Revision of the sixgill sawsharks, genus Pliotrema (Chondrichthyes, Pristiophoriformes), with descriptions of two new species and a redescription of P. warreni Regan

Recent sampling efforts in Madagascar and Zanzibar, as well as examinations of six-gilled sawsharks in several museum collections provided evidence for a complex of species within Pliotrema warreni Regan. The present manuscript contains a redescription of P. warreni involving the syntypes and additional material, as well as formal descriptions of two new species of Pliotrema Regan. All specimens of both new species were found in the western Indian Ocean. Individuals of the first new species, hereafter referred to as P. kajae sp. nov., were identified originating from Madagascar and the Mascarene Ridge. Specimens of the second new species, hereafter referred to as P. annae sp. nov., were only found off Zanzibar. Pliotrema kajae sp. nov. appears to inhabit upper insular slopes and submarine ridges at depths of 214–320 m, P. annae sp. nov. so far is only known from shallow waters (20–35 m). Both new species differ from P. warreni in a number of characteristics including the known distribution range and fresh coloration. Taxonomical differences include barbels that are situated approximately half way from rostral tip to mouth, with prebarbel length equidistant from barbel origin to symphysis of the upper jaw in P. kajae sp. nov. and P. annae sp. nov. (vs. about two thirds way from rostral tip to mouth, with prebarbel length about twice the distance from barbel origin to symphysis of upper jaw in P. warreni) and rostra that are clearly and slightly constricted between barbel origin and nostrils, respectively (vs. rostrum not constricted). Pliotrema kajae sp. nov. differs from P. annae sp. nov. in a longer snout, more numerous large lateral rostral teeth and upper jaw tooth rows, jaw teeth with (vs. without) sharp basal folds, and coloration, particularly pale to light brown (vs. medium to dark brown) dorsal coloration with (vs. without) two indistinct yellowish stripes. A revised diagnosis of Pliotrema and a key to the species are provided.

Introduction Pristiophoriform sharks (sawsharks) possess a flat, greatly elongated, and saw-like snout, which bears long ventral barbels and closely-set rows of lateral and ventral teeth [1][2][3]. Further, the anterior-most basiventral cartilages are laterally expanded and have curved, dorsally reflected margins [4,5]. Based on genetic analyses, previous studies have shown that the Pristiophoriformes form a clade with the Squaliformes and Squatiniformes but with the exclusion of the Hexanchiformes [6][7][8]. However, Naylor et al. [8] highlighted that the interrelationships between the Echinorhinidae, Pristiophoriformes, and Squatiniformes remain unclear due to weak support in the respective datasets. Interestingly, the current proposed interrelationships appear to differ significantly between published studies [9][10][11], highlighting the need to clarify understanding of the interrelationships of these groups. Regardless of the lack of detailed empirical data, there is morphological support for the interrelationships reported by Naylor et al. [6][7][8] for the Pristiophoriformes and Squatiniformes by, e.g. [12] based mainly on skeletal and myological features, as well as for the Pristiophoriformes and Squaliformes by, e.g. [3] based on characteristics of the claspers. However, Weigmann et al. [3] pointed out that, despite sharing many characteristics, the claspers of pristiophoriforms and squatiniforms differ greatly. These authors proposed that the similarity in clasper characters between pristiophoriforms and squaliforms could be regarded as a plesiomorphic character, whereas the differences in clasper morphology between pristiophoriforms and squatiniforms could be considered as autapomorphies, and the similarities in body shape and morphology of the first basiventral cartilages of the latter two orders as evidences of interrelationship.
Members of the monotypic genus Pliotrema Regan clearly differ from species of Pristiophorus Müller and Henle in their possession of six (vs. five) pairs of gill slits and serrated (vs. smooth-edged) large lateral rostral teeth [3]. Furthermore, Springer and Bullis [4] reported that the bases of the jaw teeth of species from the genus Pliotrema have 3 to 6 short but distinct ridges, somewhat stronger on the upper jaw teeth, whereas these ridges are indistinct or absent in Pristiophorus species. The only recorded species of Pliotrema, P. warreni Regan, has a known distribution from the southwestern Indian Ocean and southeastern Atlantic, and has been recorded at depths between 26 and 500 m [13,14]. It has been reported to reach a total length (TL) of up to 170 cm, making P. warreni the largest known species of sawshark [14,15]. Pristiophorus species are small to medium-sized, demersal sharks known from continental and insular shelves and upper slopes of tropical and temperate latitudes of all three major oceans, but with the center of distribution in the western Pacific Ocean and only one recently described, poorly known dwarf species in the western Indian Ocean [2,14]. Members of this genus attain maximum sizes of 62-153 cm TL and occur in depths between 0 and 1240 m [14].
While collecting data on elasmobranchs caught in small-scale fisheries in Madagascar, RHL collected two rostra of Pliotrema. Subsequently, EB, AJT and PB collected a specimen of Pliotrema from off Zanzibar during a 12-month fisheries landings observer program across the coastal regions of Kenya, Zanzibar, and northern Madagascar [16]. The rostra and the specimen from off Zanzibar turned out to represent two undescribed species of Pliotrema. During a search of museum collection, SW further identified a number of complete specimens of the same undescribed species represented by the two rostra, NJ and AJT managed to collect an additional specimen of the other new species from off Zanzibar, which was transported to the UK by PB. The availability of all these specimens enabled formal descriptions of both new species based on morphological, morphometric, and meristic data. The formal descriptions and a redescription of P. warreni based on the syntypes and additional specimens are presented herein; a revised generic diagnosis and a key to the species of Pliotrema are also given.

