The nudibranch genus Halgerda Bergh, 1880a is one of 29 recognized genera in the family Discodorididae Bergh, 1891 and has been extensively studied using morphology (Rudman 1978;Carlson andHoff 1993, 2000;Gosliner and Fahey 1998;Fahey and Gosliner 1999a, b, 2000, 2001a, b;Fahey and Healy 2003;Fahey and Carroll 2007). Currently, there are 42 recognized species of Halgerda (MolluscaBase 2021), which are predominantly found in tropical and subtropical regions on shallow reefs (Eliot 1904;Rudman 1978;Gosliner 1987;Fahey and Gosliner 2001b;Tibiriçá et al. 2018). Additional species have also been found in temperate regions (Carlson and Hoff 2000;Fahey and Gosliner 2001a) and the deep-sea including the mesophotic zone (Carlson and Hoff 2000;Tibiriçá et al. 2018) and the twilight zone (Fahey and Gosliner 2000). Furthermore, numerous undescribed species of Halgerda have been identified and range from the Indo-Pacific to the western Indian Ocean, including the Red Sea (Ono 2004;Debelius and Kuiter 2007;Yonow Communicated by C. Chen This article is registered in ZooBank under http:// zooba nk. org/ C7A7F CC8-D9F2-4A96-8D99-285FC 2BB99 97 2008;Hervé 2010;Humann and Deloach 2010;Gosliner et al. 2008Gosliner et al. , 2015Gosliner et al. , 2018)).
The genus Halgerda is characterized by having a semifirm, gelatinous body with ridges, tubercles, and no caryophyllidia; a low, smooth rhinophoral and branchial sheath; an unarmed penis and vagina; and a smooth labial cuticle and smooth, hamate inner and middle radular teeth with denticulated outer teeth (Bergh 1880a;Fahey and Gosliner 2001a). Differentiation between Halgerda species usually includes external morphology (i.e., dorsum coloration, presence of tubercles, number of branchial leaves), but due to variability in coloration as seen in Halgerda dichromis Fahey & Gosliner, 1999a and the Halgerda wasinensis Eliot, 1904 species complex, internal differences in the reproductive system (i.e., size and shape of the penis, vagina, and bursa copulatrix) and the radular teeth (i.e., number of small inner teeth and the shape of the outermost teeth) prove helpful in species identifications.
The first morphological phylogeny for Halgerda was proposed in 1999 and utilized 22 species and 48 morphological characters (Fahey and Gosliner 1999b). In 2001, Fahey and Gosliner revised their morphological phylogeny by including an additional morphological character and increasing the representation to 35 species of Halgerda. Since then, two molecular phylogenies have been published for Halgerda. In the first, Fahey (2003) combined morphology with the mitochondrial gene cytochrome oxidase I (COI) from 17 specimens representing 13 species from the central and western Pacific Ocean and Western Australia. In the second molecular study, Tibiriçá et al. (2018) sequenced three genes (COI, 16S, and histone 3 (H3)) from 32 specimens including six new species of Halgerda collected in the western Indian Ocean. Both molecular phylogenies show similar well-supported groupings; however, Tibiriçá et al. (2018) also found two species complexes with strong geographical separation: the H. wasinensis complex from the western Indian Ocean and the Halgerda carlsoni Rudman, 1978 complex predominantly from the Pacific Ocean and Western Australia. Here, we provide an updated phylogenetic analysis of the genus Halgerda with additional representatives from the western Indian Ocean, the Red Sea, and the central and western Pacific Ocean.
Ninety-nine Halgerda specimens representing 11 of 14 new species described here and 25 of 42 previously described species collected from the Central and Western Pacific Ocean and the Indian Ocean (Fig. 1) were sequenced for this study. Some specimens including the holotypes of Halgerda berberiani Donohoo & Gosliner sp. nov., Halgerda hervei Donohoo & Gosliner sp. nov., Halgerda labyrinthus Donohoo & Gosliner sp. nov., and Halgerda radamaensis Donohoo and Gosliner sp. nov. were not sequenced due to DNA degradation from prior fixation in formalin or Bouin's solution. Some Halgerda specimens initially sequenced by Fahey (2003) were resequenced to achieve longer cytochrome oxidase I (COI) fragments as well as the other genes studied here. Therefore, a total of 128 specimens, 70 newly sequenced, 12 originally from Fahey (2003), and 46 with two or more genes already published and available on Genbank, were used in the molecular analyses. Sampled specimens with the current proposed taxonomy, museum voucher numbers, locality, and GenBank accession numbers are listed in Table 1. Outgroup comparisons included Goniodorididae, Dorididae, and several members of Discodorididae based on molecular phylogenetic analysis by Hallas et al. (2017) and Donohoo and Gosliner (2020). Voucher specimens, holotypes, and paratypes are deposited in the collections at the California Academy of Sciences (CASIZ), the National Museum of Philippines (NMP), the National Museum of Natural History (MNHN), the Iziko Museum South African Museum (SAMC), and the Florida Museum of Natural History (UF).
DNA extractions were performed on a small tissue sample from each specimen's foot or mantle using the Qiagen Dneasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) spin column extraction method. Fragments from two mitochondrial (16S and COI) genes and two nuclear (28S and H3) genes were utilized to estimate our Halgerda phylogeny based upon previous sequencing success for members of Discodorididae including Asteronotus Ehrenberg, 1831; Carminodoris Bergh, 1889;Diaulula Bergh, 1878;Halgerda, Hoplodoris Bergh, 1880b;Jorunna Bergh, 1876;Sclerodoris Eliot, 1904;andThordisa Bergh, 1877 (Fahey 2003;Giribet et al. 2006;Göbbeler and Klussmann-Kolb 2010;Lindsay et al. 2016;Hallas et al. 2017;Tibiriçá et al. 2018;Donohoo and Gosliner 2020;Neuhaus et al. 2021). Each polymerase chain reaction (PCR) used gene-specific primers (Table 2) and contained the following: 2.5 μL of 10 × PCR buffer, 0.5 μL dNTPs (10 mM stock), 0.5 μL of each primer (10 μM stock), 0.25 μL DreamTaq™ Hot Start DNA Polymerase (5U/μL, Thermo Fisher), 5 μL betaine, 2 μL bovine serum albumin (BSA), 2-4 μL of template DNA, and then filled to a final volume of 25 μL with Millipore-H 2 O. An additional 1 μL of dimethyl sulfoxide (DMSO) was added to both 28S PCR amplifications to account for secondary structure and nucleotide repeats. The PCR gene-specific protocols were run on a BioRad MyCycler Thermocycler (Bio-Rad Laboratories) at the California Academy of Sciences Center for Comparative Genomics (CCG). PCR protocols are as follows: for 16S and COI, an initial denaturing for 3 min at 94 °C, followed by 40 cycles of denaturing for 30 s at 94 °C, annealing for 30 s at 46-50 °C, and extension for 45 s at 72 °C with a final extension period of 10 min at 72 °C and for H3, an initial denaturing for 3 min at 94 °C, followed by 35 cycles of denaturing for 30 s at 94 °C, annealing for 30 s at 45-54 °C, and extension for 1 min at 72 °C with a final extension period of 10 min at 72 °C. Both fragments of 28S were initially denatured for 4 min at 95 °C, followed by 40 cycles of denaturing for 30 s at 94 °C, annealing for 30 s at 52.5 °C, and extension for 2.5 min at 72 °C with a final extension period of 5 min at 72 °C. Amplified DNA was stained with ethidium bromide and examined using gel electrophoresis on a 1% TBE agarose gel. Successfully amplified products were cleaned using an ExoSAP-IT protocol (USB Scientific) before being sequenced at either ELIM Biopharmaceuticals (Hayward, CA, USA) or on an ABI3130 Genetic Analyzer in the CCG, which followed fluorescently labeled dye terminator protocols used by Donohoo and Gosliner (2020).
Successfully sequenced fragments were assembled, trimmed to remove primers, and edited using Geneious v11.1.5 (Kearse et al. 2012) and Mesquite v3.61 (Maddison and Maddison 2018). Each gene was aligned with MAFFT (Katoh et al. 2009) using the algorithm E-INS-I. Regions of ambiguous data were identified using the least stringent settings and then removed from the 16S and 28S alignments using GBlocks 0.91b (Talavera and Castresana 2007). Bayesian inference (BI) and maximum likelihood (ML) analyses were used to estimate the evolutionary relationships within Halgerda. Best-fit evolution model partition definitions for BI and ML analyses were determined for the individual genes 16S and 28S and each codon position within COI and H3 within the final four-gene concatenated dataset (16S + 28S + COI + H3) using Partition-Finder2 (Lanfear et al. 2016). The concatenated dataset was partitioned by gene and codon position for both the BI and ML analyses (Table 2). Bayesian inference was performed in MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003), and the dataset was run for 5 × 10 7 generations. Markov chains were sampled every 1000 generations, and the standard 25% burn-in was calculated before checking the convergence of the two chains using TRACER v1.7.1 (Drummond and Rambaut 2007). A 50% majority rule consensus tree of calculated posterior probabilities (pp) was created from the remaining tree estimates. Maximum likelihood was performed using randomized accelerated maximum likelihood (RAxML) v8.2.12 (Stamatakis 2014). Non-parametric bootstrap values (bs) were estimated from 5 × 10 4 fast bootstrap runs set with the evolution model GTR + GAMMA + I. Final trees were collapsed (pp ≥ 0.95) in TreeGraph 2 v2.15.0 (Stöver and Müller 2010) before final editing in FigTree v1.4.4 (Rambaut 2018) and Adobe Photoshop 2022 (San Jose, CA, USA). Tree branches were considered supported when posterior probability values were ≥ 0.95 and bootstrap values were ≥ 70 (Alfaro et al. 2003).
Species were delimited using four different approaches: (i) Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al. 2012), (ii) Assemble Species by Automatic Partitioning (ASAP) method (Puillandre et al. 2021), (iii) Bayesian Poisson Tree Process (bPTP) by Zhang et al. 2013), and (iv) General Mixed Yule Coalescent (GMYC) model (Pons et al. 2006;Fujisawa and Barraclough 2013). The ABGD method uses genetic pairwise distances to detect breaks between intraspecific and interspecific variation. An ingroup COI alignment and an ingroup 16S alignment were created in Mesquite v3.51 and uploaded to the ABGD Web-based interface (https:// bioin fo. mnhn. fr/ abi/ public/ abgd/ abgdw eb. html). We tested Jukes-Cantor (JC69), Kimura (K80), and Simple Distances as well as different gap widths to evaluate which settings were congruent with our own phylogenetic and morphological analyses (Kekkonen et al. 2015;Tibiriçá et al. 2018). The following parameters were applied for the COI ingroup: Kimura80 (K80) P.min = 0.001, P.max = 0.2, Steps = 10, NB = 20 with a relative gap width × = 1.3. The 16S ingroup used similar parameters; however, the relative gap width × was set to 1.0.
The ASAP method also uses genetic pairwise distances; however, it does not require any prior species hypotheses, and it also provides a score for each suggested partition. The previously used COI and 16S ingroup alignments were uploaded to the ASAP Web-based interface (https:// bioin fo. mnhn. fr/ abi/ public/ asap/ asapw eb. html) and tested with the following settings: Jukes-Cantor (JC69), Kimura (K80), and Simple Distances. Bayesian PTP uses a previously inputted phylogenetic tree to model the number of substitutions between branchings and uses Bayesian MCMC methods to identify groups descended from a single ancestor. This test was performed using the 16S + COI concatenated BI tree on the bPTP server (https:// speci es.h-its. org/) with the following parameters: 500,000 generations, 100 thinning, 0.1 burn-in, and 123 seeds. Convergence was checked using the ML convergence plot generated by the bPTP server.
The GMYC model is a likelihood-based method that assumes species-independent evolution, which results in differences between branching rates (i.e., diversification between species) that can then be delimited using a Yule model (Fujisawa and Barraclough 2013). We used BEAST package v 1.10.4 (Drummond et al. 2012) to estimate a COI ultrametric tree using the following priors defined in BEAUTi v1.10.4: GTR + GAMMA + I, yule speciation process, uncorrelated lognormal relaxed clock, and 10 million generations MCMC chain length sampled every 1000 steps. The resulting log files were checked for convergence using TRACER v1.7.1 (Drummond and Rambaut 2007). The first 10% of trees (burn-in) were removed using TreeAnnotator v1.10.4, and a maximum clade credibility tree was built from the remaining tree estimates. The R package Species Limits by Threshold Statistics (SPLITS, v1.0-20, Fujisawa and Barraclough 2013) was used to perform the GMYC approach using the "single threshold" model (Pons et al. 2006).
At least one specimen from each of the fourteen new species of Halgerda, as well as representatives of H. dalanghita and H. willeyi, was examined morphologically. Specimens that were dissected are indicated in Table 1 with a # symbol and noted as such in their respective Material Examined section in each species description. External features were examined and described utilizing photographs of living animals and when necessarily preserved specimens. Using a Nikon SMZ-U dissection microscope, each specimen was dissected along the center of the foot, and the buccal mass and reproductive system were removed for further study. The arrangement of the reproductive organs and the shape of the buccal mass were hand-drawn using a camera lucida drawing attachment on a Nikon SMZ-U dissection microscope. After being illustrated, the buccal mass was dissolved in 10% sodium hydroxide (NaOH) for 12-24 h, and then the labial cuticle and radula were rinsed with deionized water and mounted on glass coverslips for examination by scanning electron microscope (SEM). The SEM samples were coated with gold/palladium using a Cressington 108 Auto vacuum sputter coater before micrographs were taken using a Hitachi SU3500 scanning electron microscope at the California Academy of Sciences. Specimens and the corresponding dissected structures were deposited at the California Academy of Sciences Department of Invertebrate Zoology (CASIZ) collection, the Florida Museum of Natural History (UF), and the National Museum of Philippines (NMP).
