African Journal of Aquatic Science 2014: xx–xx Printed in South Africa — All rights reserved

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AFRICAN JOURNAL OF AQUATIC SCIENCE

This is the final version of the article that is published ahead of the print and online issue

ISSN 1608-5914 EISSN 1727-9364 http://dx.doi.org/10.2989/16085914.2014.938600

Larval development reflects biogeography in two formerly synonymised southern African coastal crabs I Papadopoulos and PR Teske* Molecular Zoology Laboratory (Aquatic Division), Department of Zoology, University of Johannesburg, Auckland, South Africa * Corresponding author, e-mail: [email protected] The southern African crab Hymenosoma orbiculare was recently split into five distinct species, of which three are estuarine/coastal and have non-overlapping distributions that are linked to temperature-defined marine bioregions. This suggests that the species’ ranges may be limited by physiological adaptations to their thermal environment. We explored this hypothesis by rearing the larvae of the warm-temperate lineage of H. orbiculare and the warm-temperate/subtropical H. longicrure at a range of water temperatures, and found clear temperaturedependent differences in the duration of larval development. Our study contributes to the growing body of evidence that stresses the importance of adaptation to regional environmental conditions, rather than physical dispersal barriers on their own, in limiting the mixing of marine species between temperature-defined biogeographic regions. Keywords: crown crab, Hymenosoma longicrure, Hymenosoma orbiculare, planktonic larval duration, range limits, temperature stress, thermal adaptation, zoea

Introduction Biogeographic disjunctions are contact zones of geographically distinct species assemblages (Seapy and Littler 1980) which often also separate distinct evolutionary lineages of the same species, or closely related species (Burton 1998). Two explanations have been invoked to explain how such distributions are maintained. The first states that physical barriers limit dispersal across the disjunctions so that regional units of populations are effectively isolated (Palumbi 1994). The second suggests that, even though there is dispersal across the boundary, each lineage or species is adapted to environmental conditions typical of a particular region. Marine biogeography is believed to be a direct result of species’ thermal tolerance ranges (Pörtner et al. 2007), so conditions in adjacent regions put physiological stress on migrants and are detrimental to their growth, reproductive output and survival (Teske et al. 2008, 2011). The coastline of South Africa presents an ideal environment for exploring the importance of thermal adaptation because its marine bioregions are defined by differences in water temperature (Lombard et al. 2004). To identify species-specific thermal adaptations, we studied the effects of temperature on the larval development of two allopatric southern African coastal crabs from the family Hymenosomatidae MacLeay, 1838. Hymenosoma orbiculare Desmarest, 1823 is particularly widespread and is one of the numerically dominant estuarine decapod crustaceans in temperate southern Africa. It occurs from Namibia on the west coast of the African continent to the south coast of South Africa (Teske et al. 2007), a region that comprises two marine provinces (cool-temperate west coast and warm-temperate south coast). The second

species, H. longicrure Dawson and Griffiths, 2012, has a comparatively narrow distribution range comprising about 150 km of coastline that is limited to the biogeographic transition zone between South Africa’s warm-temperate (south coast) and subtropical (east coast) marine provinces. In the north-east, it is replaced by a third congener, the subtropical/tropical Hymenosoma projectum Dawson and Griffiths, 2012. Until recently, H. longicrure and H. projectum were synonymised with H. orbiculare, but they are morphologically and genetically distinct (Teske et al. 2009; Dawson and Griffiths 2012). We hypothesised that H. orbiculare and H. longicrure would exhibit different fitness responses to temperature stress that reflect adaptations to each species’ thermal environment. Materials and methods Physiological experiments Crustacean larvae are more vulnerable to thermal and osmotic stress than adults (Anger et al. 2003), suggesting that adaptive differences between closely related species of Hymenosoma would be most readily identifiable by comparing the development of their larval stages. The larval development of H. orbiculare and H. longicrure can be directly compared because both species have three planktonic larval stages (Zoea 1, 2 and 3) and then metamorphose into the benthic Crab 1 stage. We conducted experiments on three broods of H. longicrure from the subtropical Qolora Estuary (South Africa; 32°37ʹ50ʹʹ S, 28°25ʹ48ʹʹ E) and compared the results with previously published data from H. orbiculare (Papadopoulos