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org: pub:6281D5F4-DC2B-4E6D-B2F9-97734E545220. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Paratypes (15) 18 Oct 2008 (taken together with 1 further specimen, which was not retained); SAIAB 189447, 1 gravid female, 3 of 6 mid-to late-term embryos (1 male: 246 mm TL; 2 females, 320 mm TL, 324 mm TL; three embryos of 243 mm TL, 318 mm TL, and 329 mm TL were donated to the ZMH, BMNH and USNM collections, respectively), and 4 early embryos Diagnosis. A large six-gilled sawshark with the following characters: barbel origin to anterior nostrils 1.4-2.3 times anterior nostrils to symphysis upper jaw; prenarial length 1.5-1.7 times prebarbel length; preoral length 2.0-2.7 times interdorsal space; pectoral-fin anterior margin 1.2-1.6 times dorsal-caudal space; mouth width 2.8-6.6 times spiracle length. First dorsal fin originates about opposite pectoral-fin free rear tips. Lateral trunk dermal denticles tricuspidate, rather flat and imbricated. Color pale to light brown dorsally with two thin yellowish longitudinal stripes; uniform white ventrally; fins with rather indistinct white posterior fin margins; dorsal rostrum surface with two distinct longitudinal dark stripes, lateral rostral teeth dark-edged. Monospondylous centra 52-57; precaudal diplospondylous centra 48-56; total vertebral centra 151-164. This new species is distinguished from its two congeners, Pliotrema warreni and the second new species, by a combination of characteristics, including most notably, a rostrum that is clearly constricted between barbel origin and nostrils. Furthermore, P. kajae has sharp folds in both upper and lower jaw teeth, as well as a posteriorly notched, teardrop-shaped dorsal fenestra of the precerebral fossa. Pliotrema kajae is further distinguished from P. warreni by barbels that are situated about half way from rostral tip to mouth, with prebarbel length about equidistant from barbel origin to symphysis of upper jaw (vs. barbels about two thirds way from rostral tip to mouth, with prebarbel length about twice distance from barbel origin to symphysis of upper jaw) and the presence of two indistinct, yellowish longitudinal stripes on the dorsal surface (vs. one pronounced yellowish longitudinal stripe). Pliotrema kajae also clearly differs from the second new species in a generally longer snout, more upper and lower jaw tooth rows, higher total large lateral rostral tooth and ventral rostral spine counts, and a pale to light brown dorsal coloration with two indistinct yellowish stripes, uniform white ventral coloration, and posterior fin margins with narrow white edges (vs. uniform medium to dark brown dorsally without longitudinal stripes, white ventrally but with few indistinct dark blotches on belly, posterior fin margins conspicuously white-edged).
Description of the holotype. Values of the paratypes are presented in parentheses, more complex differences between holotype and paratypes are described separately. Where relevant, ratios are based on horizontal measurements unless otherwise stated. Morphometric measurements and meristics are given in Table 1.
Head narrow, subtriangular and deepest at sixth gill slit, strongly depressed above eyes, head width 6.7 (6.8-8.7)% TL, 1.3 (0.9-1.9) times head height. Snout forming a very elongate, blade-like rostrum. Rostrum triangular in dorsal view; constricted between barbel origin and nostrils, sides of rostrum nearly straight from tip to barbel origin but concave in posterior part from barbel origin to origin of orbit; tip narrowly rounded; rostrum extending laterally below eyes as a well-defined suborbital ridge along ventrolateral edge of head, terminating somewhat behind level of posterior edge of spiracle (Fig 3).
Large lateral rostral teeth of prenarial portion of rostrum variable in length, curved, rather stout, serrated, longest near barbel origin and near apex of rostrum posterior to anteriormost two teeth; longest tooth immediately anterior to barbels only slightly shorter than spiracle length, length 1.1 (0.9-2.3)% TL and 0.8 (0.9-2.6) times first complete interspace anterior to barbels, width 0.2 (0.2-0.4)% TL; anteriormost two large rostral teeth on each side of the rostrum very close to snout tip, without interstitial tooth between or anterior to them; large teeth shortest near nostrils, longest rostral tooth posterior to nostrils 0.6 (0.2-0.3)% TL; large teeth absent behind nostrils but interstitial-like teeth present, short to very short and closely set, partially directed almost ventrally, particularly near mouth. Interspaces between large rostral teeth rather regularly sized, about as long as adjacent teeth, with 2-4 (1-4) smaller, variable interstitial teeth. Rostral tooth counts mostly symmetrical between left and right hand sides; left side with 21 (22-31) large teeth, right side with 21 (22)(23)(24)(25)(26)(27)(28)(29)(30)(31); anterior to barbels left side with 13 (12)(13)(14) large rostral teeth, right side with 13 (13)(14), posterior to barbels left side with 8 (9-17) large rostral teeth, right side with 8 (9-17); anterior to nostrils left side with 23 (~19-~24) ventral spines, right side with 23 (~19-~23), anterior to barbel origin left side with 13 (~11-~14) ventral spines, right side with 12 (~12-~13); one enlarged ventral spine, distinctly larger than the other ventral spines, present just in front of each nostril. Large rostral teeth (Fig 4a and 4b) with elongated crown and oval-shaped base, slightly bent to the rear and flattened towards the apex, forming anterior and posterior cutting edges at front and rear, the latter serrated by barbed hooks. Crown base with numerous short longitudinal ridges forming a pronounced transversal crest. Both, anterior and posterior faces of the root are curved outwards from the junction of crown and root towards the base of the root. The basal face shows a deep v-shaped median groove that is antero-posteriorly directed and has an oval-shaped cavity in the center. Interstitial rostral teeth (Fig 4c-4h) with blade-shaped crown and without serration (large interstitial rostral teeth serrated and similar to large lateral rostral teeth in all specimens larger than the holotype). Crown of ventral spines (Fig 4i and 4j) elongated cone-shaped with a pronounced transversal basal ridge, root with roundish and pedestal-like base. The