Three hundred and eight new sequences from 80 specimens were deposited on GenBank with the following accession numbers: 16S (MW220875-MW220954, OM780016-OM780018), 28S
(MW220114-MW220194), COI (MW223019-MW223087), and H3 (MW414939-MW415018). The final four-gene concatenated dataset was 2171 bp in length after removing 98 bp of ambiguous data. The single gene alignments without ambiguous data contained 433 bp for 16S, 750 bp for 28S, 658 bp for COI, and 330 bp for H3. The BI and ML analyses resulted in similar topologies; however, BI analysis generally resulted in better support values than the ML analysis (Fig. 2). A second Bayesian phylogenetic tree estimated from the four-gene concatenated dataset including the ambiguous data is provided in Supplementary Fig. S1. A third Bayesian tree estimated from the four-gene concatenated dataset without collapsed nodes is provided in Supplementary Figure S2. In this study, the position of Halgerda within Discodorididae remains unresolved, and a comprehensive phylogenetic study of the entire family of Discodorididae is needed to better evaluate the position of Halgerda with the other 28 recognized genera in the family.
Our analyses support the monophyly of Halgerda (pp = 1; bs = 100), which contains seven well-supported subclades (indicated and labeled with black bars in Fig. 2) and an ungrouped specimen of Halgerda leopardalis Tibiriçá, Pola & Cervera, 2018 Gosliner & Fahey, 1998;and Halgerda toliara Fahey & Gosliner, 1999a is also wellsupported (pp = 1; bs = 98); however, the two specimens of H. toliara group together with the one specimen of H. albocristata are unresolved.
The fourth subclade containing Halgerda mozambiquensis Tibiriçá, Pola & Cervera, 2018;Halgerda maaikeae Donohoo & Gosliner sp. nov.;Halgerda indotessellata Tibiriçá, Pola & Cervera, 2018;and Halgerda tessellata (Bergh, 1880b) is well-supported (pp = 1; bs = 100) but includes a polytomy between the first two species and a subclade of H. indotessellata that forms a second polytomy with a clade of H. tessellata. The fifth subclade (i.e., the mesophotic subclade) composed of Halgerda scripta Donohoo & Gosliner sp. nov.;Halgerda okinawa Carlson & Hoff, 2000;Halgerda mesophotica Donohoo & Gosliner sp. nov.;Halgerda profunda Donohoo & Gosliner sp. nov.;and Halgerda takipsilim Donohoo & Gosliner sp. nov. is well-supported (pp = 1;bs = 100).
The sixth clade which forms the H. carlsoni species complex is partially unresolved with a large polytomy and includes the following nine species: Halgerda theobroma Fahey & Gosliner, 2001b;Halgerda labyrinthus Donohoo & Gosliner sp. nov.; Halgerda batangas Carlson & Hoff, 2000;Halgerda terramtuentis Bertsch & S. Johnson, 1982;Halgerda malesso Carlson & Hoff, 1993;Halgerda diaphana Fahey & Gosliner, 1999b;H. carlsoni;Halgerda nuarrensis Tibiriçá, Pola & Cervera, 2018;and Halgerda aurantiomaculata (Allan, 1932). Within this clade, the first two species are basal to a polytomy composed of ungrouped specimens of H. batangas, a well-supported (pp = 1, bs = 95) group of two H. batangas specimens, and a well-supported (pp = 1, bs = 52) clade composed of a second polytomy of the rest of the species previously listed. The seventh subclade is also well-supported (pp = 1; bs = 0.91) and further divides into two major clades. The first major clade is unresolved, but
The COI ABGD analysis recovered six partitions (Supplementary Table S1). Eighteen groups were retrieved with a prior maximal distance p-distance of 0.02, 21 groups with 0.01, 34 groups with 0.006, 38 groups with 0.003, 41 groups with 0.002, and 47 groups with 0.001. The partitions with 18-21 groups were clearly under-divided as all 24 specimens from eight morphologically distinct species including H. labyrinthus sp. nov., H. batangas, H. terramtuentis, H. malesso, H. diaphana, H. carlsoni, H. nuarrensis, and H. aurantiomaculata were grouped together as a single taxonomic unit. The partitions with 34-47 groups also recover H. mesophotica sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov. as one taxonomic unit as well as over-dividing several Halgerda species including H. willeyi, H. paulayi sp. nov., H. batangas, and the H. wasinensis species complex; however, the partition with 34 groups also fails to separate out H. diaphana, H. malesso and H. labyrinthus sp. nov. from the H. batangas clade. The partitions with 41-47 groups successfully separate out the aforementioned species from H. batangas, but also over-divides the three specimens of H. carlsoni and one specimen of H. scripta sp. nov. The partition with 47 groups also over-divides several specimens of H. batangas. Therefore, the partition with 38 groups is the most congruent with the Bayesian and maximum likelihood analyses. This partition also over-divides H. willeyi, H. paulayi sp. nov., H. scripta sp. nov., and the H. wasinensis species complex as well as fails to recover H. labyrinthus sp. nov. separately from H. batangas; however, the latter may be attributed to the low genetic differences between the two species (maximum p-distance = 1.4%).
The 16S ABGD analysis recovered four partitions (Supplementary Table S2). One group was retrieved with a prior maximal distance p-distance of 0.006, 25 groups with 0.003, 52 groups with 0.002, and 52 groups with 0.001. The partition with one group recovers all Halgerda specimens together, while the partitions with 52 groups recover most specimens as individual taxonomic units. Therefore, the partition with 25 groups is the most likely as it successfully recovers some species; however, it fails to delimit species in several clades. The COI ASAP analysis (Supplementary Figure S3) using Kimura80 recovered 19 groups (ASAP score = 1.50); however, all species in the H. carlsoni species complex in the first clade were grouped together as one large taxonomic unit. Additionally, the seventh clade was split into two taxonomic units. The COI GYMC recovers 24 groups similar to the COI ASAP with 25 groups with a few differences. The entire H. carlsoni species complex including H. nuarrensis but excluding H. aurantiomaculata is recovered as one large taxonomic unit. All three specimens of H. paulayi sp. nov. are recovered together in one unit rather than two. The majority of the H. wasinensis species complex is recovered as one large taxonomic unit; however, H. aff. wasinensis and H. cf. formosa are recovered together in one unit outside the H. wasinensis species complex.
Order Nudibranchia Cuvier, 1817Family Discodorididae Bergh, 1891Genus Halgerda Bergh, 1880a Type species Halgerda formosa Bergh, 1880a, by monotypy Halgerda dalanghita Fahey & Gosliner, 1999a (Fig. 3a-c and 4) Halgerda dalanghita Fahey & Gosliner, 1999a: 373-374, Figs. 3b, 4a, 5, and 6;Fahey and Gosliner (2000): 496-497, Figs. 14c, 16d, 18;Ono (2004) Type locality: Bethlehem, Maricaban Island, Batangas Province, Luzon, Philippines.
External anatomy (Fig. 3a-c): Adult preserved animals 20-25 mm in length (Fig. 3b,c). Similar to adult description described in Fahey and Gosliner (1999a, Fig. 4a). Juvenile coloration described here. In small juvenile CASIZ 204807 dorsum color uniform orange with small to large, random black spots (Fig. 3a). Ridges thicker, less angled with no tubercles. Thin, semi-opaque white mantle band lines mantle edge. Mantle underside orange with numerous medium to large dark Internal anatomy (Fig. 4a-d): Buccal mass unpigmented. Similar to Fahey and Gosliner (1999a) original description. Labial cuticle smooth. Radula composed of both smooth hamate and extremely elongate teeth. Radular formula 42 × 19.0.19 in CASIZ 231295 and 30 × 20.0.20 in small juvenile CASIZ 204807. In CASIZ 231295 innermost lateral tooth (Fig. 4b) small, paddle shaped, with broad base and approximately 14 greatly reduced denticles. Middle lateral teeth 2-5 hamate, gradually increase in size and elongation (Fig. 4c). Outer lateral teeth (Fig. 4d) thin, extremely elongate, with fine tips. Outermost tooth is reduced version of outer laterals. Juvenile inner teeth denticulation (Fig. 4a) more pronounced in CASIZ 204807 with 12-13 elongate denticles (first inner tooth), 3 elongate denticles (second inner tooth), and occasionally one reduced denticle on third inner tooth.
Reproductive system: Not illustrated. Similar to reproductive system drawn and described in Fahey and Gosliner (1999a, Fig. 3b).
Geographical distribution: Known from South Africa (Gosliner 1987;Fahey and Gosliner 1999a;Gosliner et al. 2008;Strömvoll and Jones 2019), Mozambique (Tibiriçá et al. 2018), Japan (Ono 2004;Gosliner et al. 2008), the Philippines (Fahey and Gosliner 1999a;Gosliner et al. 2008), Papua New Guinea (Fahey and Gosliner 1999a;Gosliner et al. 2008), Fiji (Fahey 2000), the Marshall Islands (Debelius and Kuiter 2007), the Solomon Islands, and Hawaii (Gosliner et al. 2008).
Ecology: Found on orange sponges in shallow water between 3 and 15 m and on coral rubble at 20 m.
Remarks: Our molecular and species delimitation analyses reveal that specimens previously thought to be a new species (Halgerda sp. 4, Gosliner et al. 2018) are juveniles of the previously described species Halgerda dalanghita. Here, we have provided a second description for juvenile H. dalanghita and make morphological comparisons with adult H. dalanghita originally described by Fahey and Gosliner (1999a) and a juvenile H. dalanghita from Mozambique identified in Tibiriçá et al. (2018, Fig. 7e). Externally, the dorsum color is a uniform orange similar to that found in adult specimens; however, there are many small to large, random black spots across the entire mantle (Fig. 3a) that are similar to the Mozambique juvenile rather than the numerous minute brown and white spots seen in adult specimens (Fahey and Gosliner 1999a, Fig. 4a; present study Fig. 3b,c). The dorsum ridging is thicker and less angled in the juvenile specimen studied here, but both adults and juveniles lack tubercles at the ridge junctions. Furthermore, both adults and juveniles have numerous small to medium brown/light brown spots on the mantle underside and along the foot. In both the juvenile studied here and the Mozambique juvenile, the rhinophores and gill plume have darker, less diffused pigment than in adults; however, there are a similar number of bipinnate gill branches with opaque white glands visible in the rachises. Internally, there are fewer rows in both juvenile radulae, and the first few inner teeth have more pronounced denticles than the adult specimens (Fig. 4a). The middle and outer teeth are similar in shape in both adults and juveniles and accurately counting the thin, extremely elongate, fine tipped outer teeth is difficult.
In summary, as individuals of H. dalanghita grow, the dorsum ridging becomes narrower and more pronounced; the black spots along the mantle, rhinophores, and gills begin to diffuse and fade; and the denticles along the inner laterals of the radular teeth become reduced. The intraspecific variation between the four specimens (two adults and two juveniles studied here) collected from Papua New Guinea and three different locations in the Philippines have a range of 0.5-1.7% in the COI gene, which further supports our description of juvenile H. dalanghita.
Halgerda mango Donohoo & Gosliner sp. nov. https:// zooba nk. org/ B34FC F5A-5165-4F1C-99E5-6C7BD 089BF C5 (Figs. 3d,f,5a,6 Etymology: This species is named mango after the similar vibrant yellow-orange coloration found in the Carabao or Philippine mango, a glorious treat that has sustained many expeditions to the Philippines.
External morphology (Fig. 3d-f): Adult preserved animals approximately 20 mm in length (Fig. 3f). Body oval, rigid, and gelatinous. Dorsum color uniform lemon yellow with minute, light brown flecking across entire mantle. Ridging arranged in reticulated pattern with highly elevated tips at ridge junctions. Thin, semi-opaque white mantle band lines mantle edge. Mantle underside lemon yellow with occasional large brown spots. Gill surrounds elevated anus with six bipinnate sometimes tripinnate branchial leaves. Gill branches translucent white with light brown dorsal lining. Opaque white glands within pinnate portions of gill rachis. Gill pocket smooth with similar dorsum coloration. Rhinophores tapered and perfoliate with 16 lamellae. Rhinophoral sheath and lamellae translucent white with diffused brown subapical pigment and posterior near base splotches. Foot is broad, anteriorly notched, lemon yellow with occasional medium dark brown spots. Oral tentacles digitiform.
Juvenile coloration varies with size. In small juvenile CASIZ 204808 dorsum color uniform orange with small to large, random black spots (Fig. 3d). Ridging and ridge tips less pronounced with occasional white coloration. Thin, semi-opaque white mantle band similar to adult. Mantle underside similar to dorsum coloration with ring of small to large brown and dark brown spots. Gill branches translucent white with dark brown lining. Fewer opaque white glands within gill rachis. Gill pocket smooth with similar dorsum coloration. Rhinophores tapered and perfoliate with 12 lamellae. Rhinophoral sheath and lamellae translucent white with dark brown subapical and posterior near base splotches. Foot is broad, anteriorly notched, orange with ring of small dark brown/black spots. Oral tentacles digitiform. In a larger juvenile CASIZ 217210 dorsum color uniform pale yellow with random diffused brown spots (Fig. 3e). Ridging and ridge tips more pronounced with less white coloration. Thin, semi-opaque white mantle band less pronounced. Mantle underside pale yellow with ring of large dark brown spots. Gill branches translucent white with brown lining. More opaque white glands present within gill rachis. Rhinophores tapered and perfoliate with 14 lamellae. Rhinophoral sheath and lamellae translucent white with more diffused dark brown subapical pigment and posterior near base splotches. Foot is broad, anteriorly notched, pale yellow with ring of small dark brown/black spots. Oral tentacles digitiform.
