper evaluate to what extent there is support for the large layers to the surface, resulting in salinity increases in number of species that have recently been described the topsoil and in surface waters as well as in an and discuss the taxonomic status of four newly altered hydrology (including prolonged hydroperiod discovered (currently undescribed) lineages. Finally, of temporary waters). Consequently, freshwater using information on the autecology of lineages communities are gradually replaced by less diverse, (salinity tolerance in particular), we assess the halotolerant or halophilic species assemblages, lead- potential impact of secondary salinization on Branchi- ing to a general diversity loss (Pinder et al., 2004). nella in the South-Western Australian diversity hot- Despite significant efforts of governments and land- spot and discuss the importance of demarcating proper holders (e.g. Wallace et al., 2011), many hundreds of units for conservation management. This approach is wetlands are affected. an essential step in developing conservation strategies In the development of adequate management for Australian branchinellids, which may be an strategies and sustainable agricultural practices, the example for other taxonomic groups and regions. accurate demarcation of appropriate biological conservation units is required (Mayden & Wood, 1995). Although no absolute consensus is reached on how to Methods best approach this, for groups with often subtle morphological differences among nominal species Studied organisms such as some anostracans, delimitation of conservation units should consider cryptic genetic diversity Branchinella and five other genera (Dendrocephalus (Crandall et al., 2000; Moritz, 2002; Hebert et al., Daday, 1908, Phallocryptus Birabeń, 1951, Spirali- 2003). Presently, the taxonomy of Australian anostra- frons Dixon, 2010, Thamnocephalus Packard, 1877 cans is almost exclusively based on morphological and Carinophallus Rogers, 2006) constitute the data, which may not accurately reflect diversity of the Thamnocephalidae (Rogers, 2006). Including around evolutionary lineages in the region. As of date, 42 described species from Australia, Asia, and Africa, phylogenetic relations in Australian anostracans are Branchinella is one of the most species rich fairy poorly known, with only limited taxonomic coverage shrimp genera (Belk & Brtek, 1995; Rogers, 2006; in preliminary molecular studies on Parartemia Brendonck et al., 2008). (Remigio et al., 2001) and Branchinella (Remigio Re-evaluating earlier taxonomic work by Sayce et al., 2003). Therefore, the taxonomic status of many (1903) and Linder (1941), Geddes (1981) recognized newly discovered populations remains uncertain 16 Australian Branchinella species and divided them (Timms, unpublished data). Furthermore, as advised into three groups according to their morphology. by Ryder (1986) and Fraser & Bernatchez (2001), Later, Belk & Brtek (1995) elevated two subspecies to conservation efforts should not only be directed at species level and 14 more Branchinella species were conserving species but also at preserving unique described by Timms (2001, 2005, 2008) and Timms & 123 316 Hydrobiologia (2013) 700:313–327

Geddes (2003) over the past decade, adding up to 34 lower ethanol concentrations (70%) and from old species. All of these Australian species, 34 described samples preserved in ethanol that had been denatured and five known, undescribed, species (Timms, 2012), using methanol. Preserved specimens were dissected have been united in an endemic subgenus, Branchi- to obtain phyllopod tissue for DNA extraction. Geno- nella (Rogers, 2006). mic DNA was extracted using the NucleoSpinÒ Geddes (1981) and later also Timms (2002) and extraction kit for individual tissue samples (Mache- Remigio et al. (2003) discussed the high diversity in rey–Nagel). The partial mitochondrional 16S rDNA the genus on the Australian continent. The variety of gene was amplified by polymerase chain reaction species presumably arose as a result of large intra- (PCR), using the forward (50-CGC CTG TTT ATC continental climate gradients and variation over AAA AAC AT-30) and reverse (50-CCG GTC TGA geological periods (both in temperature and precipi- ACT CAG ATC ACG T-30) Palumbi (1996) primers. tation) and the occurrence of a wide variety of This primer set produced an approximately 470 bp different habitat types, like rock pools, creek pools fragment for six Branchinella spp. (B. nichollsi, and clay pans with different abiotic conditions such as B. wellardi, B. australiensis, B. frondosa, B. occidentalis hydrology, salinity, turbidity and pH (Timms & and B. compacta). Since most Branchinella species are Sanders, 2002; Remigio et al., 2003; Timms, 2004; rare, having been collected only once or twice, the Byrne et al., 2008). In this paper we included 27 majority of the available samples was old ([10 years) described species and four currently undescribed and often poorly preserved and stored at high temper- lineages which were presumed to represent new atures ([25°C). As a result it was difficult to amplify and species, thereby for the first time providing molecular sequence longer DNA fragments. In order to be able to data for 15 of these 31 ‘species’. sequence old and degraded DNA, Branchinella-specific internal 16S primers were designed