basal face has a large and deep roundish foramen in the center. Eyes lateral on head, large, oval, length 3.3 (2.8-5.2)% TL; skeletal interorbital space 0.9 (0.7-1.0) times eye length, 9.3 (6.1-9.5) times in horizontal preorbital length; posterior eye notches and suborbital grooves present. Spiracles moderately large, length 1.4 (0.8-1.6)% TL and 0.4 (0.2-0.6) times eye length, left spiracle with 12 (12)(13)(14)(15) folds, right one with 13 (12)(13)(14)(15); spiracles strongly crescentic, oblique, directed posteroventrally from top to bottom, located just posterior to posterior eye notch, separated by a narrow but deep vertical groove along posterior margin of orbit, shorter than eye; upper edge below level of top of eye. Gill slits small, upright, weakly pleated, lateral on head, close to ventral surface, extending slightly onto ventral surface, subequal in length, sixth slit arches around pectoral-fin origin. Mouth large, strongly inferior, broadly arched, symphysis about level with posterior edge of eye, width 4.5 (4.4-5.4)% TL and 1.5 (1.3-1.8) times in head width; upper labial furrows absent, lower furrows short, 0.4 (0.4-0.5)% TL; corner of mouth partly concealed by lateral muscles of jaw ( Fig  5). Teeth unicuspidate, in well-defined series, bases oval and flattened with short but pronounced, narrow median cusp near middle of jaw, no lateral cusps (cusps similar in paratypes except for adult male SAIAB 84096, which has distinctly longer cusps); cusps diminishing in height towards jaw angles, indistinct near jaw corners; about 4-5 series of functional teeth (Figs 6 and 7). Median cusp with labial face slightly convex and with both mesial and distal cutting edges weakly bent mesially and distally in occlusal view, respectively. The mesial and distal crown base parts somewhat curve apically. A pronounced and broad, irregularly shaped apron overlaps the junction of crown and root, building a notch at the junction with both mesial and distal crown base parts. Basal ornamentation, striae or reticulations absent, but sharp folds present in both upper and lower jaw teeth. The lingual face of the cusp is strongly convex, a well-developed uvula is present at the central crown base. The mesial/distal latero-lingual crown faces curve strongly towards the apex of the crown, partially forming a sharp notch with the uvula (Fig 6k and 6l). The root is anaulacorhizid and slightly arched without lobation. The outer surface of the root shows up to five large basal foramina, which are mostly oval-shaped. The inner face of the root shows up to six well-developed foramina along the crown-root junction at each side of the uvula. The basal face of the root is flat, partly showing some outer foramina.
Lateral trunk dermal denticles densely set and overlapping, with flat, tricuspidate crowns (Fig 8). The lateral cusps are rather weakly pronounced but situated quite far anteriorly so that the median cusp is not much longer than the lateral cusps. The median ridge is strongly pronounced and reaches the tip of the median cusp. The lateral ridges are less pronounced and do

PLOS ONE
not reach the tips of the lateral cusps. The surface of the denticles is only weakly structured by reticulations very close to base. No sexual dimorphism detectable in the morphology of the trunk dermal denticles. Dermal denticles on rostrum fan-shaped, with an obtusely angled, weakly pronounced median cusp and no lateral cusps but with 6-7 strongly pronounced ridges (Fig 9). The surface of the rostral dermal denticles is only weakly structured by reticulations very close to base (Fig 9a-9c).
Pectoral fins large, anterior margin weakly convex, 11.4 (10.3-12.2)% TL and 1.5 (1.4-2.0) times inner margin; apex narrowly rounded; posterior margin weakly concave, directed across horizontal axis at about origin of first dorsal fin; inner margin convex and strongly notched basally; free rear tip angular (Figs 3 and 10a). Pelvic fins moderately large, anterior margin almost straight to slightly convex, 6.5 (5.3-6.7)% TL, 1.6 (1.6-1.9) times in first dorsal-fin anterior margin, and 1.5 (1.3-1.7) times in second dorsal-fin anterior margin; apex narrowly rounded; posterior margin concave; inner margin weakly convex and slightly notched basally; free rear tip broadly rounded; origin distinctly posterior to level free tip of first dorsal fin and well forward of level second dorsal fin origin (Fig 10a).
First dorsal fin broad, semifalcate, anterior margin slightly convex; apex narrowly rounded; posterior margin slanting posteroventrally, slightly convex distally, strongly concave in basal three quarters; inner margin straight, free rear tip narrowly pointed; origin about opposite pectoral-fin free rear tips; insertion and free rear tip clearly anterior to level pelvic-fin origins (Fig  10a). Second dorsal fin somewhat smaller than first but of similar shape, anterior margin weakly convex, apex very narrowly rounded; posterior margin weakly convex distally, strongly concave near basal three quarters; inner margin straight, free rear tip narrowly pointed; origin clearly behind level pelvic insertions; interdorsal space 1.4 (1.3-1.7) times first dorsal-fin length, 1.7 (1.3-1.9) times dorsal-caudal space; second dorsal-fin inner margin 1.0 (0.7-1.2) times subterminal caudal-fin margin (Fig 10b).
Coloration. Fresh, prior to preservation (paratypes SAIAB 84039 and SAIAB 84096; Fig  13): ground color pale (SAIAB 84096) to light brown (SAIAB 84039) dorsally with two thin yellowish longitudinal stripes (hardly detectable in paratype SAIAB 84096); uniform white ventrally; fins translucent dusky, upper post-ventral caudal-fin and pelvic-fin posterior margins narrowly edged white, weak white edges also present at posterior margins of pectoral and dorsal fins, as well as terminal caudal-fin margin; rostrum translucent dusky, dark edged and with two distinct longitudinal stripes dorsally; lateral rostral teeth dark-edged; ventrolateral keels white. Color in preservative (holotype and paratypes; Figs 1 and 2): coloration similar to fresh coloration but specimen SAIAB 84096 with formerly pale dorsal coloration somewhat darker dorsally, ventral coloration uniform yellowish instead of white as usual, ventrolateral keels also yellowish; dark edging of rostrum and lateral rostral teeth, as well as longitudinal dorsal rostral stripes still conspicuous.