Internal anatomy (Figs. 5a, 6a-f and 7a): Buccal mass (Fig. 5a) unpigmented. Buccal bulb similar in size to oral tube. Radular sac elongate and wedge shaped. Labial cuticle smooth. Radula composed of both smooth hamate and extremely elongate teeth (Fig. 6c). Radular formula 42 × 16.0.16 in the holotype NMP 041323, 34 × 15.0.15 in young juvenile CASIZ 204808, and 34 × 23.0.23 in older juvenile CASIZ 217210. In NMP 041323 inner three lateral teeth and two to three middle lateral teeth (Fig. 6d) 6e) thin, extremely elongate, with fine tips. Some outer teeth are broken and stacked together making counting difficult. Outermost tooth reduced version of outer laterals. Juvenile radulae vary from adults in outer lateral curvature (Fig. 6f) and inner lateral denticulation (Fig. 6a,b). Juvenile inner teeth denticulation includes 12-16 elongate denticles (first inner tooth), 3-6 elongate or semi-reduced denticles (second inner tooth), and sometimes one reduced denticle on third inner tooth.
Reproductive system (Fig. 7a): NMP 041323. Triaulic. Thin preampullary duct widens into thick ampulla, then narrows into postampullary duct which splits into vas deferens and short oviduct. Vas deferens rapidly expands into wide, looped granular prostate, then narrows into long, thin ejaculatory portion and connecting thin penis. Penis shares common genital atrium with similarly sized vagina.
Narrow, elongate vagina continues into thin, elongate duct and proximally enters rounded bursa copulatrix. Thin, short duct connects bursa to slightly smaller, semi-rounded receptaculum seminis. Thin, elongate uterine duct also connects near receptaculum base and enters medium-sized, irregularly shaped female gland mass.
Geographical distribution: Known from the Philippines. Ecology: Found on shallow reefs between 5 and 15 m and on coral rubble around 20 m.
Remarks: Our molecular phylogeny shows that Halgerda mango sp. nov. is sister to the similarly patterned species H. dalanghita and species delimitation analyses reveal that smaller specimens (previously cited as Halgerda sp. 3, Gosliner et al. 2018) are H. mango sp. nov. juveniles. All four species delimitation analyses support H. mango sp. nov. as a distinct species from H. dalanghita and reveal a minimum divergence of 8.3% in the COI gene and intraspecific variation ranged from 0.8 to 1.5% Externally, adult H. mango sp. nov. have a uniform lemon-yellow coloration with minute, light brown flecking; pronounced ridging with elevated tips at the ridge junctions; six bipinnate (sometimes tripinnate) branchial leaves with a light brown dorsal stripe; and diffused brown subapical pigment along the rhinophores (Fig. 3f). In contrast, adult H. dalanghita are uniformly orange in coloration with white flecking across the mantle and less pronounced ridging which has minute brown flecking along the ridges and white spots at ridge junctions. The six branchial leaves are bipinnate with black/dark brown dorsal stripes and flecking along the rachises (Fahey and Gosliner 1999a, Fig. 4a; present study Fig. 3b,c). The rhinophores are similar to H. mango sp. nov.; however, the diffused pigment is darker and more prominent in H. dalanghita. Young juvenile H. mango sp. nov. are morphologically very similar to juvenile H. dalanghita, but there are external differences between the two species. In H. mango sp. nov., the numerous small to large spots are browner in coloration, and the dorsum ridging is less thick and more pronounced with elevated tips at the ridge junctions (Fig. 3d). In H. dalanghita, the spotting is darker and more black in coloration and the ridging thicker, more angled, and less pronounced with no elevated ridge junctions (Fig. 3a). Both species have bipinnate gills with dorsal stripes and rhinophores with dark pigment along the lamellae and splotches along the rhinophoral stalk, but in H. mango sp. nov., the dark pigment is noticeably lighter in color than that seen in H. dalanghita. The dorsum color in older juvenile H. mango sp. nov. is more yellow with the mantle spotting and rhinophore/gill pigment becoming lighter in color and more diffused (Fig. 3e).
Internally, both species have unpigmented buccal masses and similar patterns in the radular teeth including denticulation in the inner laterals and thin, extremely elongate outer teeth with fine pointed tips. In adult H. mango sp. nov., there are fewer denticles (approximately 12) in the innermost tooth and a higher number of denticles (up to 16 and 6, respectively) in the first two inner teeth in H. mango sp. nov. juveniles. In contrast, there are more reduced denticles (approximately 14) present in the first lateral tooth of adult H. dalanghita, and the inner two lateral teeth of juvenile H. dalanghita have fewer denticles (12-13 and 3, respectively) along the first two inner teeth. The reproductive systems are also similar between the two species including a narrow, elongate vagina; narrow, elongate penis and ejaculatory portion of the prostate; and thin elongate ducts between the vagina and the bursa copulatrix, the bursa and the receptaculum seminis, and an elongate uterine duct. Differences between the two species are minimal. The bursa copulatrix is twice the size of the receptaculum in H. dalanghita, and the preampullary duct abruptly expands into a thick ampulla, while in H. mango sp. nov., the bursa is only slightly larger than the receptaculum, and the preampullary duct gradually expands into a thick ampulla. The duct between the vagina and the bursa copulatrix is 1.5 × longer in H. mango sp. nov., and the ejaculatory portion of the prostate and the penis is an equal size to the vagina, rather than twice as thick as seen in H. dalanghita. Despite the similarities in adult and juvenile specimens of H. mango sp. nov. and H. dalanghita, there are external and some internal morphological differences and a strong genetic divergence between these two species. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ E505E 6C2-240E-43ED-94D4-C9EE0 4A4B5 17 (Fig. 5b,7b,8a and 9) Fig. 11 Living specimens: a-c Halgerda willeyi Eliot, 1904 External morphology (Fig. 8a): Preserved animals 35 mm in length (Fig. 8a). Body oval, semi-rigid, and gelatinous. Living animal with dorsum color grayish white with large rounded opaque white tubercles. Viscera with yellow tint visible through mantle. Wide yellow submarginal mantle band offset from thin, semi-opaque white submarginal mantle band. Mantle underside grayish white. Gill surrounds elevated anus with four unipinnate branchial leaves. Gill branches translucent white with large reddish/dark brown semi-translucent spots. Opaque white glands visible in gill branches. Gill pocket smooth with similar dorsum coloration. Rhinophores tapered, perfoliate with similar Internal anatomy (Figs. 5b, 7b and 9a-d): Buccal mass (Fig. 5b) unpigmented; however, small light brown spots present around buccal opening. Oral tube elongated, narrowed anteriorly and approximately 1.5 × longer than buccal bulb. Radular sac elongate and folded over buccal bulb. Labial cuticle smooth. Radula composed of smooth hamate teeth (Fig. 9a). Radular formula 53 × 54.0.54 in the holotype UF 456643. First three inner lateral teeth small, broad based, with a slightly curved cusp. Inner lateral teeth (Fig. 9b) 4-15 gradually increase in size with a more elongate cusp and form a shallow V in the radula center. Middle lateral teeth (Fig. 9c) larger and more elongate than inner laterals. Outer lateral teeth (Fig. 9d) similar to middle laterals with shorter, rounded cusps. Outermost tooth paddleshaped and semi-fimbriate with four small denticles.
Reproductive system (Fig. 7b): Triaulic. Thin, elongate preampullary duct widens into ampulla, then narrows into postampullary duct which splits into short, thin vas deferens and short, thin oviduct. Vas deferens thick, expands into wide, elongate, looped granular prostate, then quickly narrows into thin ejaculatory portion. Ejaculatory portion rapidly expands into short, wide penis which shares common genital atrium with vagina. Wide, elongate, nodular vagina narrows into thinner semi-elongate duct and proximally enters into large, rounded bursa copulatrix partially covered by granular prostate. Semi-elongate duct connects bursa to smaller, pyriform receptaculum seminis. Thin, elongate uterine duct also connects near receptaculum base and enters into medium-sized, pyriform-shaped female gland mass.
Geographical distribution: Known from Moorea and Tahiti, French Polynesia.
Ecology: Found on reef slopes at approximately 10-100 m. Remarks: Molecular sequencing of the holotype of Halgerda berberiani sp. nov. (UF 456643) was unsuccessful for all genes studied; however, the morphological characteristics are distinct enough to distinguish H. berberiani sp. nov. as a new species within Halgerda. Halgerda berberiani sp. nov. is morphologically similar to H. aurantiomaculata (Fig. 8b) and H. terramtuentis (Fig. 8c), and morphological comparisons between the three are made below. Externally, the dorsum coloration of H. berberiani sp. nov. is a grayish white with large opaque white tubercles, an offset wide yellow mantle band, and a thin semi-opaque white mantle band, whereas H. aurantiomaculata is an opaque white color with numerous orange spots, and a single wide yellow mantle band (Willan and Brodie 1989, Figs. 1 and2) and H. terramtuentis is translucent cream with white tipped tubercles, a network of orange lines, and an orange mantle band (Kay and Young 1969, Fig. 30;Bertsch and Johnson 1982, Fig. 15). The gill and rhinophore coloration vary slightly by species. In H. berberiani sp. nov., the gill and rhinophore spots are large with a reddish/dark brown coloration, while in H. terramtuentis the spots are smaller, more numerous, and black and in H. aurantiomaculata the brown/black spots are tiny and numerous.
The radular teeth in H. berberiani sp. nov. are similar to all Halgerda; however, the inner lateral teeth form a shallow V in the radula center, and the first three inner lateral teeth are small with a slightly curved cusp before increasing in size and developing more elongated cusps. The outermost tooth is also paddle-shaped and semi-fimbriate. Similarly, the first few inner lateral teeth in H. aurantiomaculata are smaller and less curved than the following inner teeth; however, the outermost tooth is reduced with an apical cleft (Willan and Brodie 1989, Figs. 25-32). In H. terramtuentis the radular teeth increase in length in the middle of each half-row, before decreasing to nearly "scythe-like" blades in the outermost teeth (Kay and Young 1969, as H. sp. cf. graphica, Fig. 28b;Bertsch and Johnson 1982, Fig. 16). The reproductive system also varies as H. berberiani sp. nov. has a large, rounded bursa copulatrix, a wide elongated nodular vagina, an elongated uterine duct, and a semi-elongated duct connecting the bursa copulatrix to the receptaculum seminis. In contrast, both H. aurantiomaculata and H. terramtuentis have a large, rounded bursa copulatrix ensheathed in the glandular portion of the prostate, a rounded glandular portion at the base of the duct connecting the vagina to the bursa copulatrix, a short uterine duct, and a much more elongated duct connecting the bursa copulatrix to the receptaculum seminis (Willan and Brodie 1989, Figs. 6, 7 and 8;Kay and Young 1969, Fig. 28a;respectively). Based on the unique internal and external characteristics including dorsum coloration and style of tubercles, H. berberiani sp. nov. is a distinct species within Halgerda despite the lack of molecular data for the species. Type locality: Gulf of Tadjoura, Djibouti Etymology: This species is named "biqiea" after the Arabic word for spot and refers to the numerous small brown spots along the dorsal side of the mantle.
External morphology (Fig. 8d,e): Preserved animals 4-10 mm in length. Body elongate oval, rigid, and gelatinous. Dorsum color translucent pale yellow with numerous small to medium dark brown/black spots (Fig. 8d). Smaller specimens have fewer dark spots (Fig. 8e). Light yellow low ridging arranged in irregular pattern with slightly higher peaks and no tubercles. Viscera coloration visible through mantle in larger specimens. Thin, opaque white mantle band surrounds mantle edge. Mantle underside translucent pale yellow with ring of small to large dark brown/black spots. Gill surrounds elevated anus with four unipinnate branchial leaves. Gill coloration translucent white with dorsal dark brown stripe. Gill pocket smooth with similar dorsum coloration. Opaque white glands visible in gill branches. Rhinophores elongate, perfoliate, with similar dorsum coloration, dark brown /black lateral stripes, a rounded conical tip, and 10-12 lamellae. Rhinophoral sheath smooth with similar dorsum coloration. Foot broad, anteriorly notched, and pale yellow with numerous small to large dark brown/black spots. Oral tentacles digitiform.
Internal anatomy (Figs. 5c, 7c and 10a-d): Buccal mass (Fig. 5c) pigmented on oral tube with large single black spot and small dark brown/black spots present around buccal opening. Oral tube semi-elongate and approximately 1.5 × longer than buccal bulb. Radular sac short and unpigmented. Labial cuticle smooth. Radula composed of predominantly smooth hamate teeth (Fig. 10a). Radular formula 43 × 30.0.30 in the holotype UF 455832. Inner eight lateral teeth (Fig. 10b) small, broad based, with quickly tapered, slightly curved, cusp with single, large pectinate denticle along the outer edge and form a shallow V in the center. Middle lateral teeth (Fig. 10c) large, hamate with thicker, rounded cusps. Outer lateral teeth (Fig. 10d) similar to middle laterals with shorter, less curved cusps. Five outermost teeth reduced and fimbriate.
Reproductive system (Fig. 7c): Triaulic. Thin preampullary duct widens into thick ampulla, then narrows into elongate postampullary duct which splits into vas deferens and short oviduct. Vas deferens gradually thickens into elongate, looped granular prostate, which slightly narrows distally into thick, elongate ejaculatory portion. Ejaculatory portion expands into slightly wide penis which shares common genital atrium with smaller vagina. Semi-wide vagina gradually narrows proximally into thin, elongate duct and enters rounded bursa copulatrix. Short duct connects bursa to smaller, pyriform receptaculum seminis. Short uterine duct also connects near receptaculum base and enters small irregularly rounded female gland mass.
Geographical distribution: Known from the Gulf of Tadjoura, Djibouti and the Saudi Arabian Red Sea.
