Biological Conservation 212 (2017) 256–264

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Biological Conservation

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Life-histories explain the conservation status of two estuary-associated MARK pipefishes

⁎ Alan K. Whitfielda, , Thomas K. Mkareb,1, Peter R. Teskeb, Nicola C. Jamesa, Paul D. Cowleya a South African Institute for Aquatic Biodiversity (SAIAB), Private Bag 1015, Grahamstown 6140, South Africa b Molecular Zoology Laboratory, Department of Zoology, University of Johannesburg, Aukland Park 2006, South Africa

ABSTRACT

Two endemic southern African pipefish species (Teleostei: Syngnathidae) co-occur in estuaries on the southeast coast of South Africa. The larger longsnout pipefish, Syngnathus temminckii, is abundant and has a wide range that comprises coastal and estuarine habitats in all three of the region's marine biogeographic provinces. In contrast, the smaller estuarine pipefish S. watermeyeri is critically endangered, and confined to a few warm- temperate estuaries. Here, we explore reasons for these considerable differences in conservation status. Fecundity is related to fish size, with large live-bearing S. temminckii males carrying up to 486 developing eggs/ embryos, compared to a maximum of only 44 recorded for S. watermeyeri. Loss of submerged seagrass habitats due to episodic river flooding appears to be correlated with the temporary absence of both species from such systems. Prolonged cessation in river flow to estuaries can cause a collapse in estuarine zooplankton stocks, a food resource that is important to pipefish species. The greater success of S. temminckii when compared to S. watermeyeri can be attributed to the former species' wider geographic distribution, fecundity, habitat selection and ability to use both estuaries and the marine environment as nursery areas. Genetic data indicate that this has resulted in a much smaller long-term effective population size of S. watermeyeri, a situation that has persisted since the beginning of the present interglacial period. Syngnathus watermeyeri is thus naturally more susceptible to anthropogenic disturbances, which have resulted in an alarming reduction in its contemporary population size. Possible measures to promote the conservation of S. watermeyeri are presented.

1. Introduction species at an advantage over the latter. Syngnathus temminckii and S. watermeyeri have a conservation status Fishes have over 30 reproductive guilds that can essentially be di- that also differs considerably between the two species. The former is vided into three main categories, namely non-guarders, guarders and common within its South African distributional range, from the cool bearers (Balon, 1975). Pipefishes belong to the bearer category and temperate west coast, through the estuaries and marine environment of more specifically the external bearers. Typically they exhibit parental the warm temperate southern and south-eastern coasts, reaching into care, have a low fecundity but invest a large amount of energy in each the subtropical zone on the east coast (Mwale et al., 2014). In contrast, of a small number of well-developed precocial young. The adults of S. watermeyeri has been recorded in only a limited number of estuaries such species are often specialists, have a narrow trophic niche and on the warm temperate south-east coast and, even in those estuaries, usually live in a stable and predictable environment (Bruton, 1989). the numbers are generally very low (Whitfield, 1995). Although the longsnout pipefish Syngnathus temminckii Kaup, 1856 More recently, S. watermeyeri has been listed as Critically and the estuarine pipefish S. watermeyeri Smith, 1963 fulfil many of the Endangered (CR) in the IUCN Red List (www.iucnredlist.org). The main criteria outlined above, both species occur in estuaries that are gen- threats to its existence are habitat loss, river degradation and loss of erally unstable and unpredictable environments (Whitfield, 1990). freshwater inputs to estuaries (Vorwerk et al., 2007). River inflow Fortunately for S. temminckii, it also occurs in the marine environment provides the nutrients required to stimulate planktonic productivity in that is much more stable and predictable, thus conferring this species estuaries (Grange et al., 2000), the food chain upon which this species with a distinct advantage over S. watermeyeri. This study will show that depends for its survival (Whitfield, 1995). Loss of river pulses due to this is but one of the many life-history traits that places the former excessive freshwater abstraction in the catchments leads to a reduction