Size. A large sawshark species reaching about 1430 mm TL based on photographs of an adult female kindly provided by Blue Ventures along with photographs of an adult male of 1020 mm TL. The female was caught on 19 July 2015 and landed at Andranombala, Madagascar, whilst the male was caught on 22 September 2015 and landed at Andavadoaka, Madagascar. Both specimens were not preserved. The male paratype SAIAB 84096 is adult at 970 mm TL fresh, 940 mm TL preserved, the female paratype SAIAB 84039 is gravid at 1170 mm TL fresh, 1143 mm TL preserved, containing about six eggs (based on radiographs). The size at birth is estimated at around 350 mm TL based on the four near-term embryos of 318 to 329 mm TL.
Distribution. Known from off Madagascar and the Mascarene Ridge in depths from 214 to 320 m (Fig 14). The depth range of 425-500 m, given for the holotype of Pliotrema kajae sp. nov. by Séret [26] and Compagno et al. [27] is erroneous. Pliotrema kajae sp. nov. is apparently the only species of the genus occurring in this area.
Etymology. The new species is named after Kaja Magdalena Weigmann, the daughter of the first author, who had her first contact with chondrichthyan taxonomy when observing with great interest the examination of Pliotrema specimens for the present study. The name "Kaja" also has the Frisian meaning "warrior", referring to the saw-like rostrum.
Remarks. There are several morphometric differences between the embryos and the larger type specimens of Pliotrema kajae sp. nov., which might be of ontogenetic nature, most notably, the barbel length. The differences are demonstrated in Table 2. Further ontogenetic differences can be found in the morphology of the lateral interstitial rostral teeth. In specimens larger than the juvenile holotype, larger interstitial teeth are serrated, similar to the large lateral rostral teeth. In smaller specimens up to at least 560 mm TL all interstitial teeth are unserrated.  Table 3. Paratype ZMH 26362, presumably adult female, 980 mm TL fresh, 950 mm TL 70% ethanol preserved, caught off Zanzibar in a longline at~25-35 m depth during hours of darkness, landed on 24 Feb 2017 in Kizimkazi-Dimbani, Zanzibar (two further specimens were landed at the same place but not retained on 23 Jan 2017: gravid female,~980 mm TL, with six eggs and on 25 Jan 2017: female with saw cut off,~580 mm to beginning of saw; both these specimens were also caught in a longline at~25-35 m depth during hours of darkness). Measurements taken from the fresh photographs of the not retained gravid female show that this specimen can be assigned to P. annae sp. nov. based on the main morphometric characteristics, particularly the generally shorter snout. Diagnosis. A medium-sized six-gilled sawshark with the following characters: barbel origin to anterior nostrils 1.9-2.0 times anterior nostrils to symphysis upper jaw; prenarial length 1.6-1.7 times prebarbel length; preoral length 1.5-1.7 times interdorsal space; pectoral-fin anterior margin 1.4-1.5 times dorsal-caudal space; mouth width 2.7-3.2 times spiracle length. First dorsal fin originates about opposite pectoral-fin free rear tips. Lateral trunk dermal denticles tricuspidate, rather flat and imbricated. Color uniform medium to dark brown dorsally without longitudinal stripes; white ventrally but with few indistinct dark blotches on belly; fins with pronounced white posterior fin margins, particularly caudal and pectoral fins; dorsal rostrum surface with two distinct longitudinal dark stripes, lateral rostral teeth dark-edged. Monospondylous centra 53-54; precaudal diplospondylous centra 46-49; total vertebral centra 154. Pliotrema annae is distinguished from its two congeners by a combination of characteristics, including a generally shorter snout, with head length 34.2-34.5% TL, preorbital length 21.7-22.0% TL, preoral length 24.6-25.1% TL, prebarbel length 12.6-12.7% TL, and barbel origin to symphysis upper jaw 12.1-12.3% TL. Pliotrema annae further differs from its two congeners in lower total large lateral rostral tooth and ventral rostral spine counts, and a rostrum that is slightly constricted between barbel origin and nostrils. Like in P. kajae, the barbels are situated about half way from rostral tip to mouth, with prebarbel length about equidistant from barbel origin to symphysis of upper jaw. In contrast, the barbels are situated about two thirds way from rostral tip to mouth, with prebarbel length about twice distance from barbel origin to symphysis of upper jaw in P. warreni.
Description of the holotype. Values of the paratype are presented in parentheses, more complex differences between holotype and paratype are described separately. Where relevant, ratios are based on horizontal measurements unless otherwise stated. Morphometric measurements and meristics are given in Table 3.
Head narrow, subtriangular and deepest at sixth gill slit, strongly depressed above eyes, head width 6.4 (6.8)% TL, 1.1 (1.0) times head height. Snout forming a very elongate, bladelike rostrum. Rostrum triangular in dorsal view; slightly constricted between barbel origin and nostrils, sides of rostrum nearly straight from tip to barbel origin but slightly concave in posterior part from barbel origin to origin of orbit; tip narrowly rounded; rostrum extending laterally below eyes as a well-defined suborbital ridge along ventrolateral edge of head, terminating somewhat behind level of posterior edge of spiracle (Fig 17).
