TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. Zootaxa 2838: 79–84 (2011) www.mapress.com / zootaxa/ Copyright © 2011 · Magnolia Press ISSN 1175-5326 (print edition) Article ZOOTAXA ISSN 1175-5334 (online edition) First genetic data for species of the genus Haploniscus Richardson, 1908 (Isopoda: Asellota: Haploniscidae) from neighbouring deep-sea basins in the South Atlantic SASKIA BRIX1,4, TORBEN RIEHL2 & FLORIAN LEESE3 1 Senckenberg am Meer, German Centre for Marine Biodiversity Research (DZMB), c/o Biocentrum Grindel, Martin-Luther-KingPlatz 3, 20146 Hamburg, Germany. E-mail: sbrix@senckenberg.de 2 University of Hamburg, Biocentrum Grindel, Zoological Museum, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany. E-mail: t.riehl@gmx.de 3 Ruhr University Bochum, Department of Animal Ecology, Evolution and Biodiversity, Universitaetsstrasse 150, D-44801 Bochum, Germany. E-mail. florian.leese@rub.de 4 Corresponding author Abstract The phylogenetic analysis in this study, based on a fragment of the CO1 mtDNA gene, provides first evidence in favour of Brökeland’s (2010) conclusion of recent or ongoing gene flow in Haploniscus rostratus (Menzies, 1962) across the Walvis ridge. It adds the first evidence for the presence of either restricted gene flow or potential cryptic species in the Guinea Basin. The data suggest furthermore that distinct species within the H. unicornis complex occur sympatrically in the East Atlantic deep-sea basin north of the Walvis Ridge. However, without more specimens (males in particular) and more markers, other processes that may have generated this pattern cannot be excluded. Key words: Isopoda, Haploniscidae, deep sea, CO1, DNA barcoding Introduction The abyss is of extensive dimension and difficult and expensive to access. Hence, only a small fraction of the deep sea has been studied (Rex & Etter 2010). Results of biological studies have shown that the deep-sea benthos comprises an enormous, though mostly unstudied, diversity (Hessler & Sanders 1967; Wilson 1998, Brandt et al. 2007; Ramirez-Llodra et al. 2010). Isopods are among the better known groups in the deep sea, especially with respect to their origin and evolution (Raupach et al. 2004; Rex & Etter 2010). Several new species have been described based on morphological characters; however, genetic analyses were and still are hampered due to the difficulty to obtain sufficient high-quality DNA from abyssal isopods. Thus, molecular studies on deep-sea isopods are scarce and the existing ones often used different DNA fragments. The only previous molecular study on Haploniscidae Hansen, 1916 used 16S and 18S rRNA genes besides morphology for an integrative approach to unravel a species complex (Brökeland & Raupach 2008). Overall, we found 47 CO1 sequences for four distinct asellote families in GenBank (34 of them for Munnopsidae Lilljeborg, 1864; Osborn 2009), but none for Haploniscidae. Haploniscid isopods are among the more common taxa in the deep-sea macrofauna. Brökeland (2010a, b) redescribed Haploniscus rostratus (Menzies, 1962), added information to H. unicornis Menzies, 1956 and described four new species of the H. unicornis complex (Brökeland 2010b), which now contains six species. The specimens used by Brökeland (2010a, b) were sampled during the DIVA and ANDEEP expeditions to the South Atlantic abyss (DIVA: Latitudinal Gradients of Deep-Sea Biodiversity in the Atlantic Ocean; ANDEEP: Antarctic benthic deep sea biodiversity: colonization history and recent community patterns). For a subset of this material we were able to retrieve DNA for phylogenetic analyses. The dataset comprises specimens from the Cape, Angola and Guinea Basins (Table 1) and adds the first genetic data to the morphology-based results by Brökeland (2010a, b). Accepted by J. Svavarsson: 3 Feb. 2011; published: 29 Apr. 2011 79 TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. TABLE 1. List of voucher specimens used for the present study located at the Zoological Museum Hamburg (ZMH) and all available information (GB—Guinea Basin, CB—Cape Basin, AB—Angola Basin, ARB—Argentinian Basin). Please note: All specimens used by Brökeland (2010a) are available at the ZMH under catalogue numbers K-42650 (ANDEEP), K-42649 (DIVA1) and K-42648 (DIVA2). DIVA deep-sea genus station basin species GenBank accession number ZMH catalogue number sex/stage identification number (Brökeland 2010a,b, pers. com.) 90 90 90 90 89 90 64 89 89 40 41 41 41 41 41 41 41 41 41 45 41 41 40 40 40 40 64 532 532 532 unicornis-complex unicornis-complex unicornis-complex unicornis-complex unicornis-complex