⁎ Corresponding author. E-mail address: a.whitfi[email protected] (A.K. Whitfield). 1 Present address: Kenya Marine and Fisheries Research Institute, P.O. Box 81651, Mombasa 80100, Kenya. http://dx.doi.org/10.1016/j.biocon.2017.06.024 Received 3 October 2016; Received in revised form 2 June 2017; Accepted 15 June 2017 0006-3207/ © 2017 Elsevier Ltd. All rights reserved. fi A.K. Whit eld et al. Biological Conservation 212 (2017) 256–264 in estuarine zooplanktonic resources (Froneman and Vorwerk, 2013), Bushmans and the East Kleinemonde estuaries in the Eastern Cape with dire consequences for an endangered species such as S. water- Province (Whitfield, 1995; Fig. 1), spanning a coastal distance of only meyeri. 60 km. The main aim of this paper is to examine the life-history styles and The type specimens of S. watermeyeri were collected in the Kariega population dynamics of S. temminckii and S. watermeyeri in the East Estuary during the early 1960s, but by the 1980s and 1990s this species Kleinemonde and Kariega estuaries and to equate these findings with had disappeared from that system (Whitfield and Bruton, 1996), only to the current conservation status of the two species. We use modern reappear in 2006 following river flushing of the estuary (Vorwerk et al., molecular techniques in our analysis of the population genetics of these 2007 ). It is important to note that S. temminckii remained present syngnathids (Mobley et al., 2011) and hypothesise that the long-term throughout and was sometimes abundant in the Kariega Estuary during effective population sizes of these two species should reflect their pre- the decades when S. watermeyeri was locally extinct in that system (Ter sent IUCN categorization. This would imply that S. watermeyeri, which Morshuizen and Whitfield, 1994; Whitfield and Bruton, 1996). It should is of major conservation concern, already had a smaller population size also be noted that S. temminckii has also been recorded from estuaries under natural conditions that made it particularly vulnerable to con- where submerged aquatic macrophyte beds are absent (Mwale et al., temporary habitat disturbances. Finally, we attempt to link the life- 2014). history cycles of the two pipefish species to their current status in the Both S. temminckii and S. watermeyeri have been recorded in Zostera wild, with S. temminckii showing all the attributes of a successful coastal capensis and Ruppia cirrhosa plant beds within the Kariega and East species, whereas the closely related S. watermeyeri is at considerable Kleinemonde estuaries, respectively (Fig. 1). Indeed, the type specimens risk of extinction (Whitfield and Bruton, 1996). of S. watermeyeri were captured together with S. temminckii individuals from the same eelgrass bed in 1963 (D. Galpin, pers. comm.). In es- 1.1. Study species tuarine systems elsewhere in South Africa where submerged plant beds are present, S. temminckii usually occurs on its own, although other fi Syngnathus temminckii and S. watermeyeri are two southern African pipe sh species (e.g. Hippichthys spicifer) are sometimes also recorded fi representatives of the pipefish genus Syngnathus. The former species is from these habitats (Harrison and Whit eld, 2006). The juveniles and the more abundant and widely distributed of the two, occurring in adults of both Syngnathus species are normally associated with sub- coastal waters (including estuaries) from cool-temperate Walvis Bay in merged seagrass beds in certain Eastern Cape |Province estuaries, with Namibia to subtropical waters along the east coast of South Africa S. temminckii also being associated with seaweed and reef habitats in the (Heemstra and Heemstra, 2004). In contrast, the latter species is rare coastal marine environment. and restricted to a few estuaries on the subcontinent between the Syngnathus temminckii in southern African waters was previously

Fig. 1. Published sites of occurrence for Syngnathus temminckii (•) and S. watermeyeri (*) in southern Africa (map modified from Mwale et al., 2014). The boundaries of the three estuarine biogeographic zones along the South African coast are also shown.