Large lateral rostral teeth of prenarial portion of rostrum variable in length, curved, rather stout, serrated, longest near barbel origin and near apex of rostrum posterior to anteriormost two teeth; longest tooth immediately anterior to barbels only slightly shorter than spiracle length, length 1.0 (1.0)% TL and 0.8 (0.9) times first complete interspace anterior to barbels, width 0.2 (0.2)% TL; anteriormost tooth close to tip of rostrum medium-sized, followed by a tiny tooth and the first large tooth; large teeth shortest near nostrils, longest rostral tooth posterior to nostrils 0.4 (0.3)% TL; large teeth absent behind nostrils but interstitial-like teeth present, short to very short and closely set, partially directed almost ventrally, particularly near mouth. Interspaces between large rostral teeth rather regularly sized, about as long as adjacent teeth, with 0-3 (1-3) smaller, variable interstitial teeth. Rostral tooth counts mostly symmetrical between left and right hand sides; left side with 17 (17) large teeth, right side with 17 (16); anterior to barbels left side with 10 (11) large rostral teeth, right side with 10 (10), posterior to  (15), anterior to barbel origin left side with 9 (10) ventral spines, right side with 9 (10); one enlarged ventral spine, distinctly larger than the other ventral spines, present just in front of each nostril. Large rostral teeth (Fig 18a and 18b) with elongated crown and oval-shaped base, slightly bent to the rear and flattened towards the apex, forming anterior and posterior cutting edges at front and rear, the latter serrated by barbed hooks. Crown base with numerous short longitudinal ridges forming a pronounced transversal crest. Both, anterior and posterior faces of the root are curved outwards from the junction of crown and root towards the base of the root. The basal face shows a deep v-shaped median groove that is antero-posteriorly directed and has an oval-shaped cavity in the center. Large interstitial rostral teeth similar but with somewhat less pronounced serration. Small interstitial rostral teeth (Fig 18c) with blade-shaped crown and without serration. Crown of ventral spines elongated cone-shaped with a pronounced transversal basal ridge, root with roundish and pedestal-like base. Eyes lateral on head, moderately large, oval, length 2.8 (2.7)% TL; skeletal interorbital space 0.9 (0.9) times eye length, 9.0 (8.7) times in horizontal preorbital length; posterior eye notches and suborbital grooves present. Spiracles moderately large, length 1.5 (1.3)% TL and 0.5 (0.5) times eye length, left spiracle with 10 (11) folds, right one with 10 (11); spiracles strongly crescentic, oblique, directed posteroventrally from top to bottom, located just posterior to posterior eye notch, separated by a narrow but deep vertical groove along posterior margin of orbit, shorter than eye; upper edge below level of top of eye. Gill slits small, upright, weakly pleated, lateral on head, close to ventral surface, extending slightly onto ventral surface, subequal in length, sixth slit arches around pectoral-fin origin. Mouth moderately large, strongly inferior, broadly arched, symphysis about level with posterior edge of eye, width 4.1 (4.3)% TL and 1.6 (1.6) times in head width; upper labial furrows absent, lower furrows very short, 0.3 (0.3)% TL; corner of mouth partly concealed by lateral muscles of jaw (Fig 19). Teeth unicuspidate, in well-defined series, bases oval and flattened with short but pronounced, narrow median cusp near middle of jaw, no lateral cusps; cusps diminishing in height towards jaw angles, indistinct near jaw corners; about 4-5 series of functional teeth (Fig 20). Median cusp with labial face slightly convex and with both mesial and distal cutting edges weakly bent mesially and distally in occlusal view, respectively. The mesial and distal crown base parts somewhat curve apically. A pronounced and broad, irregularly shaped apron overlaps the junction of crown and root, building a notch at the junction with both mesial and distal crown base parts. Basal ornamentation, striae, reticulations and folds absent in both upper and lower jaw teeth. The lingual face of the cusp is strongly convex, a well-developed uvula is present at the central crown base. The mesial/distal latero-lingual crown faces curve strongly towards the apex of the crown, forming a sharp notch with the uvula. The root is anaulacorhizid and slightly arched without lobation. The outer surface of the root shows large basal foramina, which are mostly oval-shaped. The inner face of the root shows well-developed foramina along the crown-root junction at each side of the uvula. The basal face of the root is flat, partly showing some outer foramina.
Lateral trunk dermal denticles densely set and overlapping, with flat, tricuspidate crowns (Fig 21). The lateral cusps are rather weakly pronounced but situated quite far anteriorly so that the median cusp is not much longer than the lateral cusps. The median ridge is strongly pronounced and reaches the tip of the median cusp. The lateral ridges are less pronounced and do not reach the tips of the lateral cusps. The surface of the denticles is only weakly structured by reticulations very close to base. Dermal denticles on rostrum fan-shaped, with an obtusely angled, weakly pronounced median cusp and no lateral cusps but with 6-7 strongly pronounced ridges (Fig 18d-18f). The surface of the rostral dermal denticles is only weakly structured by reticulations very close to base.
Pectoral fins very large, anterior margin weakly convex, 13.4 (12.7)% TL and 1.9 (1.9) times inner margin; apex narrowly rounded; posterior margin weakly concave, directed across horizontal axis at about origin of first dorsal fin; inner margin convex and strongly notched basally; free rear tip angular (Figs 17 and 22a). Pelvic fins large, anterior margin almost straight to slightly convex, 7.1 (7.0)% TL, 1.6 (1.6) times in first dorsal-fin anterior margin, and 1.4 (1.4) times in second dorsal-fin anterior margin; apex narrowly rounded; posterior margin concave; inner margin weakly convex and slightly notched basally; free rear tip broadly rounded; origin distinctly posterior to level free tip of first dorsal fin and well forward of level second dorsal fin origin (Fig 22a).
First dorsal fin broad, semifalcate, anterior margin slightly convex; apex narrowly rounded; posterior margin slanting posteroventrally, slightly convex distally, strongly concave in basal three quarters; inner margin straight, free rear tip narrowly pointed; origin about opposite pectoral-fin free rear tips; insertion and free rear tip clearly anterior to level pelvic-fin origins (Fig  22a). Second dorsal fin somewhat smaller than first but of similar shape, anterior margin weakly convex, apex very narrowly rounded; posterior margin weakly convex distally, strongly concave near basal three quarters; inner margin straight, free rear tip narrowly pointed; origin clearly behind level pelvic insertions; interdorsal space 1.5 (1.4) times first dorsal-fin length,  (Fig 22b).