Ecology: Found on shallow patch reefs between 15 and 20 m. Remarks: Our molecular phylogeny shows that Halgerda biqiea sp. nov. from the Red Sea is sister to the similarly colored Indo-Pacific species H. brunneomaculata (Fig. 8f) and part of a well-supported clade that also includes H. albocristata and H. toliara. Both of the ABGD analyses and the COI ASAP, the bPTP and the GYMC analyses within the clade support H. biqiea sp. nov. as a distinct species. There is a minimum COI divergence of 7.1% between H. biqiea sp. nov. and H. brunneomaculata, as well as a minimum divergence of 11.8% between H. biqiea sp. nov. and H. albocristata and 11.6% between H. biqiea sp. nov. and H. toliara. There was no intraspecific variation within the COI gene in H. biqiea sp. nov. specimens collected in the Gulf of Tadjoura, Djibouti (UF 455832), and on Ablo Island Reef, Red Sea, Saudi Arabia (CASIZ 192299). Morphologically, H. biqiea sp. nov. is similar to H. brunneomaculata; however, there are some noticeable differences. Externally, the dorsum coloration of H. biqiea sp. nov. is translucent pale yellow with numerous small to medium dark brown/black spots, pale yellow ridging, a ring of dark brown/black spots on the mantle underside, and a foot with numerous dark brown/black spots. In contrast, f Halgerda maaikeae sp. nov., holotype, SAMC-A094639, scale bar = 1 mm; g Halgerda pattiae sp. nov., holotype, UF 422800, scale bar = 0.5 mm; h Halgerda radamaensis sp. nov., holotype, CASIZ 173456, scale bar = 0.5 mm. Abbreviations: a, ampulla; bc, bursa copulatrix; ej, ejaculatory duct; fgm, female gland mass; p, penis; pr, prostate; rs, receptaculum seminis; ud, uterine duct; v, vagina H. brunneomaculata is translucent pale yellow with fewer large, brown spots, more vibrant yellow ridging, no spotting along the underside of the mantle, and only a handful of large brown spots on the foot which extends past the mantle (Carlson and Hoff 1993, Figs. 16, 17 and 18).
Internally, the radular teeth of H. biqiea sp. nov. are similar to H. brunneomaculata including the reduced, fimbriate five outermost teeth (Carlson and Hoff 1993, Fig. 20); however, the innermost eight teeth in H. biqiea sp. nov. are distinct. In H. brunneomaculata the innermost small tooth has a large, pectinate denticle along the inner edge (Carlson and Hoff 1993, Fig. 19), whereas in H. biqiea sp. nov., the innermost eight teeth each have a bulbous base with an abruptly tapered semi-curved cusp and a single, large pectinate denticle along the outer edge. Fahey and Gosliner (2000) noted that the buccal mass in H. brunneomaculata lacked coloration but that there were small dark spots around the buccal opening. In H. biqiea sp. nov., the buccal mass has a single large black spot on the oral tube, as well as small dark brown/black spots around the buccal opening. The reproductive system also varies as H. biqiea sp. nov. has a small female gland mass; a short, wide penis with a thick elongated ejaculatory portion of the prostate; and an elongated duct connecting a large round bursa copulatrix to a semi-elongated vagina. In contrast, H. brunneomaculata has a much longer penis, an exceptionally thin elongated ejaculatory portion of the prostate, a nodular prostate much larger than the bursa copulatrix, and a similarly thin elongated duct connecting the vagina to the bursa copulatrix and the bursa copulatrix to the receptaculum seminis, as well as a thin elongated uterine duct (Carlson and Hoff 1993, Fig. 21).
Despite their many similarities, there are clear internal and external morphological differences and a strong genetic divergence that separate H. biqiea sp. nov. and H. brunneomaculata. Additionally, there is geographical separation between the two species. To date, H. biqiea sp. nov. is only found in the Saudi Arabian Red Sea and the nearby Gulf of Tadjoura, while H. brunneomaculata has been previously documented in the western Pacific including Guam; the Northern Mariana Islands; Fiji; Okinawa; New Caledonia; the Great Barrier Reef, Australia (Carlson and Hoff 1993;Debelius 1996;Ono 1999;Fahey and Gosliner 2000;Coleman 2001;Nakano 2004;Debelius and Kuiter 2007;Hervé 2010;Humann and DeLoach 2010); and French Polynesia (present study). Eliot, 1904 (Fig. 5d, 7d, 11a-c and 12) Halgerda willeyi Eliot, (1904): 372-373, plate XXXII, Fig. 5;Eales (1938): 100-103, text-Figs. 17, 18, plate 1, Fig. 3;Gosliner et al. (1996): 160, second photograph from the bottom; Carlson and Hoff (2000): 161-162, Figs. 14, 18, 19 and 20;Coleman (2001) Bergh (1905): 124-126;taf. II, Fig. 4a, taf. XV, Figs. 29-31;Johnson and Boucher (1983): 261-262, Fig. 4; Gosliner and Fahey (1998): 348-352, Figs. 1a, 2, 3, 4, 5 and 6;Ono (1999) External morphology (Fig. 11a-c): Adult preserved animals 15-35 mm in length in specimens such as CASIZ 177248 and CASIZ 083676A (Fig. 11c) with similar adult description described for H. willeyi in Eliot (1904, plate XXXII, Fig. 5). Two additional external morphologies described here. Adult preserved specimens CASIZ 191495 and CASIZ 204790 (Fig. 11b) 13-15 mm in length. Dorsum color translucent white with low, irregular yellow and orange ridging and thick black/dark brown and orange lines within ridge valleys. Prominent yellow tubercles at ridge junctions. Central tubercles ringed with diffuse brown pigment. Small irregular spots and thin black/dark brown lines perpendicular to mantle edge. Thin, white mantle band along mantle edge. Mantle underside translucent white with short, thick black/dark brown lines perpendicular to mantle edge. Gill and rhinophores similar to Eliot (1904) description, i.e., two branchial plumes each split into three large mostly bipinnate branches. Gill coloration translucent white with dorsal and lateral brown stripes and white rachises. Opaque white glands visible in gill branches. Gill pocket smooth with similar dorsum coloration and perpendicular yellow-orange ridges. Rhinophores tapered, perfoliate, translucent white with posterior black/dark brown stripe and shorter, similarly colored lateral stripes. Lamellae translucent white with lateral black/dark brown spots number 20-25. Rhinophoral sheath smooth with similar dorsum coloration and yellow ridges. Foot broad, anteriorly notched, and translucent white with numerous short black/dark brown perpendicular lines extends past mantle edge. Oral tentacles digitiform.
Juvenile preserved specimens CASIZ 191259 and CASIZ 201944 (Fig. 11a) 6-8 mm in length with similar description described for H. elegans in Bergh (1905, taf. II, Fig. 4a). Dorsum color translucent white with low, irregular yellow-orange ridging, large black spots between ridge valleys, and thick black lines alternating thinner, short black lines perpendicular to mantle edge. Prominent yellow tubercles at ridge junctions. Thin, white mantle band along mantle edge. Mantle underside translucent white with short, black lines and some small black spots perpendicular to mantle edge. Gill and rhinophores similar to Bergh (1905) description, i.e., two branchial plumes each split into three large mostly bipinnate branches. Gill coloration translucent white with prominent dorsal and lateral brown stripes and white rachises. Opaque white glands semi-visible in gill branches. Gill pocket smooth with similar dorsum coloration and perpendicular yellow-orange ridges. Rhinophores tapered, perfoliate, translucent white with posterior dark brown stripe and short similarly colored lateral stripes. Lamellae translucent white with large black blotches near the tips number 8-12. Rhinophoral sheath smooth with similar dorsum coloration and yellow-orange ridges. Foot broad, anteriorly notched, and translucent white with spaced apart short black perpendicular lines and small black spots extends past mantle edge. Oral tentacles digitiform.
Internal anatomy (Figs. 5d, 7d and 12a-d): Buccal mass (Fig. 5d) unpigmented. Oral tube semi-elongate and approximately 1.5 × longer than buccal bulb. Radular sac slightly curved and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth (Fig. 12a). Radular formula 39 × 47.0.47 in CASIZ 083676A. Inner 25 lateral teeth (Fig. 12b) small, broad based, with elongated curved cusps form a steep V in radula center and gradually increase in size. Middle lateral teeth (Fig. 12c) larger and more elongate than inner laterals. Outer lateral teeth (Fig. 12d) similar to middle laterals with shorter cusps. Outermost two teeth are reduced and fimbriate.
Reproductive system (Fig. 7d): Triaulic. Thin preampullary duct widens into thick ampulla, then quickly narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens gradually thickens into wide, elongate, looped granular prostate, then abruptly narrows into thinner ejaculatory portion. Ejaculatory portion rapidly expands then contracts into the penis, which shares common genital atrium with vagina. Large, muscular vagina widens before proximally narrowing into thin, elongate duct and entering into large, rounded bursa copulatrix partially covered by granular prostate. Thin, elongate duct connects bursa to small ovalshaped receptaculum seminis. Uterine duct also connects near receptaculum base and enters medium-sized, pyriform-shaped female gland mass with elongated distal end.
Geographical distribution: Known from the Khuriya Muriya Islands (Eales 1938), the Ryukyu Islands, Japan (Gosliner and Fahey 1998;Ono 1999Ono , 2000Ono , 2004;;Debelius and Kuiter 2007;Gosliner et al. 2008), the Philippines (Gosliner et al. 2008; present study), Indonesia (Gosliner and Fahey 1998), Papua New Guinea (Gosliner and Fahey 1998;Debelius and Kuiter 2007;Gosliner et al. 2008; present study), Australia (Gosliner et al. 2008), Vanuatu (Gosliner et al. 2008), the Loyalty Islands (Gosliner et al. 2008), and the Marshall Islands (Johnson and Boucher 1983;Gosliner et al. 2008).
Ecology: Found on living reefs crawling in the open or under rocks feeding on sponges.
Remarks: Our molecular phylogeny reveals that Halgerda willeyi is composed of three different morphologies: the original adult morphology as described in Eliot (1904) from Fiji; a new adult morphology described here; and a juvenile morphology formerly described as Halgerda elegans in Bergh (1905) from Indonesia. In our 16S ABGD, COI ASAP, bPTP, and GYMC analyses, the five specimens of H. willeyi are grouped together into one taxonomic unit, while our COI ABGD analysis over-partitions H. willeyi into four distinct taxonomic units and the 16S ASAP analysis groups H. willeyi with rest of the entire seventh clade. The COI maximum intraspecific variation between the five specimens studied here is only 1.8% (Supplementary Table S3), and the over-partitioning may be attributed to the low genetic diversity (2.9-4.8%) between H. willeyi and other members of the subclade: H. gunnesi, H. cf. formosa, H. wasinensis, H. aff. wasinensis, H. anosy sp. nov., and H. dichromis (Table 3).
All three color morphs share several external characteristics including: low, yellow-orange ridging with yellow tubercles at ridge junctions; perpendicular brown/black lines along the top and underside of the mantle edge; a thin white mantle band; a bipinnate gill composed of two branchial plumes each split into two or three large branches with dorsal and lateral brown stripes and white rachises; and translucent white rhinophores with a posterior dark brown stripe, similarly colored shorter lateral stripes, and dark colored spots or blotches along the lamellae. Variation within the three morphs is seen predominantly in the dorsum and tubercle patterning, as well as the stripes and spots along the gill and rhinophores. Similar to the original description of H. willeyi (Eliot 1904, plate XXXII, Fig. 5), adult specimens CASIZ 177248 and CASIZ 083676A (Fig. 11c) have more complex black and orange linework in the ridges and valleys than the other two morphologies. In the second adult color morph (Fig. 11b), seen in CASIZ 191495 and CASIZ 204790, the linework in the ridges and valleys is less complex, and the pigment more concentrated and the ridge intersection tubercles are also ringed with a diffuse brown pigment not seen in the other two morphs. In juvenile specimens CASIZ 191259 and CASIZ 201944 (Fig. 11a), the dorsum and tubercle patterning are similar to that described for H. elegans in Bergh (1905, taf. II, Fig. 4a). Instead of complex linework in the ridges and valleys, there are large black spots and thicker perpendicular black lines along the mantle edge. The gill and rhinophores also have larger, more dark stripes and spots that are not quite as diffused as seen in older specimens.
Since Eliot (1904) did not provide a description of the radular teeth or the reproductive system in the original description of H. willeyi, we have dissected and described the internal anatomy of a specimen (CASIZ 083676A) that closely resembles Eliot's original external description. We disregarded the internal descriptions for H. willeyi found in Vayssière (1912) and Rudman (1978) as they are misidentifications of a new, similar species of Halgerda described and remarked upon in the Halgerda paulayi sp. nov. remarks section below. We are also unsure of which adult morph to which Eales (1938) refers, due to the lack of color from prior preservation and no external drawing; however, we are confident that Eales (1938) does refer to a true H. willeyi due to the presence of the numerous small inner radular teeth, the reduction in the shape of the outermost teeth, and the presence of a muscular vagina and large, spherical bursa copulatrix. The internal anatomy of the juvenile morphology previously described as H. elegans has a limited description in Bergh (1905). No details are given about the reproductive organs or their arrangement, but there is some description about the radular teeth (Bergh 1905, taf. XV, Figs. 29-31). Gosliner and Fahey (1998) further described the internal anatomy including the radula and reproductive system of H. elegans and was used for the internal comparisons of the three morphos. The external morphology of the H. willeyi dissected by Carlson and Hoff (2000) closely resembles the new adult morphology described here; however, it lacks details about the inner and middle lateral radular teeth. Carlson and Hoff (2000) did illustrate the outermost teeth and the reproductive system and was therefore used for some of the internal comparisons.
Internally, the buccal mass of all three morphologies lacks pigment and shares the predominantly smooth hamate teeth found in other species of Halgerda. In CASIZ 083676A, the numerous inner lateral teeth are small and broad-based with an elongated curved cusp, which is similar to the inner laterals described for H. elegans in Bergh (1905, taf. XV, Fig. 29) and the H. elegans specimen CASIZ 079348 in Gosliner and Fahey (1998, Fig. 2c-d). Gosliner and Fahey (1998) did find that H. elegans exhibits some variation in the radular teeth ranging from simple hamate to multifid middle laterals (Gosliner and Fahey 1998, Fig. 2, 3, 4 and 5). In all three morphs the outermost two to three teeth are reduced; however, the illustration of the outermost teeth in Carlson and Hoff (2000) lacks the fimbriate detail seen in the present study (Fig. 12d) and in Bergh's description of H. elegans (Bergh 1905, taf. XV, Fig. 31). In all three reproductive systems the large, spherical bursa copulatrix is partially covered by the granular portion of the prostate, the receptaculum seminis is ovalshaped and approximately half the size of the bursa, and the vagina is muscular and larger than the penis even in juvenile specimens previously considered as H. elegans (Gosliner and Fahey 1998, Fig. 6;Carlson and Hoff 2000, Figs. 18 and 19). Differences between the three reproductive systems are minimal but include the amount of the glandular prostate covering the bursa copulatrix in the second adult morph (Carlson and Hoff 2000, Figs. 18 and 19) and the penis expanding distally into an enlarged section of the ejaculatory portion of the prostate in CASIZ 083676A.
