A New Record and Species of Pygmy Pipehorse of the Genus Cylix (Teleostei, Syngnathidae) from South Africa and the African Continent

The female specimen of C. nkosi (SAMC F041935), new species, was collected in 2018 by hand via scuba diving at 22 m depth in Sodwana Bay, KwaZulu-Natal, South Africa (Figs. 1–3). The specimen was subsequently fixed and preserved in 90% ethanol and deposited in the fish collection of the Iziko South African Museum (SAM), Cape Town, South Africa. The male specimen (SAIAB 39737, https://www.gbif.org/occurrence/1230372893), originally identified as Acentronura mossambica and reidentified by the author as Cylix nkosi, new species, was collected in 1987 by trawl in 50 m depth at the mouth of the Kosi River, Kosi Bay, KwaZulu-Natal, South Africa (Fig. 1). It is housed in the fish collection of SAIAB, Makhanda, South Africa.

Head and body measurements follow Short et al. (2018) and are expressed as a percent of standard length (SL) or head length (HL). Osteocranial and postcranial skeletal terminology follow Short and Trnski (2021). External morphological characters were documented using a dissecting microscope and high-resolution digital images, which were taken using a Canon E05 5D with a 50 mm lens. Computed tomography (CT) scans of select specimens were obtained to examine osteological characters of the axial skeleton of type specimens at the University of New England, NSW, Australia CT Scanner Facility using a GE v|tome|x S industrial micro-CT scanner with a voxel resolution (slice thickness) of 8 µm. The resulting CT scan data were visualized and rendered in Horos (https://www.horosproject.org).

Genomic DNA was extracted from the right eye of the 95% ethanol-fixed holotype of C. nkosi (SAMC F041935), new species, using a DNeasy Blood and Tissue Extraction Kit (Qiagen, Inc.) in accordance with the manufacturer’s protocols and initial tissue digestion overnight at 56°C. A shotgun library was prepared from the extracted DNA using an Illumina Nextera DNA Flex library prep kit and sequenced using a MiSeq and 2 × 150 v3 reagent kit at the Ramaciotti Centre for Genomics (UNSW Sydney, Australia).

The mitochondrial genome was assembled by initially mapping reads to a reference 655 bp COI fragment from Hippocampus kuda (GenBank: EU930325) in Geneious v 10.05 (https://www.geneious.com). When no reads could be further extracted from the shotgun dataset, sequences with a minimum average phred quality score of 30 were filtered out using BBDuk and used to assemble the complete mitochondrial genome with the De Novo Assemble function in Geneious. The mitogenome was annotated by comparison to the Hippocampus kuda mitochondrial genome (AP005985) and the Find ORFs function in Geneious using the Vertebrate Mitochondrial translation table. The complete mitochondrial genome of C. nkosi, new species, is available from GenBank under accession number OM022101.

Genetic distances (uncorrected p-distances) were calculated based on COI sequences using MEGA v. 7.0.26 (Kumar et al., 2008).

Cylix nkosi, new species
urn:lsid:zoobank.org:act:A645A185-D9E1-42B2-9347-FBA8BAA9EE26
Common Name: Sodwana Pygmy Pipehorse
Figures 1–9; Tables 1–3

SAMC F041935, 45.9 mm SL, female, 2 Mile Reef, Sodwana Bay, KwaZulu-Natal, South Africa, 27°30′46.6″S, 32°41′10.4″E, 22 m depth, 18 October 2018, L. Claassens, R. Smith, scuba diving.

SAIAB 39737, 46.6 mm SL, male, the mouth of the Kosi River, Kosi Bay, KwaZulu-Natal, South Africa, 26°53′54.0″S, 32°55′30.0″E, field station ZA 10, R.V. Meiring Nande, trawl, 50 m depth, 7 June 1987.