with the online Primer 3 development tool (Rozen & Skaletsky, 1998) Sample information and the six sequences obtained using the Palumbi (1996) primers. The forward 16S_F 50-GAA GGG CCG TGG The Branchinella lineages sequenced in this study, as TAT ACT GA-30 and reverse 16S_R 50-TCG AGG well as geographic coordinates of localities and TCG CAA ACT TTT CT-30 primers were selected GenBank accession codes, are listed in Table 1. amplifying a 382 bp fragment. Localities of newly sequenced populations are plotted The PCR reaction volume of 25 ll contained 2 ll in Fig. 1d. All samples were obtained by Timms over of template DNA, 2 mM of MgCl2, 1 mM 109 the period 1985–2010 using standard sampling equip- reaction buffer, 0.2 mM dNTPs, 0.4 lM of each ment (dip nets or kick nets). Sufficiently variable primer and 1.1 U Taq polymerase. The cycle settings nuclear markers to reconstruct species phylogenies, were modified from Adamowicz et al. (2004) with an are rare for crustaceans. While we initially also initial denaturation of 3 min at 94°C, followed by five considered the available 18S nuclear marker for a liberal amplification cycles (denaturation for 1 min at total of 20 Australian Branchinella lineages, interspe- 94°C, annealing for 1.5 min at 45°C and elongation cific variation was too low (K2P distance of 0–1.6%) for 1.5 min at 72°C) and 35 more rigid cycles to make reliable phylogenetic inferences. Therefore (denaturation for 1 min at 94°C, annealing for these data are not included in this manuscript. 1.5 min at 50°C and elongation for 1.5 min at 72°C), Furthermore, variability of the available marker COI followed by a final elongation of 6 min at 72°C. was assessed as well, but it was too high to render Reaction contaminants were removed from the sam- robust reconstructions for distantly related lineages ples using the NucleoFastÒ 96 PCR Clean-Up kit such as many of the Australian Branchinella species. (Macherey–Nagel). Samples were sequenced according to the Big Dye Terminator 3.1 kit (Applied DNA isolation, molecular markers, polymerase Biosystems), following a 1/8 dilution of the Big Dye chain reaction and sequencing Terminator sequencing protocol, using the same primers as in the amplification. Finally, the products Some samples were preserved in absolute ethanol but were run on an ABI PRISM 3130 Avant Genetic DNA was also extracted from samples preserved at Analyzer automated sequencer (Applied Biosystems). 123 Hydrobiologia (2013) 700:313–327 317

Table 1 Overview and speciﬁcations of the sequenced Branchinella specimens and sequences drawn from Genbank Species Site Genbank/coordinates

1 B. afﬁnis Linder, 1941 Paroo area, NSW & Pandie Pandie, SA AF308942.1/AF527568.1 2 B. arborea Geddes, 1981 Paroo area, NSW AF308945.1 3 B. australiensis Richters, 1876 Paroo area, NSW & Claremont, QLD AF527556-7.1 4 B. basispina Geddes, 1981 Baladonia Rock, WA S 32 28 E 123 52 5 B. buchananensis Geddes, 1981 Bloodwood station, NSW S 29 28 E 144 50 6 B. buchananensis Paroo-Gidgee lake & Bloodwood station, NSW AF308943.1, AF527562.1 7 B. budjiti Timms, 2001 Paroo area, NSW & Warburton Crossing, SA AF527563-67.1 8 B. campbelli Timms, 2001 Paroo area, NSW AF527576.1 9 B. clandestina Timms, 2005 Paroo river, Currawinya National Park, QLD S 28 41 55 E 144 46 40 10 B. compacta Linder, 1941 Avon lake, NSW S 36 36 59 E 149 2 59 11 B. complexidigitata Timms, 2002 Lake Logue, WA S 29 52 5 E 115 8 28 12 B. frondosa Henry, 1924 Rockwell, QLD AF308941.1 13 B. halsei Timms, 2002 Lake Cronin, WA S 32 23 E 119 45 14 B. hattahensis Geddes, 1981 Kaponyee lake, QLD AF308944.1 15 B. kadjikadji Timms, 2002 Kadji Kadji, WA AF527569.1 16 B. lamellata Timms & Geddes, 2003 Clifton Hills Station, SA S 27 2 E 138 53 17 B. longirostris Wolf, 1911 King Rocks, WA AF527575.1 18 B. lyrifera Linder, 1941 Bloodwood station, NSW S 29 28 E 144 50 19 B. lyrifera Kaponyee lake, QLD & Bobbiemongie, SA AF527559-61.1/AF308940.1 20 B. mcraeae Timms, 2005 Onslow, WA S 21 48 42 E 115 6 36 21 B. nichollsi Linder, 1941 Kalgoorlie, WA S 30 34 12 E 121 39 17 22 B. occidentalis Dakin, 1914 Paroo area, NSW & Nilpinna, SA AF308947.1/AF527558.1 23 B. papillata Timms, 2008 Kau Nature Reserve, WA S 33 34 35 E 122 19 47 24 B. pinderi Timms, 2008 Onslow, WA S 21 48 18 E 115 6 15 25 B. pinnata Geddes, 1981 Paroo area, NSW & Rockwell, QLD AF308946.1/AF527570.1 26 B. proboscida Henry, 1924 Paroo area, NSW AF527574.1 27 B. simplex Linder, 1941 Cue, WA S 27 34 22 E 117 52 19 28 B. tyleri Timms & Geddes, 2003 Victoria River, NT S 15 26 E 131 26 29 B. wellardi Milner, 1929 Bloodwood Station, NSW S 29 32 13 E 144 52 26 30 B. wellardi Paroo area, NSW AF527571.1 31 B. sp. novum U Moora, WA S 30 28 2 E 115 58 57 32 B. sp. novum S Claremont, QLD AF527572.1 33 B. sp. novum M Moora, WA S 30 28 2 E 115 58 57 34 B. sp. novum Y Yarromere station, QLD S 21 33 E 145 49 For specimens obtained from genbank no exact coordinates are provided

All new sequences were deposited in GenBank representing 19 unique haplotypes from 16 species under accession codes (JN812771-JN812788). from GenBank, in BioEdit Sequence Alignment Editor v.7.0.0 (Hall, 1999) (Additional details and Genetic data analyses GenBank accession codes are provided in Table 1). The anostracans Streptocephalus dorothae Mackin, Thirty-three newly obtained Branchinella sequences, 1942 from North-America and Carinophallus ornata representing 26 unique haplotypes from 20 presumed (Daday, 1910) from Botswana were selected as species (Table 1), were aligned (ClustalW multi- outgroups. The alignment quality was checked ple alignment) to 25 additional 16S sequences, both by eye as well as using trimAl software 123 318 Hydrobiologia (2013) 700:313–327