Since there are low genetic differences in the five specimens studied here, no geographical separation or morphological grouping, and numerous morphological similarities, we are confident that all three morphs represent a single species. Furthermore, there are no clear differences between H. elegans and H. willeyi other than slight variations in dorsum and tubercle patterning and as such H. elegans should be considered a junior synonym of H. willeyi.
Etymology: This species is named in honor of Dr. Gustav Paulay, for his numerous contributions to molluscan research and his extensive collection of nudibranch sea slugs and other invertebrate taxa, which has greatly increased our understanding of marine biodiversity.
External morphology (Fig. 11d,e): Preserved animals 35-50 mm in length. Body oval, rigid, and gelatinous. Dorsum color bluish white with thick, black connected lines. Pronounced ridging in irregular pattern with yellow/orange coloration. Prominent yellow/orange tubercles at ridge intersections. Small yellow/orange tubercles, minute white tubercles, perpendicular black lines, and black spots along mantle edge. Mantle marginal band absent. Mantle underside bluish white with numerous short perpendicular black lines, medium black splotches, and small black spots along mantle edge. Large gill surrounds anus with four tripinnate branchial leaves split into two posterior and two anterior gill branches. Posterior gill branches further split into two large branches. Gill branches and rachises translucent white with blue tint and numerous small randomly dispersed black spots. Opaque white glands visible in gill branches. Gill pocket smooth with similar dorsum coloration and yelloworange ridges. Rhinophores tapered, perfoliate, and translucent white with blue tint, a posterior black stripe, and small to medium black splotches along stalk and lamellae. Lamellae number 20-30. Rhinophoral sheath color bluish white with yellow-orange ridges. Foot broad, anteriorly notched, and white with small black spots interspersed with short perpendicular black lines. Oral tentacles digitiform.
Internal anatomy (Figs. 5e, 7e and 13a-d): Buccal opening and buccal mass (Fig. 5e) pigmented on oral tube with small random black spots. Oral tube similar in size to buccal bulb. Radular sac curved and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth (Fig. 13a). Radular formula 43 × 40.0.40 in the holotype CASIZ 192298 and 37 × 30.0.30 in the paratype UF 476,040. In CASIZ 192298 inner eight to ten lateral teeth (Fig. 13b) small, broad based, with narrow, sharply curved cusps form a V in the center. Middle lateral teeth (Fig. 13c) larger than inner laterals with larger, less curved, slightly flared, rounded cusps. Outer lateral teeth (Fig. 13d) similar to middle laterals with shorter, unflared cusps. Outermost two to three teeth reduced, straighter than other lateral teeth with occasional single denticle.
Reproductive system (Fig. 7e): Triaulic. Thin preampullary duct gradually thickens into ampulla, then quickly narrows into postampullary duct which splits into thin, short vas deferens and thin, short oviduct. Vas deferens gradually thickens into wide, looped granular prostate, then narrows into thin, elongate ejaculatory portion. Ejaculatory portion rapidly expands into large penis, which shares common genital atrium with similarly sized vagina. Vagina abruptly narrows and gradually thins into elongate duct before entering large, rounded bursa copulatrix partially covered by granular prostate. Thin, elongate duct connects bursa to smaller, pyriform receptaculum seminis. Thin, elongate uterine duct also connects near receptaculum base and enters small, pyriformshaped female gland mass with elongated distal end.
Geographical distribution: Known from the Red Sea, including Saudi Arabia (Rudman 1978;Gosliner et al. 2008, present study), the Gulf of Aqaba (Debelius 1998), Yemen (Debelius and Kuiter 2007), and the Gulf of Tadjoura, Djibouti (Vayssière 1912).
Ecology: Found on shallow reefs and on steep slopes of fringing reef faces between 10 and 25 m.
Remarks: Our molecular phylogeny and all four species delimitation analyses reveal that Halgerda paulayi sp. nov. from the Red Sea is a distinct species within Halgerda and is separated from the similarly patterned H. willeyi by a minimum divergence of 9.5% in the COI gene. In our COI ABGD, COI ASAP, and bPTP analyses, the paratype UF 476040 collected from Jazirat Burcan, Saudi Arabia, is overpartitioned from the paratype UF 476041 also collected from Jazirat Burcan and the holotype CASIZ 192298 collected from Ablo Island, Saudi Arabia, due to a high intraspecific genetic divergence of 2.9%. Morphologically (Supplementary Figure S5), this paratype specimen is smaller than the other two specimens and has less pronounced ridging, thicker perpendicular black lines along the mantle edge, and finely tripinnate branches as a result. Internally, there are fewer inner lateral teeth and fewer rows; however, there are no discernable differences between the paratype and holotype reproductive systems. The over-partitioning may also be attributed to the low genetic diversity between H. paulayi sp. nov. and the other two members of this clade: H. jennyae and H. guahan. Within the COI gene, H. paulayi sp. nov. and H. jennyae are separated by a low genetic divergence of 2.3-2.9%, while H. paulayi sp. nov. and H. guahan are separated by a slightly higher genetic divergence of 3.4-4.2%.
Morphologically, these three species are very distinct. For example, H. guahan has low orange ridging along the dorsum with no additional dorsum coloration (Carlson and Hoff 1993, Fig. 3a), whereas H. jennyae has more pronounced ridging with high tubercles and a complex pattern of thin brown lines along the irregular ridges and valleys (Tibiriçá et al. 2018, Figs. 11d, e). In contrast, H. paulayi sp. nov. has pronounced yellow/orange ridging with thick black lines along the dorsum, ridge valleys, and mantle edge (Fig. 11d,e). These three species form a clade within the seventh subclade and share a similar spotting pattern on the rhinophores and gills that varies between stripes and solid coloration within rest of the subclade.
This species is also compared here to Halgerda johnsonorum Carlson & Hoff, 2000, described from the Marshall Islands and documented from New Caledonia (Gosliner et al. 2008). This species has low orange ridges that have low acutely pointed apices at the junction of ridges in contrast to steeper, more rounded ridge junctions found in H. paulayi sp. nov. In H. johnsonorum, the body is more translucent, and there are several fine black radiating lines between the ridges whereas the body of H. paulayi sp. nov. is opaque bluish white, and there is a single thicker line between the mantle ridges. The base of the rhinophore has numerous black spots in H. johnsonorum versus only 1-3 spots on the rhinophore base of H. paulayi sp. nov., and H. paulayi sp. nov. has a much more prominent black line on the entire posterior face of the rhinophore base. Internally, there are differences, as well. In H. paulayi sp. nov., the hamate outer lateral tooth has only a single denticle (Fig. 13d), whereas in H. johnsonorum the outer tooth is narrow with a bifid apex (Carlson and Hoff 2000, Fig. 15). Most significantly, the reproductive system of H. paulayi sp. nov. has large bulbous bases to both the vagina and penial sac (Fig. 7e), whereas these structures are both narrow and not significantly enlarged in H. johnsonorum (Carlson and Hoff 2000, Fig. 17). Halgerda johnsonorum as described from the Marshall Islands has some significant external differences to specimens recorded from New Caledonia as H. johnsonorum (Gosliner et al. 2018;110, upper right photo). In the New Caledonia specimens, the gill is much less divided and has black lines rather than pots on the branches and the narrow, more elongate rhinophores lack spotting and have only a prominent thin black line on their posterior face. A more detailed examination of New Caledonia specimens is necessary, but these differences suggest that they may represent a distinct, undescribed species.
Further morphological comparisons are made below between H. paulayi sp. nov. and the similarly patterned H. willeyi. Externally, the dorsum coloration of H. paulayi sp. nov. is bluish white with thick, black connected lines; pronounced irregularly patterned yellow/orange ridging and tubercles; and perpendicular thick black and orange lines along the mantle edge. In contrast, the dorsum coloration of H. willeyi is translucent white with low, irregular yellow and orange ridging, prominent lemon-yellow tubercles, and numerous thin perpendicular black lines along the mantle edge. The mantle underside and foot are similarly patterned between the two species with black perpendicular lines; however, in H. paulayi sp. nov., the mantle underside perpendicular lines are shorter than in H. willeyi and there are also small to medium black spots and blotches along both the mantle underside and the foot. The gill plumes are also uniquely distinct as H. paulayi sp. nov. has four tripinnate branchial leaves that further divide into six large branches which are colored translucent white with a blue tint and numerous randomly dispersedly small black spots. In H. willeyi there are two mostly bipinnate branchial leaves further divided into six large branches which are also translucent white but have dorsal brown stripes, diffused lateral brown stripes, and white rachises. The rhinophores are similar between the two species; however, H. paulayi sp. nov. has a higher number of small and large black spots along the stalk and lamellae, while H. willeyi has fewer spots along the stalk and lamellae which are browner in color. Additionally, adult specimens of H. paulayi sp. nov. studied here are generally larger in size (35-50 mm in length) than those of H. willeyi (13-35 mm in length). These two species are also geographically separated with H. johnsonorum being restricted to the western and central Pacific, possibly being endemic to the Marshall Islands whereas H. paulayi sp. nov. is restricted to the Red Sea and the Gulf of Aden. No specimens resembling either of these species have been found in any localities situated between the Red Sea and the Central Pacific.
Internally, the buccal opening and the oral tube of the buccal mass in H. paulayi sp. nov. are pigmented with small random black spots, whereas in H. willeyi neither are pigmented. The radular teeth in H. paulayi sp. nov. are similar to all Halgerda; however, there are only eight to ten small inner lateral teeth which have a more elongate stem and a sharply curved cusp and the outermost two to three teeth are reduced and straighter than the other lateral teeth. In contrast, there are 25 inner lateral teeth with a thicker elongated cusp in H. willeyi and the outermost two teeth are reduced and fimbriate. The reproductive system also varies as H. paulayi sp. nov. has a large, rounded bursa copulatrix partially covered by the granular prostate, a smaller pyriform receptaculum seminis, and a similarly sized large penis and vagina. In H. willeyi the rounded bursa copulatrix is much smaller and predominantly covered by the slightly smaller granular prostate, the receptaculum seminis is half the size of the bursa and oval-shaped, and the penis is smaller than the vagina, but tapers distally but tapers distally before expanding into the ejaculatory portion (present study, Fig. 7d).
Due to the morphological similarities between these two species, specimens of H. paulayi sp. nov. from the Red Sea have been previously misidentified as H. willeyi. Vayssière (1912) extensively described specimens from the Gulf of Tadjoura, Djibouti, but questions whether they are a variation of H. willeyi or a new species of Halgerda entirely. We are confident that these specimens are in fact H. paulayi sp. nov. based on the thick, connected black lines in coordination with the yellow/orange ridging across the mantle, the whitish tripinnate gill with numerous small black spots (Vayssière 1912, plate I, Fig. 7), the elongated stems in the first few inner radular teeth (Vayssière 1912, plate VII, Fig. 100), and the reduced, straighter outermost teeth (Vayssière 1912, plate VII, Fig. 101). Rudman (1978) described the anatomy of a specimen from Jeddah, Saudi Arabia, Red Sea; however, upon closer review this specimen is also H. paulayi sp. nov. due to the thick black, connected lines along the irregularly patterned ridging, the thick black perpendicular lines along the mantle edge, and the large, white-colored gill with numerous small black spots (Rudman 1978, Fig. 4a). Rudman also mentioned that the oral tube of the buccal mass is pigmented with spots, that there are seven small inner lateral teeth (Rudman 1978, Fig. 5a) and that the outermost two to three teeth are "simple flattened plates" or more accurately described as reduced and straight when compared with other lateral teeth (Rudman 1978, Fig. 5b), which also correlates with the morphology of H. paulayi sp. nov. described in the present study. In 1988, Frances Velkovrh collected a specimen of H. paulayi sp. nov. off the island of Cres near Baldarin Bay, Croatia in the northern Adriatic Sea. This specimen, previously identified as H. willeyi, was introduced into the Mediterranean via migration from the Red Sea through the Suez Canal or potentially through ballast water in traveling ships (Turk 2001;Rudman 2001). No further specimens have been recorded from the Mediterranean since 1988.
There are clear internal and external morphological differences, a strong genetic divergence, and even some geographical separation between H. paulayi sp. nov. H. johnsonorum, and H. willeyi. To date, H. paulayi sp. nov. is found in the Red Sea and the nearby Gulf of Tadjoura, Djibouti, while a specimen of H. willeyi has been previously documented off the coast of Oman in the Khuriya Muriya Islands, Arabian Sea it has been extensively documented throughout the western Pacific (See H. willeyi geographical distribution). Type locality: Horseshoe Cliffs, Onna Village, Okinawa, Ryukyu Islands, Japan.
Etymology: The name labyrinthus refers to the maze-like pattern of the numerous fine lines decorating the dorsum of this species.
External morphology (Fig. 11f): Preserved and living animals 84-95 mm in length. Body oval, semi-rigid, and gelatinous. Dorsum color white with numerous thin, black connected lines interspersed with yellow connected lines. Semi-pronounced ridging in irregular pattern with yellow coloration. Yellow mantle marginal band with continuing perpendicular black and yellow lines, raised yellow spots and occasional black spots near mantle edge. Mantle underside white with perpendicular lines of small black spots. Large gill surrounds anus with four tripinnate branchial leaves split into two posterior and two anterior gill branches. Posterior gill branches further split into two large branches. Gill branches translucent yellow with translucent white rachises and numerous small randomly dispersed black spots. Gill pocket smooth with white coloration and radiating yellow lines. Rhinophores tapered, perfoliate with thick stalks. White lamellae number 25-35 with small black spots. Rhinophore stalks white with small black spots and lateral black stripes. White rhinophoral sheath smooth with yellow ridges. Foot broad, anteriorly notched, and white with random small black spots and perpendicular lines with spots along foot edge. Oral tentacles digitiform.
