Intertidal submerged macrophyte beds dominated by seagrass are ecologically important habitats that provide foraging sites, shelter, and nursery grounds for many aquatic species, thereby supporting a greater density of animals per unit area than any other habitat type within estuaries and lagoons (Alfaro, 2006; Rasheed and Unsworth, 2011). However, seagrass habitats are declining globally, with 10 seagrass species (14% of all the known species) at risk of extinction (Short et al., 2011). With the loss of seagrass beds, a cascade in the loss of biodiversity and abundance of other aquatic species that depend on these habitats is expected (Jackson et al., 2001; Hughes et al., 2009). Threats to seagrasses include natural events such as flooding and siltation, which can temporarily result in the complete extirpation of seagrass beds (Riddin and Adams, 2012), but also anthropogenic factors such as coastal development, modification of shorelines, pollution and excessive bait collection (Duarte, 2002; Orth et al., 2006; Pillay et al., 2010; Stankovic et al., 2021), as well as climate change (Aller et al., 2019).

In southern Africa, the eelgrass Zostera capensis is a dominant keystone seagrass species (Bandeira and Gell, 2003; Adams, 2016). Listed as Vulnerable on the IUCN Red List, with a decreasing population trend (Short et al., 2010; Van der Colff, 2016), it has been reported in 62 of 290 South African estuaries, with all seagrass beds combined covering a total area of only between 7 and 20 km² (Bandeira and Gell, 2003; Adams, 2016). Despite the ecological significance of seagrass, only two of these estuaries (Langebaan and Knysna) have some formal protection status (Adams, 2016). Their macrophyte beds harbour animal species with high conservation status, including the Endangered Knysna seahorse, Hippocampus capensis Boulenger, 1900, which is found in Knysna and two nearby estuaries (Mkare et al., 2017), and the Critically Endangered pulmonate limpet Siphonaria compressa Allanson, 1958, which occurs in both Langebaan and Knysna (Allanson and Herbert, 2005). A third species of conservation concern, the Critically Endangered estuarine pipefish, Syngnathus watermeyeri Smith, 1963, is strongly associated with macrophytes in two estuaries (Kariega and Bushmans; Figure 1) that have not been afforded any formal level of protection (Whitfield et al., 2017).

Pipefishes, along with seahorses, pipehorses and seadragons, are part of the family Syngnathidae, which are predominantly marine teleosts (Pollard, 1984; Dawson, 1985; Gordina, 1991). Syngnathids have a low fin surface area, which limits their distributions to environments with slow water currents (Castejón-Silvo et al., 2021). They are typically small, benthic and specialised diurnal visual feeders that occupy small home ranges (especially mated males) (Foster and Vincent, 2004; Vincent et al., 2005; Kuiter, 2010). Syngnathids are often an important component of seagrass beds, mimicking macrophytes with cryptic colours and shapes. Syngnathids that are dependent on seagrass beds are important indicator species that can be used to evaluate the conservation value and health of estuarine seagrass beds (Shokri et al., 2009). The conservation of syngnathids can thus provide benefits to other species that share the seagrass habitats, as correlations with other fish species in density and spatial variations in assemblages have been found (Shokri et al., 2009).

Syngnathus watermeyeri was originally reported from three estuaries located along the warm-temperate south coast of South Africa, namely Bushmans, Kariega, and Kasouga (Figure 1; Smith, 1963; Dawson, 1986). The estuarine pipefish was recognised as a threatened species in 1987 (Skelton, 1987) before being declared provisionally extinct in 1996 (Whitfield and Bruton, 1996) after it had not been recorded for several decades. In 1997, the species was discovered in the East Kleinemonde Estuary, where it had not previously been recorded (Figure 1; Cowley and Whitfield, 2001). Twelve pipefish from the East Kleinemonde were subsequently transferred to the adjacent West Kleinemonde Estuary (Bell et al., 2003), which may already have had a viable population since the two estuaries are sometimes linked in the mouth region. Following its localised extinction in these two estuaries after a flood event in May 2003, the estuarine pipefish was rediscovered in two of its historical habitats, namely the Bushmans (in 2005) and Kariega (in 2006) estuaries (Vorwerk et al., 2007; Whitfield et al., 2017). Both estuaries are clear, marine-dominated, permanently open systems with salinity values that are close to those of the sea along most of their lengths (Teske and Wooldridge, 2003). Although these conditions are a result of excessive freshwater abstraction in the estuaries’ catchments (Bate et al., 2002), they provide ideal conditions for the growth of extensive beds of aquatic macrophytes (Adams, 2016), which provide habitat for the pipefishes.