257 fi A.K. Whit eld et al. Biological Conservation 212 (2017) 256–264 misidentified as Syngnathus acus Linnaeus, 1758, but a detailed mor- (December, January or February) and winter (June, July or August) phological and genetic study of the species (Mwale et al., 2013) con- from December 1994 to July 2014 (sampling methods are described in firmed that it was in fact S. temminckii. The snout of this species is a detail by James et al., 2008). The estuary was divided longitudinally distinguishing feature when compared to S. watermeyeri, with the into lower, middle and upper reaches, with up to 18 sites sampled on former having a snout longer than the rest of the head whereas the each occasion. A small mesh (5 mm bar) seine net (30 m × 2 m) was latter species has a snout one third of its corresponding head length used for small estuarine spawning species including pipefish. Captured (Heemstra and Heemstra, 2004). Syngnathus watermeyeri reaches a pipefish were identified, measured (mm standard length) and released maximum recorded length of approximately 15 cm SL whereas S. tem- alive at the sampling site. minckii can attain 30 cm SL (Mwale et al., 2014). However, the larger As a follow-on from littoral fish sampling in spring undertaken by adult cohorts of S. temminckii appear to be located in the marine en- Ter Morshuizen and Whitfield (1994) and Vorwerk et al. (2007),an vironment and not in estuaries (Dawson, 1986). intensive survey of the ichthyofauna in the littoral zone of the Kariega Spring and early summer reproductive activity in S. watermeyeri Estuary was undertaken annually in spring (October or November) from commences at approximately 10–12 cm SL whilst that of S. temminckii November 2012 to November 2015. As was the case in the previous two commences at approximately 12–13 cm SL (Mwale et al., 2014). All studies, 60 sites were sampled from the upper to the lower reaches on male and female S. watermeyeri are mature by 13 cm SL and all S. each occasion using a fine mesh 5 m seine net. Syngnathus species temminckii by 16 cm SL. Females of both species are reproductively caught were identified to species level, measured (mm standard length) active before the males in terms of size, and the sex ratio of both species and then released back into the estuary. At each site the development of is biased in favour of females (Mwale et al., 2014). Breeding of S. wa- Z. capensis was recorded as absent, sparse, medium or dense. termeyeri appears to be limited to estuaries but S. temminckii can breed An estimate of the maximum contemporary population of S. wa- in both the marine and estuarine environments. termeyeri was calculated on the basis of a population estimate for this species in the East Kleinemonde Estuary under optimum aquatic mac- 2. Methods rophyte conditions. According to Cowley and Whitfield (2002) there were 3584 S. watermeyeri present in 14.5 ha of macrophytes during 2.1. Main study sites 1995. Assuming similar maximum S. watermeyeri densities in the other estuaries where this species has been recorded, and a maximum total The 4 km long East Kleinemonde Estuary (Fig. 1) is an inter- macrophyte area in all these systems of 77.4 ha, we therefore an esti- mittently open system that closes off to the sea for much of the year due mate a maximum estuarine pipefish population of 19,131 individuals. to a sand bar that develops at the mouth. This estuary usually opens following heavy rains and river flooding that breaches the sand bar at 2.3. Genetic analyses the mouth (Whitfield et al., 2008). The dominant submerged macro- phyte in the East Kleinemonde in the 1990s was R. cirrhosa, which Genetic data from the mitochondrial genome were generated for occurred in a continuous band of varying width along both banks above both pipefish species collected from three and four different estuaries the road bridge (Fig. 2). for S. watermeyeri and S. temminckii, respectively (Table S1). Pipefish A major flash flood in May 2003, and the subsequent prolonged samples used for genetic analyses included either freshly acquired exposure of R. cirrhosa and Potamogeton pectinatus plants resulted in an samples or previously extracted DNA (Mwale et al., 2013). A total of 38 almost complete loss of most of these macrophyte beds (Riddin and samples, 15 from S. watermeyeri and 23 from S. temminckii, were of Adams, 2012). Recovery of the aquatic macrophytes from seed banks sufficiently good quality for genetic analyses (Table S1). was slow, with R. cirrhosa only found in small patches along both banks Genomic DNA was extracted using the cetyltrimethyl ammonium by the end of 2008 (Riddin and Adams, 2012). By 2010 the macrophyte bromide (CTAB) procedure (Doyle and Doyle, 1987, 1990), and DNA beds had expanded but never attained their pre-May 2003 abundance. sequence data from two partial mitochondrial DNA (mtDNA) frag- The 18 km long Kariega Estuary (Fig. 1) is a permanently open ments, cytochrome b gene (cytb) and control region (CR) were se- system that is dependent on the tidal prism to prevent its mouth from quenced. The two species were compared on the basis of genetic di- closing. In recent decades the estuary has received a reduced freshwater versity indices (haplotype diversity, nucleotide diversity and allelic input due to increased freshwater demand from a relatively small richness), and haplotype networks were constructed to assess genea- catchment. Average monthly flow in the Kariega Estuary is usually logical relationships among sequences. In addition, historical effective − negligible ranging from zero flow to < 1 m3 s 1 in most months, re- female population sizes during the past 10,000 years (i.e., the present sulting in high salinities throughout the system and often hypersaline interglacial period) were inferred to determine whether small con- conditions in the upper reaches (Grange et al., 2000). River flooding temporary population sizes based on survey results are a recent, po- can occur during heavy rainfall events when catchment dams are full tentially anthropogenic, development. Details of laboratory procedures − and monthly flows into the estuary are > 27 m3 s 1 (P. Nodo pers. and data analyses are provided in the Supplementary Materials section. comm.). Water temperatures in the Kariega Estuary do not deviate significantly from the natural state since considerable tidal exchange of 3. Results marine water in the lower and middle reaches prevents adverse warm conditions from developing in these regions (Allanson and Read, 1995). 3.1. East Kleinemonde estuary Zostera capensis, occurs mostly as a littoral band in the intertidal zone, mainly in the lower and middle reaches of the Kariega system but Synganthus watermeyeri was recorded consistently in catches be- extending into the upper reaches during prolonged droughts (Hodgson, tween 1996 and 2001, when macrophyte beds were present in a mostly 1987). The width of the Z. capensis band is variable, usually between 1 continuous band along the length of the estuary (Fig. 2). No individuals and 5 m but can exceed 20 m in the lower reaches where large intertidal were captured in 2002 and only one individual was sampled in June areas are present. The density of Z. capensis increases during spring and 2003 (Fig. 3) after loss of the macrophyte beds from the system. The summer and is lowest during winter. highest catch was recorded in 1998, with 26 individuals sampled in the estuary, and coincided with the development of dense R. cirrhosa beds 2.2. Pipefish population dynamics such that seine netting at these sites became very difficult. The length of S. watermeyeri caught between 1996 and 2003 ranged As part of a long-term monitoring project, the ichthyofauna of the from 58 mm to 147 mm, with mean length ranging from 91 mm in 2001 East Kleinemonde Estuary was sampled biannually in summer (single individual caught) to 129 mm ( ± 3.2 S.E.) in 2000 (Fig. 3). The

258 fi A.K. Whit eld et al. Biological Conservation 212 (2017) 256–264

Fig. 2. Bubble plots showing the occurrence and abun- dance of Syngnathus watermeyeri and associated Ruppia cir- rhosa habitat in the East Kleinemonde Estuary from 1996 to 2001.