unicornis-complex unicornis-complex unicornis-complex rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus rostratus bicuspis sp. sp. sp. JF283456 JF283454 JF283453 JF283452 HQ441212 JF283455 JF283449 JF283451 JF283475 JF283470 JF283474 JF283473 JF283472 JF283469 JF283468 JF283467 JF283465 JF283464 JF283463 JF283466 JF283461 JF283462 JF283471 JF283459 JF283460 JF283450 JF283448 JF283457 JF283458 JF283447 K-42448 K-42448 K-42448 K-42448 K-42447 K-42448 K-42446 K-42446 K-42651 K-42634 K-42635 K-42636 K-42637 K-42638 K-42639 K-42640 K-42641 K-42642 K-42643 K-42644 K-42645 K-42646 K-42647 K-42717 K-42715 K-42716 to be vouchered K-42652 K-42653 K-42654 DIVA2-HA485 DIVA2-HA488 DIVA2-HA489 DIVA2-HA490 DIVA2-HA499 DIVA2-HA486 DIVA2-HA478 DIVA2-HA496 DIVA2-HA497 DIVA2-HA465 DIVA2-HA456 DIVA2-HA458 DIVA2-HA460 DIVA2-HA466 DIVA2-HA467 DIVA2-HA468 DIVA2-HA470 DIVA2-HA471 DIVA2-HA473 DIVA2-HA469 DIVA2-HA475 DIVA2-HA474 DIVA2-HA461 DIVA2-HA462 DIVA2-HA457 DIVA2-HA459 DIVA2-HA479 DIVA3-H017 DIVA3-H020 DIVA3-H002 GB GB GB GB GB GB GB GB GB CB CB CB CB CB CB CB CB CB CB AB CB CB CB CB CB CB GB ARB ARB ARB Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Haploniscus Chauliodoniscus female/VI female/VI female/VI female/VI female/VI female/V female/VI female/VI manca female/V unknown female/IV or V female/IV or V female/V female/V female/V female/V unknown female/V unkown manca male/VI female/VI manca manca manca unknown male male unknown Methodological approach Analyses in this study comprise sequences from 18 specimens of H. rostratus and nine specimens of H. unicornis. Two specimens of a different Haploniscus species and one specimen of Chauliodoniscus sp. (outgoup) sampled during DIVA3 (cruise M79/1) were added to our dataset. The specimens belonging to the species in focus of this study were sampled during the DIVA2 expedition M63/2 with RV “Meteor” in 2005 (see Brökeland 2010a, b; station plan, sampling and fixation). Tissue from legs was extracted on board. Not more than three pereopods were taken from one side. Specimens were individually numbered and stored in 96% ethanol. During the whole process, samples, specimens and tissue were kept frozen at -20°C or cooled on ice whenever possible until tissue digestion. DNA extractions: Qiagen QIAamp® DNA Mini Kit (manufacturer’s protocol, animal tissue was digested overnight in shaking bath at 56°C /50 rpm, 50 µl elution buffer). All extractions were done at the Smithsonian Institution (Washington, D.C.). The first subunit of the mitochondrial protein-coding gene cytochrome-c-oxidase (CO1, 80 · Zootaxa 2838 © 2011 Magnolia Press BRIX ET AL. TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. ~ 650 bp) was targeted. Reagents used: Biolabproducts Taq-DNA-Polymerase and dNTPs, Crystal PCR-Mastermix (5x); dNTP-Mix Eppendorf® HotMasterMix (2.5x); Qiagen HotStarTaq Plus; FastStart PCR Master FailSafeTM package; FailSafeTM PCR PreMix Selection Kit. PCR: Folmer primers (Folmer et al. 1994), and CrustCOIF and DecapCOIR primers (Teske et al. 2006) were used. Eppendorf HotMasterMix 2.5x and Eppendorf® Perfectprep® Gel Cleanup kit were used according to the manufacturer's protocol. Cycle sequencing of purified PCR products was performed by QIAGEN, Germany or PCR and sequencing reactions were done at the Canadian Centre for DNA Barcoding (CCDB) or the Smithsonian Institution (SI). For the reactions at CCDB and SI we used aliquots of the original DNA extraction. Presently, DNA extractions are stored at the DZMB Hamburg and/or at the SI. All specimen data are managed as project “DIVA2 Haploniscidae” in the Barcode of Life Data Systems (BoLD; http://www.boldsystems.org/views/login.php). TABLE 2. PCR protocol. PCR mix ingredients ® Volume (µl) EPPENDORF HotMasterMix (2.5x) 10 ddH2O 9 Primer 1 [10, 11 or 12 µM] 1 Primer 2 [10, 11 or 12 µM] 1 Template DNA 4 Total volume 25 PCR step condition Preheated lid Yes Initial denaturation time [min] 02:00 Initial denaturation temperature [°C] 94 Denaturation time[min] 00:45 Denaturation temperature [°C] 94 Annealing time [min] 00:45 Annealing temperature [°C] 50 Elongation time [min] 01:20 Elongation temperature [°C] 72 / 65 Cycle number 35 Final elongation time [min] 07:00 Sequences were deposited in BoLD and GenBank (see table 1 for accession numbers). Sequence alignment: MAFFT (E-INS-I algorithm) (Katoh et al. 2002), deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S11144). A Bayesian phylogenetic tree was calculated with MrBayes (Huelsenbeck & Ronquist 2001) with 8 independent chains and 107 generations (samplefrequency=1000) using the HKY+I+G and