Internal anatomy (Fig. 5f, 7f and 14a-d): Buccal mass (Fig. 5f) unpigmented. Buccal bulb approximately 1.5 × longer than oral tube. Radular sac semi-elongate, club shaped, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth (Fig. 14a). Radular formula 65 × 48.0.48 in the paratype CASIZ 160949. Inner 16 lateral teeth (Fig. 14b) small, broad based, with slightly curved cusps form a shallow V shape. Middle lateral teeth (Fig. 14c) larger than inner laterals with more curved, elongate, rounded cusps. Outer lateral teeth (Fig. 14d) larger than middle laterals and slightly less curved. Outermost two teeth reduced with irregular tips.
Reproductive system (Fig. 7f): Triaulic. Thin preampullary duct gradually widens into ampulla, then narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens gradually thickens into looped, granular prostate wrapped around bursa copulatrix, before narrowing into thin, elongate ejaculatory portion. Ejaculatory portion abruptly expands into penis, which shares common genital atrium with smaller vagina. Muscular vagina abruptly narrows proximally into thin, elongate duct and enters into large, rounded bursa copulatrix hidden under glandular prostate portion. Thin, elongate duct connects bursa to smaller, irregular-shaped receptaculum seminis. Short uterine duct also connects near receptaculum base and enters small, irregularly oval-shaped female gland mass with elongated distal end.
Geographical distribution: Known from the Ryukyu Islands, Japan (present study), Kerama Islands (Ono 1999(Ono , 2000)), the Izu Peninsula, Japan (Keiu 2000 Externally, H. labyrinthus sp. nov. varies from the previous comparison between H. willeyi and H. paulayi sp. nov. (see H. paulayi sp. nov. 'remarks' section above) due to the white dorsum coloration with numerous thin, black connected lines interspersed with thin, yellow lines; semipronounced yellow ridging; and a yellow mantle marginal band with raised yellow and black spots and perpendicular black and yellow lines along the mantle edge. The mantle underside and foot are similarly patterned to H. willeyi and H. paulayi sp. nov. with black perpendicular lines; however, in H. labyrinthus sp. nov., there are fewer lines with each line composed of sequential small black spots. There are also random small black spots interspersing the short lines along the foot. The gill plume is similar to H. paulayi sp. nov. with four tripinnate branchial leaves furthered divided into six large branches patterned with numerous small black spots; however, the branchial leaves are translucent yellow rather than white and the rachises are white rather than translucent white with a blue tint. The rhinophores are also similar to H. paulayi sp. nov. with a posterior black stripe, lateral black stripes, and small black spots; however, in H. labyrinthus sp. nov., there are more lateral stripes and small black spots as well as thicker rhinophoral stalks. Additionally, adult specimens of H. labyrinthus sp. nov. are two to three times larger (> 80 mm in length) than specimens of H. paulayi sp. nov. (35-50 mm in length) and H. willeyi (13-35 mm in length) studied here, are generally less rigid when preserved, and found at depths greater than 25 m.
Internally, the buccal opening and buccal mass of H. labyrinthus sp. nov. are similar to H. willeyi (i.e., unpigmented) and the radular teeth are similar to all Halgerda; however, there are 16 small inner lateral teeth which form a shallower V in the center of the radula. The middle and outer laterals are thicker and less elongated in the cusp and the two outermost teeth are reduced with curved cusps and irregular tips rather than reduced and straight (H. paulayi sp. nov.) or reduced and fimbriate (H. willeyi). The reproductive system in H. labyrinthus sp. nov. is similar to both H. willeyi and H. paulayi sp. nov., however, the bursa copulatrix is completely covered by the granular portion of the prostate; the receptaculum is small, more elongate, and irregularly shaped; and the uterine duct is much shorter than either found in H. willeyi or H. paulayi sp. nov. The penis and vagina are both smaller in H. labyrinthus sp. nov., but the vagina is similarly muscular to the one found in H. paulayi sp. nov. (present study, Fig. 7e).
Previously, specimens of H. labyrinthus sp. nov. have been either mis-identified as H. willeyi or suggested as a variation of H. willeyi due to the similarities in dorsum coloration and patterning. This may be attributed to a lack of geographical separation between the two species, since both are found in Okinawa, Japan. There is, however, geographical separation between H. labyrinthus sp. nov. and the similarly patterned H. paulayi sp. nov. as the former is found only in Japan and Taiwan and the latter is found only in the Saudi Arabian Red Sea. Despite the similarities between these three species, there are strong morphological characteristics and a large genetic distance that separate them, making each a distinct species within Halgerda.
Given the small genetic difference between H. labyrinthus sp. nov. and H. batangas, we also compare these species to each other. Halgerda labyrinthus sp. nov. is a large species ranging from 84-95 mm, whereas H. batangas reaches a maximum length of 40 mm. Additionally, H. batangas lacks any black lines on the notum that dominate the body color of H. labyrinthus sp. nov., while H. labyrinthus sp. nov. lacks bright orange tubercles and fine orange lines found in H. batangas. Internally, the radula of H. labyrinthus sp. nov. has only the outermost tooth with a bifid apex, whereas in H. batangas the outermost tooth is strap-shaped and the adjacent two teeth are bifid (Carlson and Hoff 2000, Fig. 9). The configuration of the reproductive system also differs in the two species. In H. labyrinthus sp. nov., the vagina is narrow and muscular but lacks a ring of glandular structures immediately proximal to its opening. In H. batangas, the muscular vagina is as wide as the penis and has a ring of glandular structures immediately proximal to the vagina (Carlson and Hoff 2000, Fig. 11) Type locality: South Madagascar, Madagascar. Etymology: This species is named "anosy" after the Malagasy word for island or land of the islands and refers to the type locality of the island of Madagascar.
External anatomy: Preserved animals 20-55 mm in length (Figs. 11g-h). Body oval, rigid, and gelatinous. Dorsum color bluish white with many medium dark brown/black spots along mantle edge. Low yellow ridging in irregular pattern with semi-projected yellow tubercles at ridge intersections. A single large dark brown/black spot or two conjoined small dark brown/black spots present within ridge depressions. Viscera with dark blue/purple coloration semivisible through mantle. Smaller specimens (Fig. 11h) have less pronounced ridging with fewer, lighter colored spots and barely visible viscera. Thin white line present along mantle edge. Mantle underside bluish white with anterior mantle edge small dark brown/black spots clearly visible through mantle and larger dark brown/black spots only semivisible through mantle. Gill surrounds elevated anus with four highly bipinnate branchial leaves split into two posterior and two anterior gill branches. Posterior gill branches further split into two large branches. Gill coloration bluish white with central anterior dark brown/black stripe and bluish white rachises. Gill pocket smooth with similar dorsum coloration. Opaque white glands present within branchial leaves. Rhinophores tapered, perfoliate, with 5-6 white lamellae, then 18-22 dark brown/black lamellae, and a white apical tip. Rhinophore stalks bluish white with small posterior dark brown/black spot. Rhinophoral sheath smooth with yellow coloration. Foot is broad, anteriorly notched, bluish white with small to medium dark brown/black spots. Oral tentacles digitiform.
Internal anatomy (Fig. 5g, 7g and 15a-d): Buccal mass (Fig. 5g) unpigmented; however, small dark spots present around buccal opening. Buccal bulb approximately 1.5 × larger than oral tube. Radular sac short, rounded, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth (Fig. 15a). Radular formula 48 × 44.0.44 in the paratype CASIZ 194616B. Inner 15 lateral teeth (Fig. 15b) small, broad based, with curved, elongate, rounded cusps forming a slightly spaced V shape. Some of the first three inner teeth are bifurcated. Middle lateral teeth (Fig. 15c) are larger, thicker, and slightly more elongated than inner laterals. Outer lateral teeth (Fig. 15d) are slightly larger than middle laterals and less curved. Outermost two teeth reduced and semi-fimbriate.
Reproductive system (Fig. 7g): Triaulic. Thin preampullary duct widens into thick ampulla, then gradually narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens quickly expands into wide, elongate, looped granular prostate which abruptly narrows into thin, elongate, convoluted ejaculatory portion. Ejaculatory portion quickly expands into large penis, which shares a common genital atrium with a similarly size vagina. Vagina contains finger-like projections before proximally narrowing into thin, elongate duct and entering into rounded bursa copulatrix partially covered by granular prostate. Elongate duct connects bursa to smaller, semi-rounded receptaculum seminis. Thin, elongate uterine duct also connects near receptaculum base and enters large, semi-rounded female gland mass.
Geographical distribution: Known from South Madagascar.
Ecology: Found on patch reefs and fringing reefs between 5-50 m.
Remarks: Halgerda anosy sp. nov. is part of a large, unresolved, subclade including H. dichromis, H. aff. wasinensis, H. wasinensis, and H. cf. formosa. The COI ABGD analysis successfully separates out H. anosy sp. nov. from the previously mentioned species; however, 16S ABGD, the COI and 16S ASAP, the GMYC, and the bPTP analysis fail to recover H. anosy sp. nov. and the previously mentioned species as distinct species and instead groups them together in various large taxonomic units. This over and under grouping may be attributed to the low interspecific variation between some of the species within this clade (Table 2). There is a minimum divergence of 3.3%, 1.5%, 2.9%, and 1.1% in the COI gene between H. anosy sp. nov. and H. cf. formosa, H. dichromis, H. aff wasinensis, and the H. wasinensis complex, respectfully. There is also minimal intraspecific variation (0.0-0.6%) between the five specimens studied here from South Madagascar. Morphologically, H. anosy sp. is most similar with a specimen of H. cf. formosa (MHN-YT1682) from Mozambique and H. wasinensis. Externally, the dorsum coloration of H. anosy sp. nov. is bluish white with many dark brown to black spots along the mantle edge, low yellow ridging with semi-projected yellow tubercles at ridge junctions, and either a single large or two conjoined small dark brown to black spots present within ridge depressions. In contrast, the dorsum coloration of H. cf. formosa is whitish with a gray tinge and a handful of large dark brown to black spots along the mantle edge and occasionally present in some of the low, angled, yellow ridge depressions (Tibiriçá et al. 2018, Fig. 3a). There are no tubercles at the ridge junctions; however, there are numerous minute white tubercles along the mantle edge. Both species have a thin white mantle band along the mantle edge, semi-visibility of the dark brown to black spots through the mantle underside, and several smaller dark brown to black spots along the foot. The gill plumes are distinct as H. anosy sp. nov. has four large bluish white bipinnate gill branches, each with a central anterior dark brown to black stripe and bluish white rachises, while in H. cf. formosa the gill plume is whiter with four large tripinnate branchial leaves each with black-lined branches and black branch tips. The rhinophores are similar between the two species with dark brown to black lamellae and a white apical tip; however, in H. anosy sp. nov., the posterior dark brown to black spots along the rhinophoral stalk are much smaller than the posterior, longitudinal band seen in H. cf. formosa. Halgereda wasinensis is highly variable in the amount of black pigment present (Tibiriçá et al. 2018) but always has at least some irregular dark blotches rather than the regular spots that form circles or ovals in H. anosy sp. nov. The ridge network lines of H. anosy sp. nov. are more distinctly yellow in contrast to orange lines of H. wasinensis. The base of the gill branches in H. wasinensis is thick with a much more extensive area of opaque white than in H. anosy sp. nov. In H. wasinensis the posterior base of the rhinophores has an extensive black line that extends the entire length of the rhinophore stalk in contrast to an isolated black spot at the base of the rhinophore stalk in H. anosy sp. nov.
Internally, both H. anosy sp. nov. and H. cf. formosa have unpigmented buccal masses in contrast to that described for H. wasinensis (Rudman 1978;Tibiriçá et al. 2018), however, in H. anosy sp. nov., there are small dark spots around the buccal opening and the radular sac is short and rounded, whereas in H. cf. formosa, there is no mention of the presence or absence of pigment around the buccal opening and the radular sac is more elongate. In H. anosy sp. nov. and H. wasinensis (Rudman 1978;Tibiriçá et al. 2018), the inner lateral teeth are small with curved, elongate, rounded cusps which form a spaced-out V shape in the center of the radula, while in H. cf. formosa the inner lateral teeth are less elongate and much more tightly packed to form a steep V shape in the radula center (Tibiriçá et al. 2018, Fig. 4b). The middle teeth are larger and less elongate in H. anosy sp. nov. with more rounded tips than in H. cf. formosa or H. wasinensis, and the outermost two teeth are reduced and semi-fimbriate. In H. cf. formosa the middle teeth are also larger, but more elongate than those in H. anosy sp. nov. and the tips are more pointed (Tibiriçá et al. 2018, Fig. 4c). The three outermost teeth in H. cf. formosa are also reduced, but much more fimbriate (Tibiriçá et al. 2018, Fig. 4d) than are those of H. anosy or H. wasinensis. All three species reproductive systems share some similarities including a small receptaculum seminis, an elongate ejaculatory portion, a large penis, and deep finger-like projections in the proximal portion of the vagina. Notable differences from H. cf. formosa include a much larger female gland mass in H. anosy sp. nov. and H. wasinensis and the bursa copulatrix is only partially covered by the granular portion of the prostate. The differences in the size of the female gland mass could be easily due to different levels of maturity in the specimens examined. More significantly, in H. cf. formosa and H. wasinensis, there is a muscular sphincter at the entry to the vagina which is absent in H. anosy sp. nov. (Tibiriçá et al. 2018, Fig. 5a, b). The pronounced internal and external differences between H. anosy sp. nov., H. wasinensis, and H. cf. formosa (MHN-YT1682) supports H. anosy sp. nov. as new species within Halgerda. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ E20B6 41A-6EFF-40F4-A69C-C491B FD21A 46 (Fig. 16a,17a,18 and 19a) Halgerda sp. 4 Gosliner et al. (2015): 190, top right photograph. Halgerda sp. 11 Gosliner et al. (2018): 112, top right photograph. Halgerda sp. Stiefel (2011).