(Capella-Gutierrez et al., 2009). jModeltest 0.1.1 Integration of morphological information (Posada, 2008) selected the TPM2uf?I?G(I= in the molecular phylogeny 0.4560, G = 0.6150) model, with nucleotide frequen- cies A = 0.3325, C = 0.1035, G = 0.1359 and Owing to the highly conserved body plan among and T = 0.4281 and rate matrix (3.67, 26.29, 3.67, 1.00, observed phenotypic plasticity within Branchinella 26.29, 1.00). Model averaged phylogeny analyses in species (Zofkova & Timms, 2009), very few morpho- jModeltest 0.1.1. indicated that all 88 tested models logical characters are useful for taxonomic purposes rendered identical trees. Substitution saturation was and phylogenetic reconstruction. The Australian tested in DAMBE 5.2.13 (Xia et al., 2003; Xia and Branchinella species share similar penile characteris- Lemey, 2009). Iss was found to be significantly tics (Brendonck & Belk, 1997; Rogers, 2006) and in smaller than Iss c, indicating little saturation. Genetic all species the proximally fused basal segment of the Kimura 2-parameter (K2P) distances (Kimura, 1980) second antennae is unsclerotized (Timms, 2004). In were computed in MEGA v. 4.1 (Tamura et al., 2007). addition, resting egg morphology seems to be of little Phylogenetic analyses were carried out using neigh- taxonomic value since species-specific egg types are bour joining (NJ), maximum parsimony (MP), max- only distinguishable for 12 of the 34 Australian imum likelihood (ML), quartet puzzling (QP) methods Branchinella species (Timms & Lindsay, 2011). and Bayesian inference (BI). MP analyses were Males, however, do carry a species-specific frontal conducted in Paup 4.0b10 (Swofford, 2000) using appendage in all but eleven species (B. buchananensis, the PaupUp graphical interface (Calendini & Martin, B. compacta, B. halsei, B. hattahensis, B. lyrifera, 2005). ML analyses were run in PhyML (Guindon B. nana, B. nichollsi, B. occidentalis, B. papillata, et al., 2005) and Quartet puzzling maximum likeli- B. simplex and B. vosperi). This frontal appendage is hood analyses in TREE-PUZZLE (Strimmer & von usually composed of a basal stem and two apical Haeseler, 1996; Schmidt et al., 2002), both according branches but is often spliced into several sub- to the model and parameters selected by jModeltest. branches (Timms, 2004). Especially the structure of NJ analyses were performed in MEGA 4.1 using the the frontal appendage remains as taxonomically following settings: maximum composite likelihood, useful for species identification. However, the lim- Tamura–Nei substitution model, defined G = 0.6150 ited number of discrete character states (\10) that and 1,000 bootstrap replicates. For BI analyses can be derived from this structure are too few to MrBayes 3.1.2 (Huelsenbeck et al., 2001; Ronquist establish a robust phylogeny based on morphological & Huelsenbeck, 2003) was used. MrBayes ran for traits. We therefore refrained from constructing 3000000 generations (lset number of substitution phylogenetic trees based on morphological traits types = 6, rates = invgamma, number of rate cate- only. Instead, in order to be able to assess potential gories for the gamma distribution = 4) with a defined links between genetic groupings and different mor- outgroup (S. dorothae) and a sampling frequency of 100 phospecies, we superimposed detailed drawings of generations. In order to only include trees in which the the dorsal view of the head region and, if present, the convergence of the Markov chain had been reached, we frontal appendage on the reconstructed molecular defined a burn-in of 25%. The trees retained were used phylogeny (Timms, 2001, 2002, 2004, 2005, 2008; to construct a 50% majority consensus tree. Fig. 2). For the purpose of evaluating the taxonomic status of the 31 presumed species, we studied molecular phylogenetic data and calculated genetic distances Salinity tolerance of Australian Branchinella among monophyletic lineages. According to Adam- owicz & Purvis (2005) and Adamowicz et al. (2009) To estimate the potential threat of increasing salinities genetic distances among branchiopod species typi- through secondary salinization on the Australian cally exceed 4% for rRNA coding genes. In addition, species of Branchinella, we reconstructed the salinity we reviewed ecological and morphological character- tolerances for all species in our data set (Fig. 1c) istics of the considered lineages and compared that supplementing information from literature (Geddes, information with the position of lineages in our 1980, 1981; Timms, 2004, 2009) with newly obtained molecular phylogenetic tree. field data. 123 Hydrobiologia (2013) 700:313–327 319