Coloration. Fresh, prior to preservation (types and unretained specimens, Figs 23, 24 and 25): color uniform medium to dark brown dorsally without longitudinal stripes, white ventrally but with few indistinct dark blotches on belly; fins translucent dusky but with white posterior fin margins, particularly pronounced at the posterior pectoral-fin margin and the upper post-ventral and terminal caudal-fin margins; rostrum translucent dusky, dark edged and with two distinct longitudinal stripes dorsally; lateral rostral teeth dark-edged; ventrolateral keels white. Color in preservative (type specimens, Fig 16): coloration similar to fresh coloration, ventral ground coloration yellowish instead of white as usual but dark blotches still present, ventrolateral keels also yellowish; dark edging of rostrum and lateral rostral teeth, as well as longitudinal dorsal rostral stripes still conspicuous.
Size. A medium-sized sawshark species reaching about 981 mm TL. As one specimen of 980 mm TL (not retained) contained six eggs, the holotype and paratype are presumably also adult.
Distribution. Known only from off Zanzibar in depths of 20 to 35 m (Fig 14). All four known specimens of this new species were caught in these depths during hours of the https://doi.org/10.1371/journal.pone.0228791.g022 darkness / twilight. As both other species of Pliotrema usually occur in much deeper waters, P. annae sp. nov. possibly also occurs in deeper waters during the day but enters shallow water during the night. The area in which the specimens were caught is directly adjacent to a dropoff along the southern tip of Unguja Island. The water depth descends rapidly from~20 m to >200 m. Accordingly, deep-water sharks such as sixgill sharks and spurdogs are caught, alongside oceanic species such as mako and silky sharks and coastal species such as tiger and bull sharks, smoothhounds, and reef sharks all in the same fishery.
Pliotrema annae sp. nov. possibly also occurs off Kenya and/or Somalia following the short description of P. warreni in Gubanov [28]. Pliotrema annae sp. nov. is apparently the only species of the genus occurring in this area.  Diagnosis. A large six-gilled sawshark with the following characters: barbel origin to anterior nostrils 1.4-1.6 times anterior nostrils to symphysis upper jaw; prenarial length 1.3-1.4 times prebarbel length; preoral length 1.8-2.3 times interdorsal space; pectoral-fin anterior margin 1.4-1.5 times dorsal-caudal space; mouth width 3.1-3.9 times spiracle length. First dorsal fin originates about opposite pectoral-fin free rear tips. Lateral trunk dermal denticles tricuspidate, rather flat and imbricated. Color medium to dark brown dorsally with a pronounced yellowish longitudinal stripe; uniform white ventrally; dorsal rostrum surface with two distinct longitudinal dark stripes, lateral rostral teeth dark-edged. Monospondylous centra 53-56; precaudal diplospondylous centra 49-51; total vertebral centra 154-158. Pliotrema warreni clearly differs from both new species in a rostrum that is not constricted between barbel origin and nostrils and barbels that are situated about two thirds way from rostral tip to mouth, with prebarbel length about twice, i.e. 1.7-2.1 times, distance from barbel origin to symphysis of upper jaw (vs. barbels situated about half way from rostral tip to mouth, with prebarbel length about equal, i.e. 1.0-1.1 times, distance from barbel origin to symphysis of upper jaw), prebarbel length 60.2-68.0% vs. 49.4-52.9% of preoral length, preoral length 1.5-1.7 vs. 1.9-2.0 times prebarbel length, and prenarial length 1.3-1.4 vs. 1.5-1.7 times prebarbel length.

PLOS ONE
Description. The description is based on the intact syntype BMNH 1905.6.8.9, as well as the four specimens DMM I-E/4946, SAIAB 186452, SAIAB 189132, and SAIAB 208021. Where relevant, ratios are based on horizontal measurements unless otherwise stated. Morphometric measurements and meristics are given in Table 4.
Head narrow, subtriangular and deepest at sixth gill slit, strongly depressed above eyes, head width 6.3-7.1% TL, 1.2-1.3 times head height. Snout forming a very elongate, blade-like rostrum. Rostrum triangular in dorsal view; not constricted between barbel origin and nostrils, sides of rostrum nearly straight from tip to origin of orbit; tip narrowly rounded; rostrum extending laterally below eyes as a well-defined suborbital ridge along ventrolateral edge of head, terminating somewhat behind level of posterior edge of spiracle (Fig 27).
Large lateral rostral teeth of prenarial portion of rostrum variable in length, curved, rather stout, serrated, longest about half way from apex of rostrum to barbel origin; longest tooth immediately anterior to barbels shorter than spiracle length, length 0.5-1.3% TL and 0.5-1.7 times first complete interspace anterior to barbels, width 0.1-0.3% TL; anteriormost tooth close to tip of rostrum small, followed by the first large tooth; large teeth shortest near nostrils, longest rostral tooth posterior to nostrils 0.2-0.8% TL; large teeth absent behind nostrils but interstitial-like teeth present, short to very short and closely set, partially directed almost ventrally, particularly near mouth. Interspaces between large rostral teeth rather regularly sized, about as long as adjacent teeth, with 2-4 smaller, variable interstitial teeth. Rostral tooth counts mostly symmetrical between left and right hand sides; left side with~21-~34 large teeth, right side with~21-~34); anterior to barbels left side with~15-~17 large rostral teeth, right side with~14-~18, posterior to barbels left side with~6-~19 large rostral teeth, right side with~5-18; anterior to nostrils left side with~17-~24 ventral spines, right side with~16-~27, anterior to barbel origin left side with~11-~15 ventral spines, right side with~10-~19; one Table 4. Pliotrema warreni, morphometrics and meristics of the intact syntype BMNH 1905.6.8.9 and the four specimens DMM I-E/4946, SAIAB 186452, SAIAB 189132, and SAIAB 208021, as well as ranges and means for the five specimens. Proportional values are expressed as percentages of total length (TL) 70% ethanol preserved except for minimum, maximum, and mean of TL in mm.        enlarged ventral spine, distinctly larger than the other ventral spines, present just in front of each nostril. Large rostral teeth (Fig 28a-28c) with elongated crown and oval-shaped base, slightly bent to the rear and flattened towards the apex, forming anterior and posterior cutting edges at front and rear, the latter serrated by barbed hooks. Crown base with numerous short longitudinal ridges forming a pronounced transversal crest. Both, anterior and posterior faces of the root are curved outwards from the junction of crown and root towards the base of the root. The basal face shows a deep v-shaped median