highest catches were recorded in the middle reaches of the estuary, to 253 mm, with the mean length ranging from 116 mm ( ± 30.0 mm with S. watermeyeri only sampled at sites where submerged macro- S.E.) in 2012, with catches dominated by juveniles to 168 mm phytes were present (Fig. 2). Since June 2003 no S. watermeyeri were ( ± 9.0 mm S.E.) in 2013, with only mature individuals caught (Fig. 3). captured in the estuary but one mature (132 mm) S. temminckii was The highest catch was recorded in 2014 (n = 54), with both juvenile recorded in February 2006 in the lower reaches (Fig. 3). and mature individuals present (mean length ± S.E.; 117 ± 4.4 mm). Although S. temminckii catches were highest in the middle and lower reaches of the estuary, where Z. capensis beds were most extensive, 3.2. Kariega estuary some pipefish were also captured at sites where Z. capensis was absent or sparse (Fig. 4). During the current sampling period an episodic flood event was − recorded in October 2012 (436.6 m3 s 1) and a smaller flood in − October 2013 (30.6 m3 s 1). The 2012 flood was the largest recorded 3.3. Genetic analyses in the system for > 50 years and resulted in the loss of most of the Z. capensis leaf material from the estuary. Recovery of the eelgrass beds A total of 11 haplotypes (Syngnathus temminckii =9; S. water- from root stock in the post flood period was rapid, with some stands of meyeri = 2) were recovered for the cytb gene (Table S3) and 11 (S. Z. capensis present by November 2014 (Fig. 4). temminckii =9; S. watermeyeri = 2) for CR (Table S4). When the two Only one immature (106 mm) S. watermeyeri individual was caught fragments from the same species were concatenated, a total of 12 in the post flood period (November 2013) in the Kariega Estuary. haplotypes for S. temminckii and three for S. watermeyeri were recovered Syngnathus temminckii were recorded every year from November 2012 (Table S5). All unique haplotypes of each mitochondrial marker were to November 2015. The length of specimens caught ranged from 58 mm deposited in GenBank (Table S3 and S4). Genetic diversity indices of S.