Cylix nkosi differs from C. tupareomanaia in possessing a supraoccipital bone bearing a highly derived and distinct diamond-shaped bony protuberance (SP; vs. cup-like bony protuberance; Table 3) oriented anteriorly, rhombus-shaped in anterodorsal view, divided transversally into two sections by a ridge; knob-shaped midventral spine (CSS; vs. udder-shaped bony protuberance; Table 3) on the cleithral symphysis; blunt midventral spine (MVFTRS; vs. conical midventral spine; Table 3) on the first trunk ring between the pectoral-fin bases; thick dorsomedial crest-like ridge (FTRC; vs. thin and rugose crest-like ridge; Table 3) extending over the length of the first trunk ridge; median frontal spine (FS) at convergence of anterior edges of the supraoccipital bone, anterior margin straight-edged; rugose double lateral head spine (DHS) present below supraoccipital v; three supraorbital extensions or spines (ORE), anterior two a double spine and equal in size, the posteriormost spine large and oblong in shape. The following characters are also diagnostic, although not unique to the species: cleithrum continuous middorsally; supracleithrum present; anterior and posterior nuchal plates absent, large gap present between the supraoccipital and cleithrum; small blunt midlateral spines proximally on operculum; two oval-shaped spines on cleithral ring, one spine (CS1) present on anterior to ventral third of pectoral-fin base, the second spine (CS2) present at the ventral extent of the head; small spine (PLS) present posterolaterally on the pelvic-fin base; four subdorsal spines, forming a square, the dorsal two enlarged, the ventral two small in size.

Meristic and morphometric characters for the type specimens are listed in Table 1. Superior trunk and tail ridges discontinuous below dorsal-fin base, lateral tail ridge present, inferior trunk ridge ends at the anal ring, lateral trunk ridge confluent with the inferior tail ridge. Body slender; head large relative to body, angled ventrally approximately 25° from the principal body axis, the dorsal profile pyramidal in lateral aspect, rising steeply from snout to elevated and prominent supraoccipital bone; supraoccipital bone bearing a highly derived and distinct diamond-shaped bony protuberance (SP) oriented anteriorly well behind the eye, rhombus-shaped in anterodorsal view, divided transversally into two sections by a ridge (Figs. 6–9; referred to as the supraoccipital crest in Short and Trnski, 2021); cleithrum continuous middorsally; supracleithrum present; anterior and posterior nuchal plates absent (though overlooked by Short and Trnski, 2021 in their description of C. tupareomanaia due to the highly derived morphology of the neurocranial bones, we now note the absence of the posterior nuchal plate represents a synapomorphy of Cylix); diamond-shaped bony protuberance on supraoccipital equal in height to cleithrum, forming a large gap between the two bones; thick dorsomedial crest-like ridge present on the first trunk ridge (FTRC) directly behind the cleithrum; gill opening anterior to and supported by the cleithrum; rim of orbit elevated dorsolaterally and strongly ventrally, fluted with rugose sculpturing; opercular ridge low, angled dorsally toward gill opening; swelling of gular region posteroventrally of eye, forming a transverse pair of blunt protuberances; pectoral-fin base without distinct ridges on a strongly elevated ventrolateral bulge; dorsal-fin origin on 12^th trunk ring, fin base elevated; trunk in lateral view narrowest at 1^st and 2^nd trunk rings where angle of head forms from body axis, broadest at 5^th trunk ring; principal body ridges distinct and moderately elevated; tail rings of uniform depth over most of length, becoming progressively shorter and smaller near posterior tip; tail prehensile; scutella not evident.

Enlarged spine (SnS1) on dorsal midline of snout on the ethmoid area, at confluence with the anterior ends of supraorbital ridges, its height extended well above level of nares; two small middorsal spines (SnS2, SnS3) on the mesethmoid bone anterior to the enlarged spine on the snout; three supraorbital extensions or spines (ORE), the anterior two spines a merged double spine and equal in size, the posteriormost spine large and oblong in shape; distinct median frontal spine (FS) at convergence of anterior edges of the supraoccipital bone, protruding forward, anterior margin of spine straight-edged; lateral head spines present, rugose double lateral head spine (DHS) directly below the supraoccipital protuberance, small blunt midlateral spines proximally on operculum; knob-shaped midventral spine (CSS) on the cleithral symphysis; blunt midventral spine (MVFTRS) on the first trunk ring between the pectoral-fin bases (Figs. 2, 7, 9); two oval-shaped spines on cleithral ring, moderate-sized spine (CS1) anterior to ventral third of pectoral-fin base, large lobe-shaped spine (CS2) at ventral extent of head; small spine present (PLS) posterolaterally on the pectoral-fin base (Figs. 2, 7); superior trunk ridge spines absent; lateral trunk ridges with small blunt spines on each trunk ridge starting at the 2^nd trunk ridge; inferior trunk ridges with small blunt spines starting on the 2^nd trunk ridge; subdorsal spines four, superior trunk ridge ending with two prominent subdorsal spines, superior tail ridge commencing with two small reduced subdorsal spines, in alignment with larger subdorsal spines above; superior tail ridge spines absent.