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Sampling and laboratory procedures Ovigerous female H. longicrure with embryos in an advanced state of development (recognisable in the field by their light grey colour) were collected from sandy substrates in the Qolora Estuary, kept separately in 1-litre buckets of estuarine water to which an oxygen tablet was added and transferred to the laboratory within three days of collection. Here, they were kept in temperature-controlled environmental rooms (±0.5 °C) in 500 ml of aerated, filtered (0.1 μm), ultra-violet irradiated natural seawater (salinity  35) at 20 °C and under a 12:12 light-dark (06:00–18:00) photoperiod until the larvae hatched. Holding water was changed completely every two days. Hatching always occurred at night, commencing shortly after dark. Actively-swimming larvae were collected at 08:00 on the morning after hatching, using a wide-bore pipette. The larvae from each brood were divided into five groups of 10 individuals, and each individual was transferred to a glass vial filled with ~14 ml of water (salinity of 28 at 20 °C, hereafter referred to as culture water) and immediately fed newly-hatched (<8 h old) Artemia sp. nauplii (Argent ‘Platinum Grade’) to slight excess. Acclimation at the new salinity lasted 2 h, after which each group was acclimated to a different constant temperature (12, 16, 20, 24 and 28 °C) at a rate of 2 °C h–1. Hence, after acclimation, each temperature was represented by 30 individually reared larvae originating from three different broods. Larvae were checked daily for moult status and survival, followed by transfer to fresh culture water and feeding. This procedure was continued for 39 days, by which time most larvae had either metamorphosed to the Crab 1 stage or had died. Larvae were considered dead when opaque and/or when no visible movement of internal or external structures and appendages could be detected under a dissection microscope. Data analysis Data from different broods of H. longicrure that were kept at the same temperature were pooled. To identify adaptive differences in the larval development of the two crab species, we determined whether the mean number of days required to complete a particular larval stage differed significantly at a particular temperature by performing Mann– Whitney rank sum tests (Mann and Whitney 1947) in SigmaStat 3.1 (Systat, San Jose, California). We report only the results for the Zoea 1 stage and for the complete experiment, as the differences between the species were most obvious in these cases. Results At the lowest temperature (12 °C), the early-stage larvae (Zoea 1) of H. orbiculare from the warm-temperate province

reached the next larval stage (Zoea 2) much more quickly than those of H. longicrure. The experiment was terminated after 39 days, at which time all individuals of H. orbiculare and 19% of H. longicrure had completed this stage at 12 °C (Figure 1a). At 12 °C neither species completed development within 39 days, but while most larvae of H. longicrure had not yet reached Zoea 2, 23.5% of the larvae of H. orbiculare had reached this stage, and the remaining 76.5% had even reached Zoea 3 by this time. At 16 °C the larvae of H. longicrure took significantly longer to complete larval development than did those of H. orbiculare, whilst at the highest temperature (28 °C), all the larvae of H. orbiculare died before reaching the Crab 1 stage (Figure 1b). Discussion The distribution ranges of marine organisms are often considered to be determined primarily by water temperature (Sanford et al. 2006; Pörtner et al. 2007), with temperatures

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et al. 2006) from the warm-temperate south coast of South Africa (Kromme Estuary; 34°08ʹ26ʹʹ S, 24°48ʹ39ʹʹ E). Genetic data indicate that levels of gene flow are high along the south coast, and that H. orbiculare is represented in this region as a single evolutionary lineage (Teske et al. 2007, 2014). Therefore samples from the Kromme Estuary are representative of the species’ warm-temperate population.