groove that is antero-posteriorly directed and has an oval-shaped cavity in the center. Large interstitial rostral teeth similar but with somewhat less pronounced serration in specimens of 704 mm TL (heavily dissected syntype BMNH 1899.2.10.4) or larger. Large interstitial rostral teeth without serration in specimens of width; upper labial furrows absent, lower furrows short, 0.5-0.6% TL; corner of mouth partly concealed by lateral muscles of jaw (Fig 27). Teeth unicuspidate, in well-defined series, bases oval and flattened with short but pronounced, narrow median cusp near middle of jaw, no lateral cusps; cusps diminishing in height towards jaw angles, indistinct near jaw corners; about  : a,b,d 1 mm, c, 4-5 series of functional teeth (Fig 29). Median cusp with labial face slightly convex and with both mesial and distal cutting edges weakly bent mesially and distally in occlusal view, respectively. The mesial and distal crown base parts somewhat curve apically. A pronounced and broad, irregularly shaped apron overlaps the junction of crown and root, building a notch at the junction with both mesial and distal crown base parts. Basal ornamentation, striae or reticulations absent, sharp folds present in upper but absent in lower jaw teeth. The lingual face of the cusp is strongly convex, a well-developed uvula is present at the central crown base. The mesial/distal latero-lingual crown faces curve strongly towards the apex of the crown, forming a sharp notch with the uvula. The root is anaulacorhizid and slightly arched without lobation. The outer surface of the root shows up to four large basal foramina, which are mostly ovalshaped. The inner face of the root shows up to six well-developed foramina along the crownroot junction at each side of the uvula. The basal face of the root is flat, partly showing some outer foramina. Nostrils small, widely separated, subcircular; nostril width 0.6-1.1% TL, 3.1-5.4 times in internarial width, 4.4-6.8 times in mouth width, 5.9-9.2 times in width of rostrum at nostrils; located distinctly forward of level of anterior margin of eye; distance from anterior nostrils to symphysis of upper jaw 1.2-1.4 times internarial space, distance from barbel origin to anterior nostrils 5.5-6.6)% TL. Anterior nasal flaps well developed, leaf-like, extended ventrally beyond nostrils; incurrent and excurrent apertures surrounded by pronounced marginal lobes; no nasoral or circumnarial grooves; no dermal lobes (Fig 27).

Pliotrema
Lateral trunk dermal denticles densely set and slightly overlapping, with flat, tricuspidate crowns (Fig 28g and 28h). The lateral cusps are rather weakly pronounced but situated quite far anteriorly so that the median cusp is not much longer than the lateral cusps. The median ridge is strongly pronounced and reaches the tip of the median cusp. The lateral ridges are less pronounced and rarely reach the tips of the lateral cusps. The surface of the denticles is only weakly structured by reticulations close to base. Dermal denticles on rostrum fan-shaped, with an obtusely angled, weakly pronounced median cusp and no lateral cusps but with 6-7 strongly pronounced ridges. The surface of the rostral dermal denticles is only weakly structured by reticulations very close to base.
Pectoral fins large, anterior margin weakly convex, 10.7-12.2% TL and 1.3-1.6 times inner margin; apex narrowly rounded; posterior margin weakly concave, directed across horizontal axis at about origin of first dorsal fin; inner margin convex and strongly notched basally; free rear tip angular (Fig 26). Pelvic fins moderately large, anterior margin almost straight to slightly convex, 5.8-6.8% TL, 1.5-1.8 times in first dorsal-fin anterior margin, and 1.4-1.6 times in second dorsal-fin anterior margin; apex narrowly rounded; posterior margin concave; inner margin weakly convex and slightly notched basally; free rear tip broadly rounded; origin distinctly posterior to level free tip of first dorsal fin and well forward of level second dorsal fin origin (Fig 26).
First dorsal fin broad, semifalcate, anterior margin slightly convex; apex narrowly rounded; posterior margin slanting posteroventrally, slightly convex distally, strongly concave in basal three quarters; inner margin straight, free rear tip narrowly pointed; origin about opposite pectoral-fin free rear tips; insertion and free rear tip clearly anterior to level pelvic-fin origins ( Fig  26). Second dorsal fin somewhat smaller than first but of similar shape, anterior margin weakly convex, apex very narrowly rounded; posterior margin weakly convex distally, strongly concave near basal three quarters; inner margin straight, free rear tip narrowly pointed; origin clearly behind level pelvic insertions; interdorsal space 1.4-1.6 times first dorsal-fin length, 1.6-1.9 times dorsal-caudal space; second dorsal-fin inner margin 0.8-1.1 times subterminal caudal-fin margin (Fig 26).
Coloration. Fresh, prior to preservation (ERB 1105, ERB 1106, SAIAB 208021 and Uncatalogued; Fig 30): ground color medium to dark brown dorsally with a pronounced yellowish longitudinal stripe; uniform white ventrally; fins translucent dusky, upper post-ventral caudalfin and pelvic-fin posterior margins narrowly edged white, weak white edges also present at posterior margins of pectoral and dorsal fins, as well as terminal caudal-fin margin; rostrum translucent dusky, dark edged and with two distinct longitudinal stripes dorsally; lateral rostral teeth dark-edged; ventrolateral keels white. Color in preservative (other material examined): coloration similar to fresh coloration but yellowish longitudinal dorsal stripe not detectable in all specimens, particularly after long-time storage in ethanol; ventral coloration uniform yellowish instead of white as usual, ventrolateral keels also yellowish; dark edging of rostrum and lateral rostral teeth still pronounced in most specimens but hardly detectable in the intact syntype which is more than 100 years old; longitudinal dorsal rostral stripes still conspicuous in all specimens including the intact syntype. Fresh photographs of one specimen caught off Mozambique and kindly provided by Oddgeir Berg Alvheim, as well as a photograph of one specimen from off South Africa, taken and kindly provided by Frederik Mollen, Elasmobranch Research Belgium, are shown in Fig 30. Size. A large sawshark species reaching at least 1360 mm TL but possibly attaining 1700 mm TL [14,15]. Males are adolescent at 700 to 740 mm, mature at 830 mm and grow to at least 1120 mm TL, females are adolescent at around 950 to 1100 mm TL, are mature when over 1100 mm TL and attain at least 1360 mm TL [30]. The male specimen ERB 1106 is subadult at 945 mm TL. The size at birth is about 350 mm TL, the litter size 5-7 pups, but up to 17 developing eggs recorded [18,30].