Type material: Holotype, One specimen, NMP 041324, formerly CASIZ 193832, dissected and sequenced, 55 mm preserved, type locality: Mapating, Tingloy, Balayan Bay, Luzon Island, Philippines, 61 m depth, 11 December 2013, collected by E. Jessup.
Type locality: Mapating, Tingloy, Balayan Bay, Luzon Island, Philippines.
Etymology: The name mesophotica refers to the depth (i.e. the middle mesophotic zone) in which this species is found.
External morphology: Preserved animal 55 mm in length (Fig. 16a, Supplementary Figure S6a). Body oval, semi-rigid, and gelatinous. Dorsum color translucent white with sporadic small dark brown/black spots and extremely large, conical tubercles. Central tubercles with series of radiating thick brown lines. Outer tubercles shorter with no coloration. Thin, white mantle band along mantle edge. Mantle underside translucent white with random small to medium brown and dark brown spots near foot. Elevated gill surrounds anus with four branchial leaves split into two posterior and two anterior gill branches. Each posterior branch splits into two-three large secondary branches. Gill branches long, thin with few very short pinnae extending from each branch. Leaves and rachis translucent white with thick brown dorsal stripe. White glands visible in gill branches. Gill sheath smooth with similar dorsum coloration and radiating series of brown lines. Rhinophores long, tapered, and perfoliate with 34 cream-colored lamellae. Rhinophoral stalk and lamellae translucent white with posterior dark brown stripe. Rhinophoral sheath smooth with similar dorsum coloration and brown lines. Foot is broad, anteriorly notched, and translucent white. Oral tentacles digitiform.
Internal anatomy (Fig. 17a, 18a-d and19a): Buccal mass (Fig. 17a) unpigmented. Buccal bulb 1.5 × longer than elongate oral tube. Radular sac elongate, irregular shaped, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 18a). Radular formula 41 × 74.0.74 in the holotype NMP 041324. Inner 31 lateral teeth (Fig. 18b) small, broad-based with elongated stems, sharply curved elongated cusps form a V in the center. Middle lateral teeth (Fig. 18c) larger than inner laterals with more curved, elongated, rounded cusps. Outer lateral teeth (Fig. 18d) larger and thicker than middle laterals. Outermost three teeth reduced versions of the outer laterals with more pointed tips.
Reproductive system (Fig. 19a): Triaulic. Thin preampullary duct quickly widens into thick ampulla, then narrows into postampullary duct which splits into the vas deferens and oviduct. Vas deferens rapidly expands into wide, elongate, and curved granular prostate which abruptly narrows gradually into thin, elongate, curved ejaculatory portion. Ejaculatory portion rapidly expands into large, wide penis and shares common genital atrium with similarly sized vagina. Large, wide vagina abruptly narrows proximally into very thin, elongate duct and enters large, rounded bursa copulatrix partially covered by granular prostate. Very thin, elongate duct connects bursa to smaller, tapered receptaculum seminis. Thin, semi-elongate uterine duct connects near receptaculum base and enters medium-sized, irregularly pyriform-shaped female gland mass.
Geographical distribution: Known from the Philippines. Ecology: Found on deep reefs in the mesophotic zone at approximately 60-70 m (Steifel 2011).
Remarks: In our molecular phylogeny, Halgerda mesophotica sp. nov. is part of a well-supported mesophotic clade including H. scripta sp. nov., H. okinawa, H. profunda sp. nov., and H. takipsilim sp. nov. The COI and 16S ABGD, the COI ASAP, the GYMC, and bPTP analyses successfully separates H. mesophotica sp. nov. from H. okinawa and H. scripta sp. nov.; however, the 16S ASAP analysis does group H. mesophotica sp. nov. with one specimen of H. scripta sp. nov. and the rest of the mesophotic clades. There are genetic differences greater than 5.5% in the COI gene; however, all four delimitation analyses group H. mesophotica sp. nov. with H. profunda sp. nov. and H. takipsilim sp. nov. This may be attributed to the low genetic divergences (0.9-1.5%) in the COI gene between the three species; however, there are strong morphological differences in each species (summarized in Table 4). Therefore, H. mesophotica sp. nov. is morphologically compared with H. profunda sp. nov. and H. takipsilim sp. nov. in the H. takipsilim sp. nov. remarks section below. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ 2FC8E F5F-4198-4DDC-9F70-D0E36 71CF9 5D (Fig. 16b,17b,19b and20 Etymology: This species is named "profunda" after the Latin word for deep and refers to the deep depths of the lower mesophotic zone in which this species is found.
External morphology: Preserved animal 60 mm in length (Fig. 16b). Body oval, rigid, and gelatinous. Dorsum color translucent white with small black spots along mantle edge. Irregular dark black ridging with large, conical light yellow-tipped tubercles. Thin, white mantle band along mantle edge. Mantle underside translucent white with small dark brown/black spots near mantle edge. Elevated gill surrounds anus with four highly bipinnate branchial leaves split into two posterior and two anterior gill branches. Each posterior branch splits into two-three large secondary branches each bearing an elongate lateral pinnae. Leaves and rachis translucent white with dorsal thin black stripes and small black spots. Thin area of white glands visible in gill branches. Anal papilla with black pigment at apex. Gill sheath smooth with similar dorsum coloration and small black spots. Rhinophores tapered, and perfoliate with 24 cream-colored lamellae. Rhinophoral stalk and lamellae translucent white with posterior black stripe. Rhinophoral sheath smooth with similar dorsum coloration and radiating black lines. Foot is broad, anteriorly notched, translucent white with small dark brown/black spots and medium dark brown/black blotches. Oral tentacles digitiform.
Internal anatomy (Fig. 17b, 19b and 20a-d): Buccal mass (Fig. 17b) unpigmented; however, dark brown/black blotches present around buccal opening. Buccal bulb 2 × longer than oral tube. Radular sac rounded, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 20a). Radular formula 48 × 57.0.57 in the holotype NMP 041325. Inner 15 lateral teeth (Fig. 20b) small, broad-based with thin elongated stems and thin elongated cusps form a V in the center. Middle lateral teeth (Fig. 20c) larger and thicker than inner laterals with more sharply curved cusps and rounded tips. Outer lateral teeth (Fig. 20d) larger and thicker than middle laterals with shorter curved cusps. Outermost three teeth reduced, thinner, and straighter than other teeth with slightly curved tips.
Reproductive system (Fig. 19b): Triaulic. Thin, elongate preampullary duct gradually widens into semi-thick ampulla, then quickly narrows into postampullary duct which splits into vas deferens and oviduct. Vas deferens quickly expands into wide, elongate, and looped granular prostate which abruptly narrows into thin, very elongate, and looped ejaculatory portion. Ejaculatory portion rapidly expands into large, wide penis which shares common genital atrium with slightly thinner, similarly sized vagina. Large, wide vagina quickly narrows proximally into gradually thinning duct and enters large, rounded bursa copulatrix partially covered by granular prostate. Thin, elongate duct connects bursa to small, thin oblong receptaculum seminis. Short, thin uterine duct connects near receptaculum base and enters large, semi-pyriform shaped female gland mass.
Geographical distribution: Known from the Philippines (Gosliner et al. 2015(Gosliner et al. , 2018;;present study).
Ecology: Found on deep reefs in the mesophotic zone at approximately 90 m.
Remarks: In the mesophotic clade, Halgerda profunda sp. nov. is sister to H. takipsilim sp. nov. and is successfully separated from H. okinawa and H. scripta sp. nov. during all but the 16S ASAP analyses. The COI ABGD revealed genetic differences greater than 5.8% in the COI gene between H. profunda sp. nov. and the previously mentioned species. Since there are low genetic differences between H. profunda sp. nov., H. mesophotica sp. nov., and H. takipsilim sp. nov. all three species are morphologically compared below in the remarks section following H. takipsilim sp. nov and summarized in Table 4. Type locality: South Pinnacle, Verde Island, Luzon Island, Philippines.
This species is named "takipsilim" after the Filipino word for twilight and refers to the great depths (i.e., the lower mesophotic zone) in which this species is found.
External morphology: Preserved animal 30-45 mm in length (Fig. 16c). Body oval, semi-rigid, and gelatinous. Dorsum color semi-translucent white with randomly distributed large black spots. Irregular brown ridging with large, conical bright yellow-tipped, rounded tubercles. Thin, white mantle band along mantle edge. Mantle underside semi-translucent white with sporadic small brown spots. Elevated gill surrounds anus with four highly bipinnate, branchial leaves split into two posterior and two split anterior gill branches. Each posterior branch splits into two-three large secondary branches. Leaves and rachis semitranslucent white with small brown dotted dorsal lines and numerous small single brown spots. White glands visible in gill branches. Anal papilla without dark pigment. Gill sheath smooth with similar dorsum coloration. Rhinophores elongated, tapered, and perfoliate with 37-40 light yellow lamellae. Rhinophoral stalk semi-translucent white with posterior dark brown stripe and small black spot. Rhinophoral sheath smooth with similar dorsum coloration. Foot is broad, anteriorly notched, and semi-translucent white. Ring of small dark brown/black spots near foot bottom. Ring of large brown blotches near top of foot connecting to mantle. Oral tentacles digitiform.
Internal anatomy (Figs. 17c,: Buccal mass (Fig. 17c) unpigmented; however, small brown flecks/spots present around buccal opening. Oral tube 1.5 × longer than buccal bulb. Radular sac semi-elongate, irregularly rounded, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 21a). Radular formula 48 × 52.0.52 in the holotype NMP 041327. Inner 17 lateral teeth (Fig. 21b) small, broad-based with short, highly curled cusps form a V in the center. Middle lateral teeth (Fig. 21c) larger and thicker than inner laterals with more elongated, sharply curved cusps. Outer lateral teeth (Fig. 21d) larger and thicker than middle laterals with shorter, rounded cusps. Outermost three teeth reduced, thinner, and slightly more curved than other lateral teeth.
Reproductive system (Fig. 19c): Triaulic. Thin, elongate preampullary duct gradually widens into slightly wide ampulla, then gradually narrows into postampullary duct which splits into short oviduct and short vas deferens. Vas deferens gradually expands into wide, elongate, looped granular prostate and then abruptly narrows into thin, elongated, and curved ejaculatory portion. Ejaculatory portion rapidly expands into large, wide penis which shares common genital atrium much smaller and thinner vagina. Slightly wide, elongate vagina narrows proximally into thinner, elongated duct and enters large, rounded bursa copulatrix partially covered by granular prostate. Very thin, elongate duct connects bursa to thin, elongate, oblong-shaped receptaculum seminis. Short, thin uterine duct connects near receptaculum base and enters small-medium sized irregularly rounded female gland mass.
Geographical distribution: Known only from the Philippines.
Ecology: Found on deep reefs in the mesophotic zone between 80-90 m.
Remarks: In all of our delimitation analyses, H. mesophotica sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov. are grouped together as a single taxonomic unit due to low genetic differences. All three species were found originally in the Philippines; however, H. mesophotica sp. nov. was found in Balayan Bay at a slightly shallower depth (60 m), H. profunda sp. nov. was found at Lubang Island at 90 m in depth, and H. takipsilim sp. nov. was found at Verde Island at 80 m depth and at Lubang Island at 90 m in depth. Externally, all three species have a semi-translucent/ translucent white dorsum with sporadic small black spots; large, rounded to conical tubercles; spots along the mantle underside and foot; and a thin white mantle band; however, there are distinct patterns in the ridging and tubercles. In H. profunda sp. nov., the irregular ridging is dark black in color with large, yellow-tipped conical tubercles, whereas in H. takipsilim sp. nov., the irregular ridging is brown, and the bright yellow-tipped tubercles have a broader base and a quickly elongating, more rounded tip. In H. mesophotica sp. nov., there is no ridging, and each tubercle has a series of brown lines radiating from the tip. The rhinophores and gills also vary between the three species. All three have the dark posterior stripe along the rhinophoral stalk, but it varies in color. In H. mesophotica sp. nov. and H. takipsilim sp. nov., the stripe is dark brown, while in H. profunda sp. nov., the stripe is black. Additionally, the lamellae in H. takipsilim sp. nov. are light-yellow in color, rather than cream as seen in H. mesophotica sp. nov. and H. profunda sp. nov. All three species have a gill composed of four bipinnate leaves with two anterior branches and two posterior branches with two to three large secondary branches; however, the branching of the gill, coloration, and patterning vary. In H. mesophotica sp. nov., the gill branches are thin and elongate with very short pinnae as is found in H. scripta sp. nov. In H. profunda sp. nov., the gill branches are elongate and wide with elongate lamellae, whereas in H. takipsilim sp. nov., the gill branches are short with fewer pinnae that have very faint dark pigment. In H. mesophotica sp. nov., the leaves and rachises are translucent white with a thick brown dorsal stripe and radiating brown lines along the gill sheath, while in H. profunda sp. nov., the leaves and rachises are translucent white, and the dorsal stripes are thin and black with extra small black spots. In H. takipsilim sp. nov., the leaves and are semi-translucent cream with small brown dotted lines and numerous single brown spots along the dorsal side.