Results species united in clade B 10.9% and species from both clades diverged on average 11.6% from each other. Our Australian Branchinella data set included mtDNA data from 27 of the 34 accepted species and four Species groupings according to frontal appendage currently undescribed lineages which were presumed morphology to represent species based on their morphological and ecological characteristics. The 27 described Australian Branchinella species were divided into four groups according to the general Genetic variation structure of their frontal appendage (Fig. 2). A first morphogroup contains ten species, without or with The final 16S rDNA data set comprised 47 sequences, very small frontal appendages: B. simplex, B. halsei, all representing unique haplotypes, with a total of 104 B. lyrifera, B. occidentalis, B. compacta, B. buchan- (39%) polymorphic sites of which 82 (31%) were anensis, B. hattahensis, B. nichollsi, B. papillata and found to be parsimony-informative. one of two B. australiensis morphotypes. Secondly a Among the 31 presumed species in our data set, the grouping of six species with frontal appendages number of nucleotide differences ranged from one consisting of a trunk and two simple branches with between Branchinella specimens from Yarromere sta- short spines and papillae was defined: B. affinis, tion in Queensland (QLD) and B. wellardi (correspond- B. clandestina, B. mcraeae, B. proboscida, B. longi- ing with a K2P distance of 0.4%) to 47 between B. rostris, and one of the two B. australiensis morpho- longirostris from King Rocks (WA) and B. sp. novum types. A third group contains seven species: U from Moora (WA) (corresponding with a K2P B. wellardi, B. tyleri, B. arborea, B. pinnata, distance of 26.6%). The overall average genetic diver- B. basispina, B. pinderi and B. frondosa, with more gence between nominal species was 11.4 ± 3.2%. A elaborate frontal appendages consisting of a trunk complete list of K2P distances between the studied bearing two approximately tubular branches which in genetic lineages can be consulted in the electronic turn bear a few or several (\15) digitiform ramified supplementary material, Table S1. sub-branches which may bear papillae and do not usually terminate in spines. Finally, the fourth group unites five species: B. lamellata, B. budjiti, Phylogenetic reconstructions on the basis B. campbelli, B. complexidigitata and B. kadjikadji,of of 16S rDNA which the frontal appendage consists of a trunk bearing two lamellate branches with numerous ([20) In general, the five different methods of phylogenetic digitiform processes, usually terminating in spines. inference (ML, MP, NJ, QP and BI) consistently retrieved a similar tree topology (Fig. 2). All considered Australian lineages were grouped together mon- Discussion ophyletically. The most basal Australian Branchinella ‘clade’ in our phylogeny includes two species: Evolutionary relationships within the Australian B. proboscida and B. longirostris. Subsequently, four Branchinella clades appear to have diverged: a first clade containing B. campbelli and B. basispina, a second clade We constructed a molecular phylogeny for 31 presumed harbouring B. arborea and B. pinnata, a diverse clade Australian Branchinella species based on the 16S containing B. wellardi, B. fondosa, B. tyleri, mtDNA region (Fig. 2) in order to better resolve the B. papillata and two currently undescribed species taxonomy and the evolutionary affiliations within the and finally a clade containing all remaining lineages. genus. Complemented with available morphological Most diversity seems to be restricted to this clade and autecological information, this provides back- which comprises two large monophyletic groupings ground information for conservation management. (labelled A and B) and one additional lineage, Since all considered lineages grouped monophyletically representing a currently undescribed species. The 11 in our molecular phylogenetic reconstructions, their species in clade A diverged on average 6.4%, the six placement into the same (sub)genus, as suggested by 123 320 Hydrobiologia (2013) 700:313–327 morphological data (Timms, 2004;Rogers,2006), is Fig. 2 Consensus phylogram of the 31 considered Australianc supported. None of the Australian lineages seem to bear Branchinella based on QP quartet puzzling, ML maximum likelihood, MP maximum parsimony, NJ neighbour joining and close phylogenetic affinities to the African C. ornata BI Bayesian inference analyses of the 16S mtDNA region. with an average K2P distance of 32.5% (min: 23.9%, Statistical support values are given for the respective nodes. The max: 38.7%). This exceeds the average distance of scale bar indicates the estimated number of changes per site. 11.4% (min 0.4%, max 26.6%) between the considered Species with current dubious taxonomic status are highlighted in dark grey boxes, genetic lineages that deserve species rank in Australian lineages and is just slightly smaller than the light grey boxes. Species threatened by secondary salinization average K2P distance of 33.7% (min: 27.9%, max: in the Wheatbelt area of Western Australia are marked using an 43.5%) between Australian Branchinella species and anostracan as symbol. A detailed drawing of the second the non congeneric S. dorothae. Consequently, the antennae and, if any present, the frontal appendage structure is provided for every species. Numbers provided along with the repositioning of C. ornata into another genus (Rogers, figures correspond to the species identities in Table 1.Acircled 2006) is supported by our genetic data. number (1–4) indicates to which of the four morphological Since four