Male paratype SAIAB 39737 exhibits brood pouch dimorphism (Figs. 1, 4, 6). Brood pouch positioned along the ventral midline of the tail below the anteriormost six tail rings. It is enclosed by six arcuate bony pouch plates (Fig. 6), which extend ventrolaterally from the anterior ventral plate ridges of the tail, progressively reducing in size posteriorly. Second to fifth brood pouch plates consist of double arcuate bony extensions merged into one bony extension. Anterior- and posteriormost pouch plates consist of single bony extensions. The posteriormost pouch plate diminutive in size.

Holotype in life (Fig. 3), head, trunk, and tail reddish brown; ventrolateral margin of trunk brown; dorsum of head speckled with translucent red dots; snout white. Thin red filaments are present on the dorsum of the trunk. Other individuals of C. nkosi observed at the type location exhibit background coloration uniformly solid red, mustard yellow, or light brown, respectively (Fig. 4A–D), which apparently relates to the coloration of local sessile invertebrate growth. In alcohol, the holotype is dark brown in coloration (Fig. 1). Fins hyaline. Coloration of preserved paratype in Figure 1. Specimen lacks pigment in head, trunk, and tail, light tan in coloration.

Cylix nkosi is currently recorded from two locations in KwaZulu-Natal, South Africa: Sodwana Bay and Kosi Bay (Fig. 5). These two sites are situated within the boundaries of the iSimangaliso Wetland Park, a UNESCO World Heritage Site that extends from Lake St Lucia in the south to the Mozambique border in the north, encompassing the north coast of the KwaZulu-Natal province. The new species likely has a wider distribution along the tropical Indian Ocean coast of East Africa and remains undetected, perhaps attributable to its cryptic behavior and diminutive size, low abundance throughout its range, a rarity in the regions where surveys have been conducted, or misidentification as members of Acentronura or Hippocampus. Future ichthyofaunal surveys, as well as observations by recreational scuba divers, may confirm its occurrence in northern regions such as Mozambique, Tanzania, Kenya, and offshore areas towards Madagascar.

Africa’s southernmost coral reefs are situated in northern KwaZulu-Natal Province, South Africa, which are dominated chiefly by soft coral and branching stony corals of the genus Acropora and Pocillopora (Riegl et al., 1996; Schleyer and Celliers, 2002, 2003; Schleyer et al., 2005, 2008; Porter and Schleyer, 2017, 2019; Schleyer and Porter, 2018). The coast of KwaZulu-Natal is regularly subjected to open ocean and episodic storm swells that cause the breakage and dislodgement of these stony corals (Riegl et al., 1996; Schleyer and Porter, 2018; Porter and Schleyer, 2019). At the type locality in Sodwana Bay, the marine ecosystem consists of flat sandstone-based coral reefs in addition to unique topographic features comprising low pinnacles, shallow drop-offs, and sandy gullies (Ramsay, 1994). The holotype and other observed individuals of C. nkosi in situ (Fig. 4A–D) were observed in protected sandy basins in otherwise exposed areas with accumulated debris near flat rocky reefs and observed loosely associating with sandy worm casts, sponges, and small octocorals (Fig. 4), using the sessile invertebrates as a holdfast for their prehensile tails. The holotype was not observed in close proximity to other individuals of C. nkosi at the time of collection, although their cryptic nature may have prevented other individuals from being observed.

The species epithet is derived from the Nguni or Zulu term for chief due to the crown-like nature of the highly derived bony protuberance on the supraoccipital bone. A noun in the genitive. New English Names: Sodwana Pygmy Pipehorse is proposed here for Cylix nkosi.

Cylix nkosi and C. tupareomanaia share numerous derived characters, including angled head, rim of orbit elevated dorsad and ventrad, slender body, body ridge configurations, prominent supraoccipital, continuous cleithrum, midventral spines on the cleithral symphysis and on the first trunk ring between the pectoral-fin bases, dorsomedial crest-like ridge on the first trunk ridge, two spines on the cleithral ring, spine present posterolaterally on the pelvic-fin base, four subdorsal spines, presence of dimorphic brood pouch plates in males that consist of arcuate bony extensions located under the anteriormost tail rings, prehensile tail, and absence of a caudal fin (Figs. 6–9; Table 2). In addition, the two congeners share certain morphological features of the neurocranium, including a prominent supraoccipital bone bearing a highly derived bony protuberance and absence of anterior and posterior nuchal plates as revealed by micro-computed tomography (Figs. 6–8; Table 2).