Recent estimates based on survey data suggest that the current population size of S. watermeyeri may be considerably larger than the 100–250 individuals reported in the IUCN Red List entry (Pollom, 2016), and may actually be in the order of tens of thousands of individuals if all known estuaries where they have occurred are occupied (Whitfield et al., 2017). However, total population size may fluctuate considerably, depending on estuarine conditions and pipefish occurrences prevailing at the time. The effects on genetic diversity resulting from severe population bottlenecks that result from either local reduction of pipefish populations, or the founder events associated with recolonisation of habitats where the species has become extinct, are currently unknown. Such information is necessary to develop conservation measures to minimise the risk of this species becoming extinct in the wild.

In the present study, we used genome-wide data from individuals sampled non-destructively to investigate genetic diversity and population structure in the last two remaining populations of this Critically Endangered species. Our work serves as the first step toward the development of a conservation plan for S. watermeyeri. Specifically, we hypothesised that each of the two remaining populations has unique genetic diversity, which would indicate that translocations of individuals between estuaries can counteract the negative consequences of inbreeding by maximising the genetic diversity of both populations.

The sequencing run produced an average of 1,203,529 quality filter sequences per sample. The ipyrad assembly assigned 18,001 single nucleotide polymorphic sites across 34 individuals (Supplementary Table 1). After additional filtering steps, the final dataset used to assess genetic structure (which retained SNPs that departed from Hardy-Weinberg expectations) contained 4,883 SNPs, and the selectively neutral dataset contained 3,769 SNPs (Supplementary Table 2). The percentage of missing SNPs per individual for each dataset is shown in Supplementary Table 3.

No genetic structure was found between estuaries, with F_ST = 0.01 (P = 0.36) and DAPC (Supplementary Figure 1A) identifying a single population cluster. A DAPC scatterplot enforcing K = 2 recovered the individuals from each estuary as a single cluster, but with three outlier individuals (Supplementary Figure 1B). Although the marginal likelihoods from the fastSTRUCTURE analysis were slightly higher for K = 2 than for K = 1 (Supplementary Figure 1C), this was not confirmed by an ancestry assignment barplot, where the same three outlier individuals as in the DAPC scatterplot were partially assigned to a second population that showed no clear affiliation with either estuary (Supplementary Figure 1D).

The population-level inbreeding coefficients (F_IS) were significantly positive in both estuarine populations (Table 1). In both cases, observed (H_O) heterozygosity estimates were smaller than expected (H_E) heterozygosity estimates, indicating heterozygote deficiencies typical of inbred populations. The 95% confidence intervals of both F_IS and mean allelic richness broadly overlapped for the two estuarine populations, indicating that they did not differ significantly. Tajima’s π, on the other hand, was greater for Kariega.

The average inbreeding (F_ab) and relatedness (r_ab) coefficients between pairs of individuals were strongly influenced by the estimated values for the Δ₄ and Δ₆ coefficients (the probability of identity-by-descent of two alleles within an individual), and point to a considerable level of heterozygote deficiency in both populations. The inbreeding coefficients were significantly positive for both populations, and this was also true when F_ab was compared between individuals from different populations (Figure 2A). Moreover, many individuals were found that were closely related (Figure 2B). Within estuaries, a total of 71% (Bushmans) and 20% (Kariega) of the pairs of individuals had a relatedness coefficient that exceeded the relatedness expected between second cousins, and in 4% (Bushmans) and 1% (Kariega) of pairs of individuals, r_ab exceeded that expected between first cousins. High relatedness was also found between pairs of individuals from different estuaries, and thus corroborates the finding that the two populations are not distinct, with 34% having an r_ab that exceeded the level of relatedness between second cousins, and 2% having an r_ab that exceeded relatedness between first cousins.