GTR+I+G model as suggested by the analysis using jModeltest (Posada 2008). The first 10,000 trees were discarded as burnin. In addition, a Maximum Likelihood bootstrap tree (10,000 replicates) was calculated with RAxML (Stamatakis 2006). A parsimony analysis was conducted with 1000 Bootstrap replicates using Paup (win paup 4b 10, Swofford 1998). Results Haploniscus rostratus (Menzies, 1962) The known distribution of H. rostratus is restricted to abyssal depths of 4577–5647 m, geographically extending from the southern part of the Cape Basin over the Angola Basin to the Guinea Basin. The type locality of the species is in the Cape Basin. Using a morphological approach, Brökeland (2010a) studied specimens of H. rostratus collected north and south of the Walvis Ridge. The role of the ridge as a potential barrier to gene flow and hence allopatric divergence or speciation has been discussed (Brandt et al. 2005). For H. rostratus, Brökeland (2010a) found no significant morphological differences across the Walvis Ridge and consequently stated that populations FIRSTGENETIC DATA FOR HAPLONISCUS SPECIES Zootaxa 2838 © 2011 Magnolia Press · 81 TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. on either side do not represent allopatric sibling species but rather are members of a single species distributed across the ridge. However, since distinct species may be morphologically very similar or even indistinguishable (e.g. Held & Wägele 2005; Raupach & Wägele 2006), genetic data provide important independent information to test species hypotheses. The data of the 658 bp CO1 alignment from this study adds genetic support in favor of Brökeland´s (2010a) conclusion of gene flow across the Walvis Ridge. Our data resolve a single, well-supported H. rostratus group (posterior probability 0.95, ML Bootstrap 98) including both, the specimen from the Angola Basin (DIVA2-HA469, station 45) as well as those from the Cape Basin (16 sequences from stations 40 and 41). Interestingly, the single specimen available for analysis from the Guinea Basin (DIVA2-HA497, station 89), which is far more to the north, forms the sister group to the Cape and Angola Basin group and is genetically distinct (about 7% uncorrected p-distance; Fig. 1). This specimen was clearly identified as H. rostratus by Brökeland (pers. comm.), indicating possible barriers to gene flow among the southern basins and the northern Guinea Basin or even the presence of yet overlooked species. We conclude that although our dataset is rather small, it supports Brökeland’s (2010a) hypothesis of recent or ongoing gene flow across the Walvis Ridge as specimens from the Angola Basin and the Cape Basin share a single CO1 haplotype. In addition, our data support the possible existence of cryptic or yet overlooked species occurring in the deep Guinea Basin. FIGURE 1. CO1 Bayesian phylogenetic tree based on the 658 bp alignment. Branch support shows the ML / MP bootstrap values / posterior probabilities, respectively. (CB) = Cape Basin, (AB) = Angola Basin, (GB) = Guinea basin, (ARB) = ArgentinianBasin. Haploniscus unicornis Menzies, 1956 complex Brökeland (2010b) described four new species belonging to the H. unicornis complex, i.e. H. bihastatus Brökeland, 2010, H. monoceros Brökeland, 2010, H. machairis Brökeland, 2010, and H. angolensis Brökeland, 2010. All 82 · Zootaxa 2838 © 2011 Magnolia Press BRIX ET AL. TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. species were described based on male characters. Brökeland (2010b) stated that it was difficult or impossible to assign females, juveniles and manca stages to one of the six species belonging to the complex by morphological traits. In case of the H. unicornis complex we have only sequences of female specimens available, which thus cannot be assigned to one of the species mentioned above, neither using morphological nor genetic characters. The type specimen of H. unicornis was collected from the Puerto Rico trench in the western Atlantic while the specimens analyzed by Brökeland (2010b) were sampled in the Guinea and Angola Basins in the southeastern Atlantic. In contrast to H. rostratus, H. unicornis could not be recorded from the Cape Basin south of the Walvis Ridge. Thus, its distribution is hypothesized to be limited by the Walvis Ridge, in contrast to H. rostratus (see Brökeland 2010a and results above). Of the six species in the H. unicornis complex, H. monoceros, H. bihastatus and H. machairis were described (based on males) to occur sympatrically in the Guinea Basin (Stations 89 and 90 of the DIVA2-cruise; Brökeland 2010b). In the