Although exhibiting a high degree of morphological similarity in external appearance, a suite of anatomical features of the head and first trunk ring readily distinguish the two members of Cylix from each other (Figs. 6–9; Table 3), including the shape of the supraoccipital bony protuberance, which differs markedly in morphology in the form of complex anatomical shapes (diamond-like in C. nkosi vs. cup-like in C. tupareomanaia); the shape of the midventral spines on the cleithral symphysis (knob-like in C. nkosi vs. udder-like in C. tupareomanaia); the shape of the midventral spines on the first trunk ring between the pectoral-fin bases (blunt in C. nkosi vs. conical in C. tupareomanaia); the shape of the dorsomedial crest-like ridge on the first trunk ring (thick in C. nkosi. vs. short and rugose in C. tupareomanaia). The members can be further distinguished by the number and shape of the male brood pouch plates. Cylix nkosi possesses six plates in the form of double arcuate extensions, whereas C. tupareomanaia possesses eight plates in the form of single arcuate extensions (Fig. 6).

A total of 7,259,439 forward and reverse reads were recovered from the shotgun library of which 15,273 were used to assemble the mitogenome of C. nkosi (coverage: mean = 117.1, SD = 20.3). The mitogenome of C. nkosi is 16,534 bp, consisting of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region (D-loop). Table 4 summarizes uncorrected p-distances at the mitochondrial CO1 gene between C. nkosi and the previously sequenced pygmy pipehorses C. tupareomanaia, A. breviperula, A. tentaculata, I. australe, and I. lumnitzeri (Hamilton et al., 2017; Short and Trnski, 2021). Cylix nkosi differs from C. tupareomanaia by 10.0%, A. breviperula by 16.6%, A. tentaculata by 20.6%, I. australe by 18.1%, and I. lumnitzeri by 18.8%. According to the Reece et al. (2010) study, the reported mitochondrial DNA (mtDNA) clock rates in marine teleosts suggest a divergence rate of approximately 1.2% per million years. Applying this rate indicates that the divergence between C. nkosi and C. tupareomanaia may have occurred approximately 8.3 million years ago.

Cylix comprises two recognized species that occur in the Southern Hemisphere. Cylix nkosi occurs along the northeast coast of South Africa in the Western Indian Ocean, whereas C. tupareomanaia occurs off the North Island of Aotearoa New Zealand in the South Pacific (Short and Trnski, 2021). A representative of Cylix was recorded in Fiji, located in the Western Pacific, by the liveaboard scuba diving company Nai’a in 2010. These records can be found at the following online sources: https://inaturalist.ala.org.au/observations/34691744 and https://www.youtube.com/watch?v=RowELvDg7jY&ab_channel=Saminfiji. Furthermore, a recent observation of Cylix was recorded at Mafia Island, Tanzania in 2022, expanding the known distribution of the genus in eastern Africa (https://inaturalist.ala.org.au/observations/143646333). Similarly, a representative of Cylix was recently recorded in the Maldives in March 2023, marking the first confirmed observation of the genus in the Northern Hemisphere. (https://www.inaturalist.org/observations/150749541).

Although Cylix exhibits a wide distribution spanning the Indo-Pacific region, from the Western Indian Ocean to the Central and South Pacific, its members are at present recorded as short-range endemics restricted to their respective type localities. This restricted range is likely a result of morphological factors such as their diminutive size and adaptations for crypsis. Additional contributing factors may include geographical isolation, ocean currents, and specific habitat preferences (Smith, 1963; Briggs, 1999; Randall, 2001; Letourneur et al., 2004; Gordon et al., 2010). The distribution of Cylix encompasses three well-established marine biogeographic provinces, all recognized as biodiversity hotspots for nearshore marine ichthyofauna: the tropical western Indian Ocean (based on specimen records), Indo-Polynesian Provinces (based on photographic observations), and the temperate Auckland Province (based on specimen records; Smith, 1963; Briggs, 1974, 1999; Gordon et al., 2010; Briggs and Bowen, 2012; Kulbicki et al., 2013; Toonen et al., 2016). Marine biogeographic provinces of the Indo-Pacific, based on distinct marine floras and faunas, are defined by >10% endemism and recognized habitat discontinuities in coastal and shallow marine habitats (Briggs, 1974; Brown and Lomolino, 1998; Bellwood and Wainwright, 2002; Robertson et al., 2004; Floeter et al., 2008; Briggs and Bowen, 2012; Kulbicki at al., 2013; Bowen et al., 2016; Samoilys et al., 2022). In addition, Cylix spans both the warm-temperate and tropical regions of the Indo-Pacific, which distinguishes it from most of the 57 currently described syngnathid genera (Dawson, 1985; Fricke et al., 2023). Seven other genera, including Campichthys, Festucalex, Hippocampus, Maroubra, Nannocampus, Syngnathus, and Urocampus (Dawson, 1985; Kuiter, 2004; Short and Trnski, 2021; Araki and Motomura, 2022), share this distribution across both regions of the Indo-Pacific. It is important to note that Cylix was only identified within the past decade, so our knowledge of its distribution and behaviors remains limited.