259 fi A.K. Whit eld et al. Biological Conservation 212 (2017) 256–264

S. watermeyeri from many Eastern Cape estuaries with similar habitat and physico-chemical conditions to the Kariega and East Kleinemonde systems (e.g. Beckley, 1983, 1984; Vorwerk et al., 2001), suggests that S. watermeyeri has a narrow range and is less environmentally tolerant than S. temminckii. Similarly, S. watermeyeri has never been recorded in the high wave action marine environment off the Eastern Cape coast, despite numerous studies in various littoral marine habitats having yielded catches of only S. temmminckii from this environment (Whitfield and Pattrick, 2015). There is a possibility that S. watermeyeri is more dependent on aquatic macrophyte beds than S. temminckii, a species that is also known to occur in relatively large numbers in estuaries without sub- merged plants, e.g. the Great Fish Estuary (Mwale et al., 2014). Long- term monitoring of fish populations in the East Kleinemonde Estuary showed that a breeding population of S. watermeyeri was present during a macrophyte-dominated phases between 1998 and 2003, but absent from catches during a macrophyte-senescent period (post major flood event) from 2004 to 2009. Recovery of the macrophytes in this estuary did not lead to re-colonization of the system, suggesting that S. water- meyeri were not present in the estuary during the macrophyte-senescent phase. In addition, S. watermeyeri were only recorded at East Kleine- monde sites where submerged macrophytes were present. Similarly, in the Kariega Estuary Vorwerk et al. (2007) found that Fig. 3. The mean length (mm SL ± S.E.) of Syngnathus watermeyeri and Syngnathus peaks in the abundance of S. watermeyeri were recorded at stations with temminckii caught in a) the East Kleinemonde Estuary between 1995 and 2014 and b) the dense stands of macrophytes. Sampling during this study in the Kariega Kariega Estuary between 2012 and 2015. Numbers of individuals caught are shown per fl year. Estuary during a post-episodic ood event in 2012 resulted in the capture of only a single individual of S. watermeyeri. In contrast, S. watermeyeri were low compared to those of S. temminckii (Table S5). temminckii was recorded throughout the study period at sites where This is also evident on the basis of the haplotype networks, which in- submerged macrophytes were abundant, as well as some sites where dicate that the haplotypes of S. watermeyeri are separated by com- macrophytes were sparse or absent. The dispersal capabilities of the larger S. temminckii are con- paratively few mutational steps (Fig. 5). Allelic richness (Ar) was always higher for S. temminckii (Table S5). siderably greater than that of S. watermeyeri. Not only does the former Trends in effective female population sizes estimated using BSP species have a much wider geographic distribution, it is also able to indicate that both species had relatively stable population sizes since breed and disperse via both the estuarine and marine environments. In the onset of the present interglacial period (~10,000 years ago), with contrast, the smaller S. watermeyeri does not appear to breed within the that of S. temminckii being on average ~5 times as high as that of S. marine environment but it may sporadically disperse from one suitable fl watermeyeri (Fig. 6). This result was confirmed by the LAMARC ana- estuary to another following river ood events in the Eastern Cape lyses, which identified a significantly larger long-term population size Province. The apparent absence of S. watermeyeri from the Kowie for S. temminckii (~10 times higher than that for S. watermeyeri), with Estuary, which is in the middle of its distributional range (Fig. 1)is 95% confidence intervals that did not overlap for the two species, thus puzzling, especially as the larvae, juveniles and adults of S. temmminckii fi indicating that these differences are significant. The 95% credibility are well represented in the same system (Whit eld et al., 1994). fi intervals for the growth parameter g encompass zero (Table S6), which Fecundity is related to sh size, with large S. temminckii males suggests long-term stability in population size for both species. It is carrying up to 486 developing eggs/embryos (Mwale et al., 2014), possible that individual populations of both species experienced genetic compared to a maximum of only 44 recorded for the much smaller S. fi fi bottlenecks that reduced their genetic diversity, but it seems unlikely watermeyeri males (Whit eld, 1995). There is a signi cantly positive that these occurred range-wide. The inclusion of individuals from dif- relationship between the size of S. temminckii and the number of oocytes ferent populations may mitigate localised demographic stochasticity on in the female and eggs/embryos carried by the male (Mwale et al., ffi overall genetic diversity to some extent (Grant, 2015), and particularly 2014), thus implying that reproductive e ciency is higher in this when populations recovered quickly after a bottleneck, such subtle species when compared to the smaller S. watermeyeri. In the absence of demographic changes would not be detectable using mitochondrial any evidence to suggest that survivorship of S. watermeyeri larvae is DNA sequences (Mourier et al., 2012). higher than that of S. temminckii larvae, we must surmise that the latter species is at a considerable advantage in terms of populating new or existing habitats. 4. Discussion Another possibility for the differential success of the two syng- nathids is that there is competition for food between adults of S. tem- The distribution and abundance of S. temminckii and S. watermeyeri minckii and S. watermeyeri, especially under conditions of little or no in Eastern Cape estuaries provides strong evidence that the former river flow which result in poor zooplankton stocks (Grange et al., species is more widespread and successful than the latter. Clearly the 2000). Syngnathids feed on small invertebrates associated with sub- broad biogeographical distribution of S. temminckii, together with its merged plant beds or in the water column (Garcia et al., 2005), so there occurrence in both estuaries and the coastal marine environment is a distinct possibility that direct dietary competition exists, but this (Mwale et al., 2014), implies that this fish is physiologically tolerant of assumption needs to be tested. Given the larger mouth size and longer a wide range of physico-chemical conditions. Direct and indirect evi- snout of S. temminckii when compared to S. watermeyeri, it is probable dence suggests that this species can tolerate a range of salinities from 8 that adults of the former species can suck a wider range of small in- to at least 35 (Whitfield et al., 1981) and water temperatures from vertebrates into its buccal cavity than the latter (Bergert and approximately 9 to 30 °C (Russell, 1994). Wainwright, 1997). Thus, in a highly competitive situation, S. water- Conversely, the limited geographical distribution and absence of the meyeri would be less effective at securing prey than S. temminckii, which

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Fig. 4. Bubble plots showing the occurrence and abun- dance of Syngnathus temminckii and associated Zostera ca- pensis habitat in the Kariega Estuary. A single specimen of Syngnathus watermeyeri was captured in 2013, the locality of which is shown on the map by a star.

might then be forced to find an alternative habitat or perish. contrast, S. temminckii is most abundant in permanently open estuaries Both pipefish species breed mainly in spring and early summer and, when present in small temporarily open/closed estuaries, is (September – November) (Mwale et al., 2014), which enables the ju- usually recorded as isolated individuals (Harrison and Whitfield, 2006). veniles to benefit from increased planktonic food resources during the Indeed, during this study only a single S. temminckii was recorded in the productive summer months (Jerling and Wooldridge, 1991). Competi- East Kleinemonde Estuary over more than a decade. In contrast, for a tion for zooplanktonic prey by juveniles occupying the same habitat is period of five years prior to loss of the submerged macrophyte beds, S. likely, but studies have not been conducted on this aspect. The lower watermeyeri was regularly recorded in this system. recorded densities of both fish species during the winter months (Mwale The reason why S. watermeyeri does not occupy all estuaries within et al., 2014), when food resources are likely to be more limiting (Jerling its limited distributional range that have submerged macrophyte beds and Wooldridge, 1991), may be real or an artefact of reduced sampling (Vorwerk et al., 2001) is unknown. The consistent abundance of S. frequencies during this time of the year. It is perhaps also significant watermeyeri at selected submerged macrophyte sites in the East Klei- that S. temminckii has been reported to also breed during the remainder nemonde Estuary over many years suggests that this species is well of the year, although not as frequently as in summer, whereas that for S. adapted to both the oligohaline and mesohaline conditions that pre- watermeyeri is restricted to the spring and summer months (Mwale vailed at different times within this system (Whitfield et al., 2008). The et al., 2014). healthy population between 1998 and 2003, with both juvenile and Syngnathus watermeyeri can occur in both permanently open and mature individuals present, suggests that breeding and the rearing of temporarily open/closed estuaries, sometimes in moderate numbers. In the juveniles was also successful during the predominantly closed phase