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Figure 1: Duration of larval development in Hymenosoma orbiculare and H. longicrure at five different temperatures; (a) development of the first larval stage (Zoea 1) and (b) total development time until the adult (Crab 1) stage was reached. Standard deviations are shown as error bars, and significant differences in the duration of larval development are indicated as asterisks (*p < 0.05, **p < 0.01). The experiment was terminated after 39 days, by which time not all individuals of H. longicrure had reached the Zoea 2 stage at 12 °C (Figure 1a), and neither species had reached the Crab 1 stage (Figure 1b)

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beyond a species’ thermal tolerance range resulting in a mismatch between oxygen demand and their capacity to supply the tissues with oxygen (Pörtner 2001). Temperaturedependent range limitations are, however, often difficult to disentangle from the isolating effects of physical dispersal barriers (Teske et al. 2008). Data on the duration of development of the larvae of the two species of Hymenosoma reared at five different temperatures indicate that differential adaptation may be at least partially responsible for maintaining the abrupt distribution break found on the south-east coast of South Africa. Slower development of the larvae of H. longicrure at colder temperatures suggests that metabolic rates in this species are significantly reduced, which in vivo will result in increased mortalities due to their inability to capture prey and the increased chances of predation. Although neither species was able to complete larval development and reach the Crab 1 stage at 12 °C within 39 days, the larvae of H. orbiculare developed much more quickly, which may be an adaptation to periods of cold-water upwelling in the warm-temperate province. In contrast, the fact that H. orbiculare was unable to complete development at 28 °C suggests that warmer water in the biogeographical transition zone may present a significant barrier preventing this species from establishing itself in this region, because the higher temperatures exceed its physiological tolerance. Interestingly, the eastern distribution limit of the invasive mussel Mytilus galloprovincialis is in almost the same location as the eastern distribution limit of H. orbiculare (Zardi et al. 2007), suggesting that the warmer temperatures further east prevent both these temperate species from spreading further. Distribution limits in coastal south-eastern Africa have been reported for numerous marine species (Teske et al. 2011), and are particularly compelling when closely related species, or evolutionary sister lineages of the same species, are largely confined to adjacent marine provinces. These disjunctions have often been attributed to physical barriers such as river discharge (Ridgway et al. 1998), summer upwelling (Maree et al. 2000) and ocean currents (Zardi et al. 2011). However, the ranges of sister lineages can overlap over hundreds of kilometres (Teske et al. 2007; Zardi et al. 2007), and localised or seasonal oceanographic features on their own are unlikely to explain such distribution gradients. Moreover, many species from the warmer east coast can readily bypass any potential physical dispersal barriers to settle along the warm-temperate coast during summer, but are unable to establish themselves there in the long term (Beckley 1985). The present study supports the idea that thermal adaptation is important in limiting the exchange of closely related marine species or recently diverged populations of the same species across adjacent, temperaturedefined biogeographic regions. The role of adaptation in maintaining genetic structure is currently the subject of much interest in evolutionary biology because of technical advances that allow for the identification of gene regions that are under diversifying selection between bioregions (i.e. next-generation sequencing) (Kirk and Freeland 2011). However, there is a risk that processes other than selection can create the illusion of an adaptive cline (Gilchrist and

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Meats 2010), and comparatively simple experiments like the one reported here, will prove very useful to strengthen the conclusions of the more sophisticated genetic studies. A combination of these approaches will be particularly useful to establish whether marker-specific genetic resolution that is insufficient to detect recently evolved genetic structure explains the finding that approximately half the region’s planktonic dispersers seem not to be affected by either physical or physiological barriers which, for the remaining species, limit the ranges of distinct evolutionary lineages (Teske et al. 2014). Acknowledgements — We gratefully acknowledge the Zoology Department, Nelson Mandela Metropolitan University, for the use of its climate-controlled rooms. This research was supported by the PADI Foundation (Grant No. 10981).

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Manuscript received 23 April 2014, revised 27 April 2014, accepted 19 June 2014 Associate Editor: MM Coke