phylogenetic groupings, containing all groups each of the Branchinella species belongs Australian Branchinella species except for B. proboscida and B. longirostris, are depicted as a single were positioned as a sister-clade to all other branch- polytomy (comb-like structure) in our reconstructions, inellids in Remigio et al. (2003), they emerged along rapid cladogenesis seems to have taken place. Five out with B. nichollsi, nested within the large diversified B of six species commonly occurring in saline systems clade in our analysis. are united in a monophyletic group within Clade B. Therefore the saline environments were probably Mismatches between morphological groupings colonized by a common ancestor of the species within and molecular phylogeny this grouping. On the other hand, B. simplex, the only other Branchinella that occurs in saline environments, Up until now only a very limited morphological is positioned well away (in the A clade) from these (Geddes, 1981) and molecular (Remigio et al., 2003) salt-tolerant species. This species is the most salt phylogeny of the diverse Australian Branchinella tolerant of all known Australian Branchinella (it has species group was available. Geddes (1981)divided even been collected in hypersaline waters with a the by then 16 known species into three groups, hereby salinity of [50 g/L). It also is morphologically suggesting at least some evolutionary affiliations within atypical with males lacking a frontal appendage the genus using the frontal appendage morphology as altogether and females having long, bent and band- one of the main discriminating characters. Later, like second antennae and a transverse brood chamber Remigio et al. (2003) constructed a molecular phylog- (Timms, unpublished manuscript). Possibly, its large eny for 14 of the 27 Branchinella species described at phenotypic divergence from other Branchinella spe- that time and identified major discrepancies with cies, which arose over a relatively short evolutionary Geddes’ morphological work. When comparing our period, can be linked to the hypersaline environment it extended molecular phylogenetic reconstructions for 31 inhabits. Accelerated evolution in saline environments presumed Branchinella species to the newly composed has previously been described in the Australian salt morphological data set there seems to be correspon- specialist genus Parartemia by Hebert (2002). dence as well as disparity between morphological and Our phylogenetic reconstructions for 31 Branchi- molecular variation. nella species largely comply with the reconstructed The first Branchinella morphogroup contains ten evolutionary patterns for the limited set of 16 species, all without (or with very small) frontal Branchinella species by Remigio et al. (2003). appendages. Although the species of this group do Differences appear to be largely attributable to our not cluster together in one monophyletic clade in our extension of the data set. It was, for example, only molecular reconstructions, all but one (B. papillata), possible to compare groupings of terminal tree are positioned within clades A and B in a total of three branches, as intermediate and deep portions within different monophyletic subclades: one clade contain- the phylogeny were not resolved in Remigio et al. ing B. simplex, B. halsei and B. lyrifera, another (2003). One major difference did emerge from our grouping B. occidentalis and B. australiensis and one analysis; whereas B. hattahensis and B. buchananensis with B. compacta, B. buchananensis, B. hattahensis 123 Hydrobiologia (2013) 700:313–327 321

123 322 Hydrobiologia (2013) 700:313–327 and B. nichollsi. These species appear to have (largely) Taxonomic status of the Australian Branchinella lost their frontal appendage. The evolutionary reduc- lineages tion of the structure within these three groups likely arose through convergent evolution. Remarkably, at We offer supplementary molecular support for the least six of the ten species with reduced frontal species status of 26 of the 31 studied Branchinella appendages typically inhabit saline waters and no lineages which, based on ecological and morpholog- saline species with elaborate frontal appendages are ical differences, were either considered different known. As such, the reduction of the frontal append- species or for which the species status was dubious. age, decreasing the surface to volume ratio of the Our phylogenetic analyses, following ML, MP, NJ, animal, could be adaptive, reducing osmotic stress in QP and BI searches, supported the monophyly of all these environments. This explanation, however, lineages except for B. affinis and B. wellardi, which remains hypothetical and should be experimentally appear paraphyletic with B. clandestina and B. sp. tested. The species within this first group are generally novum Y (QLD), respectively. also of a large size (22–60 mm). From our phyloge- Three currently undescribed lineages appear to netic reconstructions, B. occidentalis and B. austral- deserve separate species status based on the here iensis appear to be evolutionary more related to much obtained genetic divergences (KP2 distances [4.6%) smaller species (typically 8–16 mm) such as B. affinis, and phylogenetic reconstructions and, to lesser extent, B. clandestina and B. budjiti, belonging to morpho- their ecology