The overall effective population size of the remaining populations of Syngnathus watermeyeri was estimated at 199 individuals (95% CI: 153–280) using a 0.05 threshold for the lowest allele frequency, or 114 individuals (95% CI: 105–125) when a 0.02 threshold was used.

The estuarine pipefish, Syngnathus watermeyeri, is one of the rarest fish species in Africa, and the fact that extensive recent surveys have documented it in only two estuaries highlights the urgency of comprehensive conservation initiatives to reduce its extinction risk. Previous genetic research based on maternally inherited mitochondrial DNA found that genetic diversity in S. watermeyeri is low compared to that reported for the closely related congener S. temminckii Kaup, 1856, a species that is abundant throughout temperate South Africa and that also occurs in the Bushmans and Kariega estuaries (Whitfield et al., 2017). The present study based on more informative biparentally-inherited, genome-wide markers not only confirmed low genetic diversity in S. watermeyeri, but assessments of inbreeding, pairwise relatedness, and genetic structure indicate that the species’ extinction risk is considerably greater than was previously thought.

Population-level inbreeding (F_IS) and mean pairwise individual-based (F_ab) coefficients of inbreeding were significantly positive in both populations of S. watermeyeri, indicating a heterozygote deficiency resulting from mating between close kin. This result not only suggests that this species likely suffers from reduced fitness because of the accumulation of deleterious alleles (i.e., inbreeding depression), but also that its evolutionary potential to adapt to future ecological and climatic challenges is diminished (Frankham, 1997, 2005).

Genome-wide measures of relatedness are considered to be suitable alternatives to traditional pedigree-based estimates in wild populations for which long-term genealogical data that are required to reconstruct a pedigree are not easily obtainable (Lopes et al., 2013; Taylor, 2015). In cases where the individuals that gave rise to the present generation were themselves inbred, they are potentially even superior because pedigree-based methods assume that the founders are unrelated (Speed and Balding, 2015; Hanghøj et al., 2019).

Population-level inbreeding coefficients found in S. watermeyeri (F_IS = 0.10–0.12) were similar to those of several other threatened fish species, such as the Chinese sturgeon, Acipenser sinensis (F_IS = 0.148; Liu et al., 2018) and red-tailed barb, Gonoproktopterus curmuca (F_IS = 0.115; Musammilu et al., 2014), and they exceeded those of the Sichuan taimen, Hucho bleekeri (F_IS = 0.003; Zhang et al., 2020). The magnitude of mean coefficients of inbreeding based on pairs of individuals (F_ab) in S. watermeyeri (F_ab = 0.31–0.40) was similar to that reported for the endangered Pyrenean desman, Galemys pyrenaicus (0.33; Escoda et al., 2017), and mean relatedness (r_ab = 0.02–0.05) approached that for the critically endangered harlequin frog Atelopus varius (0.049; Byrne et al., 2021).

Not only are estimates for inbreeding and relatedness in S. watermeyeri high, but the estimated effective population size for the species is low. This suggests that the long-term population size is closer to the conservative IUCN estimate of ∼250 individuals rather than the thousands of individuals calculated by Whitfield et al. (2017), although it is possible that high census population sizes may be attained temporarily when conditions are favourable. Repeated population bottlenecks during the recent evolutionary history of S. watermeyeri may have eroded genetic diversity to the point of creating two genetically depauperate populations.