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A B macrophyte beds had recovered and the S. temminckii population then consisted of both juveniles and mature individuals, many of which may have recruited from the adjacent marine environment. In contrast, al- though S. watermeyeri may recolonise estuaries via the sea, there is no evidence to suggest that there is a constant marine population of this species ready to colonise vacant estuaries within its distributional range. Hence, when this fish species becomes locally extinct (Whitfield and Bruton, 1996), there is less probability for marine populations to readily replenish an estuary, even though conditions are once again favourable for settlement. Recolonization of estuaries that have lost their S. watermeyeri po- pulation are dependent on river flood events that wash individuals into the sea from nearby systems, a process that is not guaranteed to be successful given the vagaries of coastal ocean currents and the poor swimming ability of syngnathids. The recolonization of the Kariega Estuary by S. watermeyeri following river flooding in 2006 (Vorwerk et al., 2007) was almost certainly facilitated by individuals that had Fig. 5. Statistical parsimony networks showing the evolutionary relationships of the been washed out of nearby estuarine systems such as the East and West haplotypes of Syngnathus temminckii (A) and S. watermeyeri (B). Circles represent haplo- Kleinemonde. The re-establishment of healthy zooplankton stocks in types, and their sizes are proportional to the number of sequences collapsing into that the Kariega Estuary following this river pulse event (Froneman and haplotype. Colour represents sampling localities, except for black, which represents un- Vorwerk, 2013) was probably a major reason why S. watermeyeri was sampled or extinct interior node haplotypes. Vertical cross bars represent mutational steps. Both networks were reconstructed using concatenated mitochondrial DNA cyto- recorded in subsequent years within this system. Their depletion and chrome b and control region data. eventual loss from the Kariega Estuary followed a major episodic river flood in 2012, an event that destroyed almost all the submerged mac- rophytes within this system. The loss of the eelgrass beds also had an adverse impact on S. temminckii but, in contrast to S. watermeyeri, the populations of S. temminckii could be easily replenished from the sea once the submerged macrophyte habitat in the estuary became re-es- tablished. Both pipefish are endemic to southern Africa and belong to a common clade within the genus Syngnathus (Mwale et al., 2013). However, whilst S. temminckii is a widespread southern African en- demic in the mould of the bay pipefish Syngnathus leptorhynchus from northwest America (Wilson, 2006), S. watermeyeri is a much more re- stricted endemic. It is perhaps significant that the estuarine pipefish occurs in a region where endemic fish species are most common (Turpie et al., 2000) and this may be attributed to the localised current and weather patterns that create the isolation needed for speciation in this area (Teske et al., 2005). In terms of life-history strategies, Mwale et al. (2014) suggested that ff Fig. 6. Bayesian Skyline plots showing trends in e ective female population size (Nef, S. watermeyeri and S. temminckii have evolved a reproductive strategy median estimate) of the two endemic southern African species of Syngnathus from the that falls within the r-range of the r–K continuum. They highlighted onset of the present interglacial period (~10,000 years ago) to the present. Estimates were based on a combined dataset of mitochondrial DNA cytochrome b and control region that both species exhibit some K-selection traits, such as being bearers, fragments. allocating time and resources to ensure the survival of embryos during gestation, and producing young that are fully developed and in- of this estuary. dependent. However, both species provide no parental care after birth, In the Kariega Estuary, no S. temminckii juveniles were recorded in which contrasts to most K-selected organisms but is consistent with 2013 following major flood events in 2012 and 2013. By 2014, the many fish species that show diverse attributes within the r–K

Table 1 Selected life-history traits that, on balance, render an advantage to either the longsnout pipefish Syngnathus temmincki or the estuarine pipefish S. watermeyeri. Traits where both species have similar attributes are not included in this summary.

Life-history trait Syngnathus temmincki Syngnathus watermeyeri Advantage

Biogeographical distribution Cool temperate, warm temperate and subtropical Warm temperate S. temmincki Ecosystem occupation Estuaries and coasts Estuaries S. temmincki Estuary type occupation Mainly large permanently open estuaries Permanently open and temporarily open/closed estuaries S. watermeyeri Habitat occupation Submerged macrophyte beds, turbid estuarine littoral and Submerged macrophyte beds S. temmincki marine reefs Reproductive strategy Estuarine and marine spawning Estuarine spawning S. temmincki Reproductive output High fecundity and large brood size Low fecundity and small brood size S. temmincki Reproductive timing Mainly spring and summer but breeding can occur in any Spring and summer S. temmincki month Physiological tolerances Tolerance of a wide range of both salinities and water Tolerance of a wide range of salinities but a moderate range of water S. temmincki temperatures temperatures Mobility and connectivity More mobile due to large adult size Less mobile due to small adult size S. temmincki Genetic diversity High Low S. temmincki