phylogenetic tree (Fig. 1) the female specimens form no single monophyletic group but cluster in four distinct groups (1: HA490; 2: HA478; 3: HA485, HA486, HA489, HA496, HA499; 4: DIVA2-HA488) separated by 9% in case of group 1 and 2, otherwise by 18–20% sequence divergence (uncorrected p-distance) each. Similarly high pairwise sequence divergence values are found between members of these H. unicornis groups and other distinct species (H. bicuspis, H. rostratus). The high between-group divergence is contrasted by a withingroup variability of below 1.8%. The outgroup, a specimen from the haploniscid genus Chauliodoniscus, is separated from the ingroup taxa by 25–28%. A distance of 23.2–24.5% separates Haploniscus sp. from the H. unicornis complex, H. rostratus and H. bicuspis. Comparable values for intra- and interspecific divergences are found also for other asellote isopods (Osborn 2009; see also supplement 1). This comparison further supports that the H. unicornis groups found in our analysis (Fig. 1) represent different species within the H. unicornis complex and may very likely represent three of the four newly described species described by Brökeland (2010). Discussion Despite tremendous efforts and more material potentially available for this study, we were only able to obtain 30 sequences for our dataset (18 H. rostratus, 8 H. unicornis, 4 outgroups). This highlights the general problem that sampling the deep sea is not only time consuming and expensive but also that a lot of impediments need to be overcome to produce high quality molecular data from the few specimens caught. In addition, the abundance of males is often much lower than of females, making phylogenetic and biogeographic studies that rely upon taxonomic information from male specimens more difficult. Still, the genetic data provided in this study reveals the power molecular tools have when morphological analyses are limited: For H. rostratus, our phylogenetic analysis supports Brökeland’s (2010a) conclusion of recent or ongoing gene flow in H. rostratus across the Walvis ridge but furthermore indicates the presence of either restricted gene flow or potential cryptic species in the Guinea Basin. Similarly high values as those observed between the specimen from the Guinea Basin and those from the Cape and Angola Basins are in the range observed among closely related congeneric species. When comparing all available asellote isopod CO1 data from GenBank (accessed December 13th 2010) it becomes obvious that values lower than 2% uncorrected pairwise distances are usually found within species, between 5 and 10% between closely related species and above 18% among distantly related species (see Osborne 2009 and supplement 1). In the case of the H. unicornis complex, even without males in our dataset, we can document four divergent groups of females belonging to the H. unicornis complex and therefore reveal an existing genotypic diversity that is hidden within the uniform female morphology. These four groups may well represent the species described by Brökeland (2010b). However, the inclusion of males is needed for verification and correct assignment. Our data suggest that distinct species within the H. unicornis complex occur sympatrically in the East Atlantic deep-sea basin north of the Walvis Ridge. Two morpho-types of females were present and there may be some distinct morphological traits for species assignment, which were not detected with traditional analysis methods (W. Brökeland, pers. comm.). In conclusion, our data indicate that CO1 may be a powerful tool for species identification and delimitation. However, without more specimens (male specimens for correct species assignment in particular) and more markers, other processes that may have generated this pattern cannot be excluded. In the case of asellote isopods, the general usefulness of CO1 for phylogenetic inferences within a genus or a family is still under investigated. Hence, our study adds only few but highly important data for future comparative studies. FIRSTGENETIC DATA FOR HAPLONISCUS SPECIES Zootaxa 2838 © 2011 Magnolia Press · 83 TERMS OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website is prohibited. Acknowledgements We would like to thank all pickers and sorters on bord of RV Meteor during the DIVA-2 expedition (M 63/2). Special thanks go to Dirk Steinke for helping with managing the BoLD projects. Without the help of Amy Driskell and Andrea Ormos from the Smithsonian Institution many of the sequencing work would not be finished yet. 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