and morphology (Fig. 2). They are all groups two and three, rather than to the large bodied awaiting formal description. Firstly, this is the case for clade containing B. compacta, B. nichollsi, B. hattah- the B. sp. novum M (WA, code 33 in Table 1) since ensis and B. buchananensis. The morphological sim- they have diverged at least 9.4% and are positioned ilarity between both clades, containing species with well away in our phylogenetic reconstructions from generally large and sturdy bodies, probably also arose other species in the data set. In addition, although independently through convergent evolution. Large morphologically they somewhat resemble B. affinis bodied fairy shrimp are usually superior competitors from eastern Australia, they are distinct in having pairs (Jocque et al., 2010) and produce more offspring than of conspicuous knobs on many of their thoracic smaller species but generally only occur in long lived, segments. Secondly, the newly discovered Branchi- predictable, nutrient rich and turbid systems where nella, which first seemed a West Australian population growth is not food-limited and predation risk is low of B. compacta, from Moora and Unicup (code 31 in (Hamer, 1999). Hardly any congruence was found Table 1, named B. sp. novum U) also appears to between the molecular phylogeny and the second represent a distinct evolutionary lineage with a K2P morphogroup which unites species with a frontal distance of 12.8% from B. compacta. Although appendage consisting of a long trunk and two simple morphologically similar in general habitus to B. branches with at most short spines and papillae. The compacta, female specimens from the West Australian third morphogroup comprises species with more lineage consistently exhibit various dorsal and lateral complex frontal appendages. All members of this tumidities attached to their genital segments. Interest- group, except for B. pinderi, are positioned on the large ingly, given the monophyly of the two ‘‘compacta- polytomy within the molecular phylogenetic recon- like’’ lineages, the morphological resemblance despite struction. B. pinnata and B. campbelli are the only substantial genetic divergence seems indicative of species that do not belong to this third group and are morphological stasis (Schluter, 2000). Thirdly, with a positioned as terminal branches in this polytomy. Two minimum K2P distance of 4.6% to all other species, monophyletic clades unite species from the third the presumed species status, on morphological morphogroup in the molecular reconstructions; B. grounds, of a new lineage from QLD (code 32 in wellardi and B. frondosa on one hand and B. pinnata Table 1, B. sp. novum S) seems to be confirmed as and B. arborea on the other. Finally, of the fourth these distances are still in range of typical interspecies morphogroup, containing species with the most elab- K2P distances within Branchinella as well as those orate frontal appendages, only B. complexidigitata and reported in other branchiopods (Adamowicz et al., B. kadjikadji emerge as more closely related to each 2009). Finally, we address the species status of B. other than to species of other morphogroups. hattahensis and B. buchananensis, which has been 123 Hydrobiologia (2013) 700:313–327 323 disputed in the past (Geddes, 1981; Belk & Brtek, 16S K2P distance amounted only 0.4% between both 1995; Remigio et al., 2003; Timms, 2005). Although and therefore is a firm indication for conspecificity. Geddes (1981) described both as subspecies within B. The strongly opposing results from the considered nichollsi, Belk & Brtek (1995) assigned them species lines of evidence, however, urge for further investi- status but this suggestion was later challenged (Rem- gation before a definite conclusion can be drawn. igio et al., 2003) because of low levels of genetic Finally, the species status of B. pinderi and B. mcraeae differentiation between B. nichollsi, B. buchananensis (Onslow, WA) should also be further evaluated since and B. hattahensis. In 2005, Timms redescribed B. both differ by only 1.2% on 16S from B. lamellata buchananensis and B. hattahensis and designated (Clifton Hills Station, SA). All three of these evolu- lectotypes. We report 16S K2P distances of 4.7–5.2 tionary lineages, however, are clearly morphologi- and 4.1% between B. nichollsi and B. buchananensis cally distinct and the limited habitat data available and B. hattahensis, respectively. Although B. buchan- suggest they utilise different types of water bodies anensis and B. hattahensis are separated by slightly (Timms & Geddes, 2003; Timms, 2005, 2008). lower genetic distances (3.2–3.7%) compared to typical species level differentiation in crustaceans at Synergy of molecular and morphological this gene (typically [4%), the reported level of taxonomy for the establishment of conservation divergence in combination with their morphological units and ecological distinctness (Timms, 2005) pleads in favour of retaining their taxonomic status as separate Conservation implications of the reconstructed molec- species. ular phylogeny are twofold. First of all, our results On the other hand, genetic data (K2P distances highlight that a number of the studied Branchinella \2.4%) disclosed a questionable species status for lineages may be more important than previously five Branchinella lineages (Fig. 2). Firstly, although assumed since they are substantially diverged from Branchinella specimens from Yarromere station in other lineages, representing different species. Queensland (QLD) share some morphological char- Secondly, our results illustrate that some species acteristics with B. wellardi, they were considered a may show important adaptive variation, worthy of distinct species based on some important differences preserving in the light of ongoing environmental such as the absence of a bilobed outgrowth between change. Despite the low 16S divergence of B. sp. the two branches of the frontal appendage, fewer novum Y, B. clandestina, B. halsei, B. pinderi and subbranches attached to the frontal appendage and an B. mcraea from other species, all of them represent expanded basal medial subbranch (Timms, personal morphologically distinct evolutionary lineages. In observations). The Yarromere population, however, addition, they occupy non-overlapping ranges or genetically differs from B. wellardi populations substantially dissimilar habitats such as rock pools, (Bloodwood Station, NSW and Paroo area, NSW) by salt lakes, freshwater clay pans, tree dominated a K2P distance of, respectively, only 0.4–2.4% and swamps, grassy pools and other temporary aquatic was consistently nested within B. wellardi in our habitats (Timms, 2004). Consequently, the investi- phylogenetic reconstructions, indicative of conspeci- gated lineages are likely to harbour unique adaptive ficity. The opposing results from morphological and potential, regardless of whether their species status genetic analyses, however, urge for further investiga- could be confirmed or not. Some of the more tions into the matter. Secondly, B. clandestina (Paroo, generalistic species (e.g. B. affinis) probably are, at QLD) and B. affinis (Paroo, NSW) only differ by a least to some extent, also phenotypically plastic with K2P of 1.8%. As intermediate specimens between the regard to their environment. In this respect, all of the two ‘morphotypes’ were found recently (Timms, 31 lineages included in this study should be regarded personal observations), B. clandestina should be as separate ESUs and treated as distinct units for considered a junior synonym of B. affinis rather than conservation. In addition, given that most Branchi- a separate species. Thirdly, although B. halsei (Lake nella species comprise several discrete and morpho- Cronin, WA) and B. simplex (Cue, WA) are very logically diverse evolutionary lineages, occurring over different morphologically (Timms, 2002, 2004) and wide geographic and/or ecological ranges, we wish to inhabit, respectively, freshwaters and salt lakes, the stress the necessity to also recognise a number of 123 324 Hydrobiologia (2013) 700:313–327

ESUs within nominal species, as nicely exemplified by Branchinella species managed to adapt to a variety Branchinella longirostris. Populations of this species, of temporary aquatic habitats, the majority of species which are separated by genetic distances of up to 10%, is absent from saline waters and the possibility of rapid most likely evolved for several millions of years in local adaptation to this changing environment remains isolation, under distinct selection regimes, and most indefinite. An overview of the known salt tolerance of likely harbour unique adaptive variation at the Australian anostracans is provided in Fig. 1c. In regional scale (Zofkova & Timms, 2009). Other clear general, most ‘species’ (22 spp.) from our data set examples are provided by B. sp. novum Y and inhabit freshwaters (0–3 g/L) while three, B. buchan- B. clandestina which are separated by minimal genetic anensis, B. compacta and B. simplex typically distances of, respectively, only 0.4 and 1.8% from inhabit hypo- (3–20 g/L) or sometimes mesosaline B. wellardi and B. affinis. Despite the close (phylo- (20–50 g/L) systems. Only the latter species has been )genetic and, at least for B. clandestina, likely collected in hypersaline ([50 g/L) water (Hammer, taxonomic relationship, the lineages are clearly dis- 1986; Timms, 2009). B. nichollsi and B. hattahensis similar both in morphology and habitat preference. are known to inhabit lakes that become hyposaline Hence, in order to conserve adaptive potential, shortly before they dry out (Geddes, 1981) and the protection of both lineages is recommended. former species has been collected at a salinity of 11 g/L (Chapman & Timms, 2004). Although they are Anostracan conservation in the light of ongoing more typical of freshwater systems, B. affinis and environmental change: the threat of secondary B. papillata have also been reported in salt lakes at salinization salinities of, respectively, 7 and 16 g/L (Timms, 2009). Finally, both undescribed species from Moora, Since temporary aquatic habitats in Australia are B. sp. novum U and B. sp. novum M, were collected at currently threatened by a variety of anthropogenic salinities of up to 12 g/L. Nonetheless, few species are pressures, of which secondary salinization is one of the likely to be able to adapt to the hypersaline conditions most ominous, proper delimitation of targets for (typically[100 g/L) often reached through secondary conservation is essential. Seven species from our data salinization (Short & McConnell, 2001) and, based on set, B. complexidigitata, B. halsei, B. kadjikadji, known