262 fi A.K. Whit eld et al. Biological Conservation 212 (2017) 256–264 continuum (Pianka, 1970). References Table 1 summarizes some of the major reasons why S. temminckii has been so successful, whilst S. watermeyeri is possibly threatened with Allanson, B.R., Read, G.H.L., 1995. Further comment on the response of Eastern Cape extinction. The extent to which human-induced change of the coastal Province estuaries to variable freshwater flows. S. Afr. J. Aq. Sci. 21, 56–70. Balon, E.K., 1975. Reproductive guilds of fishes: a proposal and definition. J. Fish. Res. environment in the Eastern Cape Province has exacerbated this trajec- Bd. Can. 32, 821–864. tory towards extinction is not fully understood. However, given the Beckley, L.E., 1983. The ichthyofauna associated with Zostera capensis Setchell in the relative disadvantages of some of the life-history traits of S. watermeyeri Swartkops estuary, South Africa. S. Afr. J. Zool. 18, 15–24. ff Beckley, L.E., 1984. The ichthyofauna of the Sundays estuary, South Africa, with parti- (Table 1), it is likely that these di erential trajectories were already in cular reference to the juvenile marine component. Estuaries 7, 248–258. place since the evolution of both species. This idea is supported by fact Bergert, B.A., Wainwright, P.C., 1997. Morphology and kinetics of prey capture in the that the demographic trends reconstructed using genetic data indicate a syngnathid fishes Hippocampus erectus and Syngnathus floridae. Mar. Biol. 127, – long-term effective female population size of S. watermeyeri which has 563 570. Bruton, M.N., 1989. The ecological significance of alternative life-history styles. In: been relatively stable over the past 10,000 years, and which has re- Bruton, M.N. (Ed.), Alternative Life-History Styles of Animals. Kluwer Academic mained much lower than that of S. temminckii. This indicates that S. Publishers, Dordrecht, pp. 503–553. fi fi watermeyeri was already at a greater risk of extinction prior to any Cowley, P.D., Whit eld, A.K., 2002. Biomass and production estimates of a sh com- munity in a small South African estuary. J. Fish Biol. 61 (Suppl. A), 74–89. human impacts. Dawson, C.E., 1986. Family no. 145: syngnathidae. In: Smith, M.M., Heemstra, P.C. Current census data for S. watermeyeri suggests a maximum total (Eds.), Smiths' Sea Fishes. Macmillan, Johannesburg, pp. 445–458. population size of approximately ~10% of the species' long-term ef- Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11–15. fective population size of about 190,000. In reality, considerable loss of Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15. submerged macrophyte areas has occurred in most of these systems Froneman, P.W., Vorwerk, P.D., 2013. Response of the plankton to a freshwater pulse in a over the past two decades (e.g. Riddin and Adams, 2012) which would freshwater deprived, permanently open South African estuary. J. Water Resource Prot. 5, 405–413. have resulted in a much smaller actual S. watermeyeri population than Garcia, A.M., Geraldi, R.M., Vieira, J.P., 2005. Diet composition and feeding strategy of under the optimal conditions used to determine the maximum abun- the southern pipefish Syngnathus folletti in a Widgeon grass bed of the Patos Lagoon dance of this species per estuary. It is thus likely that excessive river Estuary, RS, Brazil. Neotrop. Ichthyol. 3, 427–432. ff Grange, N., Whitfield, A.K., de Villiers, C.J., Allanson, B.R., 2000. The response of two water abstraction and other anthropogenetic e ects have merely ex- South African east coast estuaries to altered river flow regimes. Aquat. Conserv. 10, acerbated an existing historical trend of increasing vulnerability to 155–177. natural and, more recently, human driven environmental change in Grant, W.S., 2015. Problems and cautions with sequence mismatch analysis and Bayesian – these estuaries. skyline plots to infer historical demography. J. Hered. 106, 333 346. Harrison, T.D., Whitfield, A.K., 2006. Estuarine typology and the structuring of fish communities in South Africa. Environ. Biol. Fish 75, 269–293. Heemstra, P.C., Heemstra, E. (Eds.), 2004. Coastal Fishes of Southern Africa. National 5. Conservation recommendations Inquiry Services Centre and South African Institute for Aquatic Biodiversity, Grahamstown. Evidence suggests that S. watermeyeri is vulnerable to localised ex- Hodgson, A.N., 1987. Distribution and abundance of the macrobenthic fauna of the – tinction when adverse environmental perturbations occur in a parti- Kariega estuary. S. Afr. J. Zool. 22, 153 162. James, N.C., Whitfield, A.K., Cowley, P.D., 2008. Long-term stability of the fish assem- cular estuary (Whitfield and Bruton, 1996). In addition, it is likely that blages in a warm-temperate South African estuary. Estuar. Coast. Shelf Sci. 76, river pulse events are becoming less frequent due to increased river 723–738. impoundment and freshwater abstraction for both urban and agri- Jerling, H.L., Wooldridge, T.H., 1991. Population dynamics and estimates of production for the calanoid copepod Pseudodiaptomus hessei in a warm temperate estuary. Estuar. cultural users in the Eastern Cape Province (Schlacher and Wooldridge, Coast. Shelf Sci. 33, 121–135. 