The three members of Idiotropiscis are restricted to the Southern Hemisphere and recorded exclusively in Australia within the temperate Southeastern and tropical Southwestern Australian Provinces (Dawson, 1985; Kuiter, 2004; Briggs and Bowen, 2012; Toonen et al., 2016; Short and Trnski, 2021). Idiotropiscis lumnitzeri is a short-range endemic restricted to New South Wales (NSW) where it occurs from Bawley Point in the south to Cabbage Tree Bay, Manly. Idiotropiscis australe occurs over a broader range extending from the Fleurieu Peninsula in South Australia (https://www.inaturalist.org/observations/17644477, https://www.inaturalist.org/observations/87369954) to southern Western Australia (https://www.inaturalist.org/observations?place_id=6827&subview=map&taxon_id=89079; Dawson, 1985). Types specimens of I. larsonae are recorded in northern Western Australia (Dawson, 1984, 1985), within the southern extent of the Indo-Polynesian Province.

In contrast, members of Acentronura exhibit a wide distribution that extends across both the Northern and Southern Hemispheres. This genus occupies a significant range within the Indo-Pacific region, encompassing four distinct marine biogeographic provinces (Dawson, 1985; Short and Trnski, 2021). Acentronura breviperula is distributed throughout the Indo-Polynesian Province, extending into the northern boundaries of the Southeastern Australian Province (specifically, Stradbroke Island, QLD; Richard Smith, pers. obs., 2020). Acentronura tentaculata occurs within the Indo-Polynesian and Western Indian Ocean Provinces (Dawson, 1985), while A. gracilissima is restricted to the Sino-Japanese Province in central and southern Japan (Dawson, 1985). Given these findings, it is highly probable that targeted surveys within the aforementioned marine biogeographic provinces across the Indo-Pacific would uncover numerous additional species records for Cylix.

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We extend our sincere gratitude to the research colleagues who made invaluable contributions to this study. We would like to acknowledge the following individuals for their specific roles: Christo van Jaarsveld and Petro van Jaarsveld from SeaXplore dive centre and lodge, Sodwana Bay, for assisting with scuba logistics and identifying the holotype of C. nkosi; David Delport for providing field assistance; Peter Teske from the University of Johannesburg for assisting with collection permits; Michael Stat from the University of Newcastle, Callaghan, NSW, Australia, for his expertise in mitochondrial genome sequencing and assembly of C. nkosi; Christopher Goatley from the University of New England, Australia, for conducting μCT scans of the type specimens of C. nkosi; Adam Summers from Friday Harbor Laboratories, University of Washington, for granting access to the lab and CT visualization software; Albé Bosman, Collections Manager, Marine Biodiversity Collections, National History Department, Iziko South African Museum, for valuable curatorial assistance; Nkosinathi Mazungula and Roger Bills, Collections Manager, South African Institute for Aquatic Biodiversity, for their support in curatorial matters; iSimangaliso Wetland Park and Enzemvelo KZN Wildlife for granting the necessary authorizations for specimen collection; Amanda Hay, Kerryn Parkinson, and Sally Reader from the Department of Ichthyology, Australian Museum, for exceptional curatorial assistance. We express our appreciation for the generous support that contributed to the success of this research. A research permit was received from the iSimangaliso Wetland Park Authority for specimen collection in Sodwana Bay. Additionally, this research was conducted under a national permit (Permit no: RES2017/103 extension to 2018) approved by the South African Department of Environmental Affairs and obtained ethical clearance from the University of Johannesburg (Permit no: 2017-10-02). We acknowledge the funding received from the National Science Foundation (NSF; DBI-1759637, DEB-170166), awarded to Adam Summers at Friday Harbor Laboratories, University of Washington, which played a pivotal role in facilitating this study through access to the CT scanner. Furthermore, we are grateful for the financial support provided by the 2020–2021 AMF/AMRI Visiting Collections Fellowship from the Australian Museum in Sydney, Australia. We would also like to extend our appreciation to Daniel Roozen for his generous contribution toward covering the publication costs associated with this study.