Distribution. Known from off South Africa and southern Mozambique in depths from 26 to 500 m (Fig 14). However, the maximum depth of 500 m is apparently based on erroneous data for the holotype of Pliotrema kajae sp. nov., indicating that the verified maximum depth of P. warreni was 430 m [30]. Nevertheless, P. warreni can be found in waters shallower and deeper than this based on specimens ERB 1105 (caught in 10-25 m depth) and one specimen from 915 m depth in the iSAM collection (SAM 33308, catch location 35.035˚S 24.0217˚E). Accordingly, the updated depth range for P. warreni is 10-915 m, albeit the species is usually found in 60-430 m depth [27,30]. Pliotrema sp. possibly occurs down to 1080 m depth [28] but it is impossible to assign this maximum depth to a certain species nor any verified specimen of Pliotrema. The northernmost verified records of P. warreni are from off southern Mozambique at about 22˚S latitude.
Remarks. There are several morphometric differences between the small and large examined specimens of Pliotrema warreni, which might be of ontogenetic nature. However, the number of specimens and coverage of different sizes is too small to reliably detect ontogenetic differences. Nevertheless, like in P. kajae, the total number of large lateral rostral teeth and the number of large lateral rostral teeth posterior to barbels clearly differ between the smaller (405.9-456.4 mm TL) and larger (785-925 mm TL) specimens of P. warreni (total number 34-34/34-34 vs. 21-23/21-23; number posterior to barbels 18-19/18-18 vs. 6-6/ 5-7). Like in P. kajae, the large interstitial rostral teeth are serrated in large specimens of P. warreni (704 mm TL or larger), whereas all interstitial teeth are unserrated in specimens of 456.4 mm TL or smaller.

Discussion
The three species of Pliotrema are apparently allopatric with the distribution of P. kajae being restricted to Madagascar and the Mascarene Ridge, P. annae known only from off Zanzibar, and P. warreni occurring off South Africa and southern Mozambique. They can further be differentiated based on numerous morphological, morphometric, and meristic characteristics.
Both new species differ from Pliotrema warreni in barbels that are situated about half way from rostral tip to mouth, with prebarbel length about equal to distance from barbel origin to symphysis of upper jaw (1.0-1.1 times in P. kajae and 1.0 times in P. annae) vs. barbels about two thirds way from rostral tip to mouth, with prebarbel length about twice, i.e. 1.7-2.1 times, distance from barbel origin to symphysis of upper jaw in P. warreni, prebarbel length 49.4-52.9% of preoral length in P. kajae and 50.7-51.1% in P. annae vs. 60.2-68.0% in P. warreni, preoral length 1.9-2.0 and 2.0 vs. 1.5-1.7 times prebarbel length, prenarial length 1.5-1.7 and 1.6-1.7 vs. 1.3-1.4 times prebarbel length.
The Pliotrema warreni syntype BMNH 1905.6.8.9 (Figs 26 and 27) was used as reference for characterizing the true P. warreni. The heavily dissected P. warreni syntype BMNH 1899.2.10.4 (Fig 31) could be identified as the same species despite its condition (only skeletal parts and remains of flesh and fins still exist), based on the measurements of horizontal prebarbel length (138 mm) and an estimation for the distance barbel origin to the symphysis of the (missing) upper jaw (~74 mm), resulting in a ratio of 1.9. This value is in line with P. warreni and clearly different from P. kajae and P. annae. The identification of the heavily dissected syntype is further evidenced by its catch location (False Bay, Cape of Good Hope), where both new species apparently do not occur. The identity of Pliotrema specimens occurring off Mozambique and, thereby, the possible presence of one of the new species in this area was investigated by examination of specimens from the DMM, iSAM, and USNM collections. As all specimens examined were clearly identified as P. warreni based, amongst other things, on the rostral morphology, it is assumed that P. kajae and P. annae do not occur off Mozambique and have a distribution allopatric to that of P. warreni. Nevertheless, the occurrence of P. annae off Zanzibar, close to the African mainland, indicates that the species may be distributed along the eastern African coast and overlap with P. warreni appears possible. It remains unclear if Pliotrema indeed occurs off Kenya and/or Somalia at all, as described for P. warreni in Gubanov [28]. Due to the proximity to Zanzibar it is conceivable that Pliotrema (i.e. P. annae) might be found in these areas but nevertheless would likely be very rare. Pliotrema annae appears to be rare also off Zanzibar and P. kajae also appears to be rare within its distribution area. The great catch depth of 915 m of one single specimen of P. warreni in the iSAM collection (and the possible maximum depth of 1080 m from Gubanov [28]) indicates that Pliotrema possibly enters depths beyond most fishing operations but such records appear to be very rare and both new species are likely affected by fishing operations in most of their bathymetric ranges. This assumption combined with the limited range and apparent rarity of both new species raises concerns that they are vulnerable to overfishing and might be in continuing decline, as has been previously suggested for P. warreni [2]. This could be particularly alarming for P. annae due to its very small known range, rarity and occurrence in shallow waters (the species is only known from depths of 20 to 35 m).