occurrences of species along salinity gradients, B. lyrifera, B. papillata and the two new species, most are expected to be negatively affected when B. sp. novum U and B. sp. novum M from Moora (WA), salinities increase further (Timms, 2009). On the other occur in the salt-threatened regions in the Wheat- hand, two of the endemic West Australian species, belt (Fig. 1d). The conservation status of at least B. longirostris and B. basispina, are rock pool B. papillata should be assessed against statutory specialists and as such occupy groundwater indepen- criteria (e.g. IUCN, 2001, 2003, 2008), as its range dent habitats which are unaffected by secondary appears to be restricted to one site, which lies within an salinization (Pinder et al., 2000). Lastly, even Austra- area under immediate threat of salinization. B. sp. lian anostracans that are tolerant to hypersaline novum M is restricted to a number of lakes in the conditions (e.g. Parartemia spp.) do not appear to be Coomberdale area which, for the time being, seem to colonizing the secondarily saline wetlands, and are be stable with regard to salinization but as the species even disappearing from natural salt lakes affected by range is so limited, conditions should be monitored ongoing salinization and altered hydrology (Timms, carefully and priority listing the species for conserva- 2009, personal observations). It has therefore been tion purposes may be advisable. In addition, although suggested that a combination of factors, such as the other five species are, at least to some extent, more increased salinity, change in quality of the salts, widely distributed, the loss of evolutionary lineages acidity and nutrient content and extended hydroperiod from the Wheatbelt would almost certainly result in a of the temporary salt lakes is responsible for the failure loss of adaptive variation within these species and also of Parartemia to colonize (or sometimes persist in) urges for accurate assessment of their conservation wetlands affected by salinization (Timms, 2009). This status within the threatened regions. observation that Parartemia is failing to utilise In view of ongoing salinization, salt tolerance is a salinized habitat suggests that Branchinella will even crucial factor in species survival. Even though be less likely to do so. 123 Hydrobiologia (2013) 700:313–327 325

Conclusions and Maarten H. D. Larmuseau for their valuable insights during preparation of the manuscript and technical assistance. We thank the Department of Environment and Conservation of Western We have constructed a large scale molecular phylog- Australia for issuing permits. eny for 27 of the 34 described Australian Branchinella species and four currently undescribed lineages, which were presumed new species, hereby for the first time providing genetic data for 15 of these ‘species’. The References discrepancies between our molecular phylogeny and Adamowicz, S. J., P. D. N. Hebert & M. C. Marinone, 2004. groupings according to frontal appendage morphol- Species diversity and endemism in the Daphnia of ogy, suggest that although these traits are taxonomi- Argentina: a genetic investigation. Zoological Journal of cally valuable to distinguish between species, they the Linnean Society 140: 171–205. often carry little phylogenetic signal. Likely at least Adamowicz, S. J. & A. Purvis, 2005. How many branchiopod crustacean species are there? Quantifying the components some of the differentiation on these traits arose of underestimation. Global Ecology and Biogeography 14: through differential selection pressures acting on them 455–468. through the course of evolution or they are to some Adamowicz, S. J., J. K. Colbourne, J. D. S. Witt & P. D. N. extent plastic with regard to the environment. By Hebert, 2009. The scale of divergence: a phylogenetic appraisal of intercontinental allopatric speciation in a combining our phylogenetic and genetic distance passively dispersed zooplankton genus. Molecular Phy- analyses with morphological and ecological informa- logenetics and Evolution 50: 423–436. tion from literature and field surveys, we found solid Belk, D., 1998. Hotspots of inland water crustacean biodiver- confirmation for the taxonomic status of 26 of the sity. Newsletter of the Species Survival Commission 30: 50. investigated ‘species’. As indications for both conver- Belk, D. & J. Brtek, 1995. Checklist of the Anostraca. Hydro- gent evolution and morphological stasis throughout biologia 298: 315–353. the evolutionary history of the genus emerged, we Brendonck, L., 1997. The anostracan genus Branchinella wish to stress the importance of the use of genetic (Crustacea: Branchiopoda), in need of a taxonomic revision; evidence from penile morphology. Zoological Jour- markers for phylogenetic and taxonomic purposes in nal of the Linnean Society 119: 447–455. groups characterized by subtle morphological differ- Brendonck, L. & D. Belk, 1997. On potentials and relevance of ences among nominal species. Furthermore, our study the use of copulatory structures in anostracan taxonomy. shows the value of phylogenetic approaches in the Hydrobiologia 359: 83–92. Brendonck, L., D. C. Rogers, J. Olesen, S. Weeks & W. R. Hoeh, delimitation process of conservation units within such 2008. 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