The absence of genetic structure between the two populations may indicate that levels of gene flow are sporadically high and/or ongoing, as the two estuaries are separated by a narrow 2.5 km stretch of coastline. However, the combination of low genetic structure and low genetic diversity may point to a recent founder event. Failure to find this species in the Kariega Estuary over several decades of regular sampling suggests potential recolonisation by individuals originating from either the populations that previously resided in the East and West Kleinemonde estuaries, or from the Bushmans Estuary, which was not regularly surveyed and where a small population may have survived unnoticed for several decades. Whitfield et al. (2017) hypothesised that occasional flooding may have facilitated the dispersal of S. watermeyeri between adjacent estuaries by washing individuals into the sea. Such flood events are sporadic (Jordaan et al., 2017) and, given the poor swimming ability of pipefishes and the erratic nature of the region’s nearshore currents, are unlikely to result in large numbers of estuarine pipefishes reaching suitable habitats in nearby estuaries (Whitfield et al., 2017). For the same reason, unauthorised translocations by well-intentioned citizen scientists cannot be ruled out to explain the presence of S. watermeyeri in the Bushmans and Kariega estuaries, but this is unlikely.

Barring gene flow from potentially unreported populations of S. watermeyeri, the current low level of genetic diversity is at risk of declining even further unless concrete conservation measures are taken. In addition to the fixation of deleterious alleles and the species’ reduced adaptive potential, stochastic events like floods, as well as pollution events, the introduction of diseases, alien predators, and competitors, can singly, or in combination, drive these pipefish populations to extinction. Perhaps the greatest threat to the estuarine pipefish in both the Bushmans and Kariega estuaries is a prolonged drought, together with the impact of catchment impoundments, that results in a complete loss of river flow for an extended period such that the zooplankton food chain which supports the pipefish population collapses (Whitfield et al., 2017).

Translocation of individuals represents a suitable management strategy when genetic diversity in individual populations is low (Whiteley et al., 2015), and the risk of outbreeding depression is not a valid concern in this case because of the evident lack of genetic structure between populations (Frankham et al., 2011). However, given the fact that both estuaries are essentially inhabited by the same inbred population, conservation efforts need to focus on preventing any further loss of genetic diversity. Although both populations potentially comprise thousands of individuals (Whitfield et al., 2017), floods can result in the partial or complete loss of macrophyte beds (Riddin and Adams, 2012; Whitfield et al., 2017), while prolonged droughts result in reductions in the abundance and diversity of zooplanktonic prey (Whitfield et al., 2017). As such, both floods and droughts can potentially result in very significant reductions in population size, and even localised extinction. Preventing the occurrence of prolonged periods during which the estuarine pipefish populations remain small appears to be the only means of reducing the risk that the remaining genetic diversity will be further reduced by inbreeding and genetic drift to a degree that may be detrimental for the species’ survival.

While conservation translocations do not represent a suitable means of improving the genetic health of the two remaining pipefish populations, they need to be considered to both maximise the species’ overall effective population size, and to minimise the impact of local extinctions on the erosion of the remaining genetic diversity. These aims could be achieved by reintroducing individuals to additional estuaries within the species’ historical range, in addition to captive breeding. The success of such conservation measures requires that the cause of past population declines and their impacts on the remaining populations be investigated, and that feasible steps be undertaken to mitigate such factors (Semlitsch, 2002; George et al., 2009; Ayres et al., 2012; Grant et al., 2019). In the case of the estuarine pipefish, these causes are primarily natural phenomena that cannot be readily controlled or prevented, although impoundments in the catchment areas of both the Bushmans and Kariega estuaries accentuate the climatic issues. Additional research about the species’ ecology, behaviour and dietary preferences may provide valuable information on how to identify suitable locations and times for release that will maximise the chances of successful rehabilitation of this species throughout its historical range.

The raw read datasets generated and analyzed in this study were submitted to the NCBI Sequence Read Archive (SRA) repository and are available under BioProject PRJNA777762 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA777762).

This animal study was reviewed and approved by the Faculty of Science Ethics Committee at the University of Johannesburg (Ethics Reference Number: 2020-02-06/Teske_Weiss).

PC, HK, AW, and PT conceived the research. PC and PT generated funding and obtained permits. S-EW, AE-K, PC, NJ, and PT participated in sampling. S-EW, AE-K, HK, and PT analysed the data. S-EW, HK, and NJ created figures. HK, BJvV, and PT supervised the student. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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