1996). If the above pipefish attributes and river flow scenarios are true, Mobley, K.B., Small, C.M., Jones, A.G., 2011. The genetics and genomics of Syngnathidae: fi – this means that S. watermeyeri populations are unlikely to naturally pipe shes, seahorses and seadragons. J. Fish Biol. 78, 1624 1646. Mourier, T., Ho, S.Y.W., Gilbert, M.T.P., Willerslev, E., Orlando, L., 2012. Statistical repopulate all systems where they previously occurred due to a re- guidelines for detecting past population shifts using ancient DNA. Mol. Biol. Evol. 29, duction in water volume entering the marine environment. Under such 2241–2251. circumstances it can be argued that translocations of individuals into Mwale, M., Kaiser, H., Barker, N.P., Wilson, A.B., Teske, P.R., 2013. Identification of a uniquely southern African clade of coastal pipefishes, Syngnathus spp. J. Fish Biol. 82, previously occupied estuaries is an acceptable conservation strategy, 2045–2062. particularly as the current known population of S. watermeyeri is limited Mwale, M., Kaiser, H., Heemstra, P.C., 2014. Reproductive biology and distribution of to the Bushmans Estuary and no captive individuals of the species exist. Syngnathus temminckii and S. watermeyeri (Pisces: Syngnathidae) in southern Africa. Afr. J. Mar. Sci. 36, 175–184. There is also strong evidence to suggest that excessive freshwater ab- Pianka, E.R., 1970. On r- and K-selection. Am. Nat. 104, 592–597. straction has a negative influence on the zooplanktonic food resources Riddin, T., Adams, J.B., 2012. Predicting macrophyte states in a small temporarily open/ of S. watermeyeri (Wooldridge, 2007). Therefore dams in the Eastern closed estuary. Mar. Freshwat. Res. 63, 616–623. fi Cape Province need to have a freshwater release policy to ensure the Russell, I.A., 1994. Mass mortality of marine and estuarine sh in the Swartvlei and Wilderness lake systems, southern Cape. S. Afr. J. Aq. Sci. 20, 93–96. ecological sustainability of downstream estuaries, thereby promoting Schlacher, T.A., Wooldridge, T.H., 1996. Ecological responses to reductions in freshwater the long-term conservation of the critically endangered S. watermeyeri. supply and quality in South Africa's estuaries: lessons for management and con- servation. J. Coast. Conserv. 2, 115–130. Ter Morshuizen, L.D., Whitfield, A.K., 1994. The distribution of littoral fish associated Acknowledgements with eelgrass Zostera capensis in the Kariega estuary, a southern African system with a reversed salinity gradient. S. Afr. J. Mar. Sci. 14, 95–105. Teske, P.R., Hamilton, H., Palsboll, P.J., Choo, C.K., Gabr, H., Lourie, S.A., Santos, M., Financial support was provided by the National Research Sreepada, A., Cherry, M.I., Matthee, C.A., 2005. Molecular evidence for long-distance Foundation of South Africa (Grant No. 85373). The genetic research colonization in an Indo-Pacific seahorse lineage. Mar. Ecol. Prog. Ser. 286, 249–260. was partly funded by the Rufford Foundation (Small Grant 14490-1). Turpie, J.K., Beckley, L.E., Katua, S.M., 2000. Biogeography and the selection of priority areas for conservation of South African coastal fishes. Biol. Conserv. 92, 59–72. We are grateful for accommodation provided by Sibuya Game Reserve Vorwerk, P.D., Whitfield, A.K., Cowley, P.D., Paterson, A.W., 2001. A survey of selected during the pipefish surveys of the Kariega Estuary. We also thank two Eastern Cape estuaries with particular reference to the ichthyofauna. Ichthyol. Bull. anonymous referees whose reports were of great assistance in im- J.L.B. Smith Inst. Ichthyol. 72, 1–52. Vorwerk, P.D., Froneman, P.W., Paterson, A.W., 2007. Recovery of the critically en- proving an earlier draft of this manuscript. dangered river pipefish, Syngnathus watermeyeri, in the Kariega Estuary, Eastern Cape Province. S. Afr. J. Sci. 103, 199–201. Whitfield, A.K., 1990. Life-history styles of fishes in South African estuaries. Environ. Biol. Appendix A. Supplementary data Fish 28, 295–308. Whitfield, A.K., 1995. Threatened fishes of the world: Syngnathus watermeyeri Smith, 1963 Supplementary data to this article can be found online at http://dx. (Syngnathidae). Environ. Biol. Fish 43, 152. Whitfield, A.K., Bruton, M.N., 1996. Extinction of the river pipefish Syngnathus doi.org/10.1016/j.biocon.2017.06.024.

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