Internally, the buccal mass, radular teeth and the reproductive systems are similar, but there are noticeable differences. The buccal mass of H. mesophotica sp. nov. and H. profunda sp. nov. is larger than the oral tube and the radular sac is elongate in H. mesophotica sp. nov. but short in H. profunda sp. nov. In contrast, H. takipsilim sp. nov., the buccal mass is much shorter than the oral tube and the radular sac is moderately elongate. In H. mesophotica sp. nov., there are no markings around the buccal opening, while there are dark brown/black blotches around the buccal opening in H. profunda sp. nov. and small brown flecks/spots in H. takipsilim sp. nov. The radular teeth in H. mesophotica sp. nov. include 31 small inner lateral teeth with elongated stems and sharpy curved cusps, while in H. profunda sp. nov., there are only 15 small inner lateral teeth with thin elongated stems and thin elongated cusps, and in H. takipsilim sp. nov., there are only 17 small inner lateral teeth with elongated stems and short, highly curved cusps. All three species have similar middle and outer lateral teeth (i.e., larger and elongated); however, in H. mesophotica sp. nov., the three outermost teeth are reduced versions of the outer laterals with more pointed tips. In contrast, the three outermost teeth in H. profunda sp. nov. are reduced, thinner, straighter, and only slightly curved at the tips. The outermost three teeth in H. takipsilim sp. nov. are also reduced and thinner but more curved than in H. profunda sp. nov. and less elongated than those in H. mesophotica sp. nov.
In all three species the reproductive system is composed of a large, spherical bursa copulatrix partially covered by the granular portion of the prostate; a large penis approximately half the width of the bursa; and a much smaller receptaculum seminis. In H. mesophotica sp. nov. and H. profunda sp. nov., the vagina is a similar size to the penis, but in H. takipsilim sp. nov., the vagina is much smaller and more elongated. The ejaculatory portion of the prostate is elongated in all three species, but in H. mesophotica sp. nov. and H. takipsilim sp. nov. it only curves once or twice and in H. profunda sp. nov. it is looped and twice as long. The size and length of the ducts connecting the proximal end of the vagina to the bursa copulatrix and the bursa to the split into the receptaculum seminis/uterine duct also varies between species. In H. mesophotica sp. nov., both ducts are thin, elongated and a similar length, whereas in H. profunda sp. nov., the vagina to bursa duct is initially wide and then gradually narrows and the bursa to receptaculum duct is consistently thin. In H. takipsilim sp. nov. both ducts are a similar length; however, the vagina to bursa duct is twice as thick as the bursa to receptaculum duct. The female gland mass is a similar size in H. mesophotica sp. nov. and H. takipsilim sp. nov., but twice as large in H. profunda sp. nov.
Despite the similarities between H. mesophotica sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov., there are consistent, unique internal and external characteristics 4) that separate these three species from one another and their distinctness as species within Halgerda. Examination of five specimens H. scripta sp. nov. below demonstrates the consistency of color pattern in species of this clade and further supports the distinctness of the other members of this clade, based on features of their color pattern. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ 54C1B FC7-21E9-447E-8B74-3FDA9 4C7D5 74 17d,19d Etymology: The name scripta refers to the fine, script like linework ornamenting the dorsum of this species.
External morphology: Preserved animals 35-50 mm in length (Fig. 16d,e). Body oval, rigid, and gelatinous. Dorsum color bluish white with numerous medium black spots along mantle margin. Large conical tubercles with radiating thin black lines. Thin, white mantle band along mantle edge. Mantle underside bluish white with random small to medium brown/dark brown spots. Elevated gill surrounds anus with four bipinnate branchial leaves split into two posterior and two anterior gill branches. Each posterior branch splits into two-three large secondary branches. Leaves and rachis semi-translucent white with dorsal black stripe and random small black spots. White glands visible in gill branches. Anal papilla unpigmented or with black pigment at apex. Gill sheath smooth with similar dorsum coloration and radiating series of thin black lines. Rhinophores long, tapered, and perfoliate with 32-35 cream-colored lamellae. Rhinophoral stalk semi-translucent white with posterior black stripe and small lateral black spots. Rhinophoral sheath smooth with similar dorsum coloration and radiating thin black lines. Foot is broad, anteriorly notched, and bluish white with small random dark brown/black spots. Oral tentacles digitiform.
Internal anatomy (Figs. 17d,: Buccal mass (Fig. 17d) unpigmented; however, small dark brown/ black spots present around buccal opening. Buccal bulb approximately 2.5 × larger than oral tube. Radular sac elongate, semi-folded over, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 22a). Radular formula 50 × 70.0.70 in the holotype NMP 041326 and 45 × 50.0.50 in the paratype CASIZ 193823. In NMP 041326 inner 27 lateral teeth (Fig. 22b) small, broad-based with elongated sharply curved cusps form a steep V in the center. Middle lateral teeth (Fig. 22c) are larger, thicker than inner laterals with longer curved cusps and rounded tips. Outer lateral teeth (Fig. 22d) are larger, thicker, and less curved than middle laterals. Outermost three to five teeth gradually reduced with slightly curved tips.
Reproductive system (Fig. 19d): Triaulic. Thin preampullary duct gradually widens into thick ampulla, then gradually narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens rapidly expands into wide, extremely elongate, fairly straight granular prostate which abruptly narrows into thin, elongate, and looped ejaculatory portion. Ejaculatory portion quickly expands into very large, wide penis, which shares common genital atrium with slightly smaller vagina. Wide vagina, half-length of penis, abruptly narrows proximally into thin elongate duct and enters into large, rounded bursa copulatrix partially covered by granular prostate. Very thin, elongate duct connects bursa to small, oval-shaped receptaculum seminis. Short, very thin uterine duct connects near receptaculum base and enters large female gland mass.
Geographical distribution: Known from the Verde Island Passage, Philippines.
Ecology: Found on deep reefs in the mesophotic zone between 65-80 m.
Remarks: Our molecular phylogeny places Halgerda scripta sp. nov. at the base of a mesophotic clade including H. okinawa, H. mesophotica sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov. The species delimitation analyses clearly indicate that H. scripta sp. nov. is a distinct species within the mesophotic clade with a genetic divergence of 6.7-8.2% in the COI gene. However, our 16S ABGD and ASAP analyses grouped one specimen of H. scripta sp. nov. with H. sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov. and our COI ABGD and bPTP analyses over-partitioned the same specimen of H. scripta sp. nov. collected on Maricaban Island (CASIZ 193823) from the other four specimens including the holotype NMP 041326 collected on Verde Island. Externally, this paratype specimen is smaller than either the holotype or the other paratypes. Internally, there are fewer inner lateral teeth and fewer rows; however, there are no discernable differences between the paratype and holotype reproductive systems (Supplementary Figure S7). Ong et al. (2017) suggested that there may be genetic isolation within the Verde Island Passage (VIP) after they found an intraspecific genetic divergence of 1.7% in the COI gene between two Gastropteridae specimens collected 22 km apart on the north and south sides of the VIP. This may explain the high maximum intraspecific genetic divergence of 2.2% between the H. scripta sp. nov. specimen CASIZ 193823 and the holotype NMP 041326 since there is a 21 km separation between Maricaban Island and the Verde Island.
Halgerda scripta sp. nov. shares some morphological similarities with other members of the mesophotic clade (Table 4); however, it most similar with H. jennyae in its color pattern. Externally, the dorsum coloration of H. scripta sp. nov. is bluish white with prominent conical tubercles and radiating thin black lines, numerous medium black spots along the mantle margin with a thin white mantle band, small random dark brown/black spots along the foot, and small to medium brown/dark brown spots along the underside of the mantle. In contrast, H. jennyae has a translucent white dorsum with high tubercles, a complex pattern of thin brown lines along the irregular ridges and valleys, a thin white mantle band, and the foot and mantle underside have numerous small to medium brown spots (Tibiriçá et al. 2018, Figs. 11d, e). There are also differences in the coloration of the rhinophores and gills. In H. scripta sp. nov., the rhinophores are semi-translucent white with cream lamellae and a posterior black stripe and small lateral black spots, whereas in H. jennyae the rhinophore stalks are translucent with yellowish lamellae and brown lines on the dorsal and lateral sides. The gill in H. jennyae is translucent white the numerous brown spots, while in H. scripta sp. nov., the gill is semi-translucent white with a dorsal black stripe and random small black spots.
Internally, the buccal mass of H. scripta sp. nov. is unpigmented and the radular teeth are similar to other Halgerda; however, there are 27 small inner teeth which from a sharp V in the center of the radula. Additionally, the middle and outer lateral teeth have long curved cusps with rounded tips and the outermost three to five teeth gradually reduce in size and curvature. In H. jennyae the buccal mass is pigmented and the radular teeth are pigmented in yellow or gold. The inner 16-18 lateral teeth are similar to H. scripta sp. nov.; however, the middle and outer lateral teeth are much thicker the entire length of the curved cusp (Tibiriçá et al. 2018, Fig. 15a-c). The outermost three to five teeth are also slightly more curved than those in H. scripta sp. nov. (Tibiriçá et al. 2018, Fig. 15d). The reproductive system varies as H. scripta sp. nov. has a small receptaculum seminis; a large penis connected to an extremely elongate and looped ejaculatory portion; a slightly shorter, vagina; and a large, rounded bursa copulatrix which is only slightly covered by a portion of the elongated granular prostate. In contrast, the receptaculum seminis in H. jennyae is slightly larger though similarly shaped to H. scripta sp. nov.; the penis and vagina are small, similarly sized and the ejaculatory portion is much shorter; and the granular prostate completely covers the slightly smaller bursa copulatrix (Tibiriçá et al. 2018, Fig. 14b).
There are some morphological similarities between H. scripta sp. nov. and H. jennyae; however, our molecular analyses clearly indicate that these are two distinct species as they are found in completely different clades and separated by a minimum divergence of 7.5% in the COI gene. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ C5A09 FCC-573C-4FB8-AF4C-3D3C0 560B7 1F (Fig. 16f,17e,19e and 23) Halgerda sp. 1 Hervé (2010): 197-198 Type locality: La Joliette, Thio, New Caledonia Lagoon, New Caledonia.
Etymology: This species is named in honor of Jean-François Hervé, who collected and photographed the type specimen from New Caledonia.
External morphology: Preserved animal 40 mm in length (Fig. 16f, Supplementary Figures S6b-d). Body oval, semirigid, and gelatinous. Dorsum color semi-translucent white with sporadic small black spots. Irregular dark brown/black ridging with large, pronounced tubercles at junctions. tubercles semi-translucent white with series of connected radiating dark brown/black lines. Thin, white mantle band and perpendicular dark brown/black lines along mantle edge. Mantle underside semi-translucent white with occasional small to medium light brown spots. Elevated gill surrounds unpigmented anus with two bipinnate, branchial leaves split into two posterior and two anterior gill branches. Upper onethird of leaves and rachis translucent white with thin dark brown/black dorsal stripes. Lower two-thirds of leaves translucent yellow with ventral dark brown/black spots. White glands visible in gill branches. Gill sheath smooth with similar dorsum coloration and radiating series of black lines. Rhinophores long, tapered, and perfoliate with 26 lamellae. Rhinophoral stalk and lamellae translucent white with posterior dark brown/black stripe. Small black spots present near stalk base. Rhinophoral sheath smooth with similar dorsum coloration, small black spots, and radiating black lines. Foot is broad, anteriorly notched, and semi-translucent white with sporadic small to medium light brown spot. Oral tentacles digitiform.
Internal anatomy (Figs. 17e,: Buccal mass (Fig. 17e) unpigmented; however, small light brown spots present around buccal opening. Buccal bulb 2.5 × longer than short, rounded oral tube. Radular sac elongated, rounded, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 23a). Radular formula 45 × 47.0.47 in the holotype CASIZ 186494. Inner 19 lateral teeth (Fig. 23b) small, thick with sharply curved cusps form a steep almost vertical V in the center. Middle lateral teeth (Fig. 23c) larger and thicker than inner laterals with a more elongated, curved cusp with rounded tips. Outermost lateral teeth (Fig. 23d) are slightly larger and thicker than middle laterals with shorter curved cusps. Outermost two teeth are small, paddle-shaped, and semi-fimbriate.
Reproductive system (Fig. 19e): Triaulic. Preampullary duct quickly widens into thick ampulla, then quickly narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens rapidly expands into wide, elongate, and curved granular prostate which abruptly narrows into thinner, elongate, and looped ejaculatory portion. Ejaculatory portion rapidly expands into large, wide penis which shares common genital atrium with slightly smaller, slightly longer, vagina. Large, wide vagina abruptly narrows proximally into thinner semi-elongate duct and enters large, rounded bursa copulatrix partially covered by granular prostate. Thin, short duct connects bursa to smaller, rounded receptaculum seminis. Short, thin uterine duct connects near receptaculum base and enters medium-sized, irregularly oval-shaped female gland mass.
Geographical distribution: Known from New Caledonia. Ecology: Found on shallow to moderately deep reefs rich in sediments between 15-40 m (Hervé 2010;Bas 2022).
Remarks: Molecular sequencing of the holotype of Halgerda hervei sp. nov. (CASIZ 186494) was unsuccessful for all genes studied; however, the morphological characteristics are distinct enough to distinguish H. hervei sp. nov. from the similarly patterned H. jennyae and H. mesophotica sp. nov. Morphological comparisons of H. hervei sp. nov. to H. jennyae and H. mesophotica sp. nov. are made below. Externally, H. hervei sp. nov. is semi-translucent white with small black dots, an irregularly dark brown/black ridging with large, pronounced tubercles; and radiating dark brown/ lines from the tips of the tubercles and similarly colored lines perpendicular to the edge of the mantle. Halgerda jennyae is also translucent white with high tubercles; however, there is a complex pattern of thin brown lines along the ridges and valleys of the irregular ridging and numerous small to medium brown spots along the foot and dorsum (Tibiriçá et al. 2018, Figs. 11d, e). Halgerda mesophotica sp. nov. is also translucent with sporadic black dots, but the extremely large and conical tubercles have a series of radiating thick brown lines and there is no linework along the edge of the mantle. The rhinophores are similar between H. hervei sp. nov. and H. mesophotica sp. nov. due to the translucent white coloration, cream-colored lamellae, and posterior dark stripe along the rhinophoral stalk; however, there are also small black spots and radiating black lines along the rhinophoral sheath in H. hervei sp. nov. that are absent in H. mesophotica sp. nov. In contrast, the rhinophores in H. jennyae are translucent with yellow lamellae and brown lines along the dorsal and lateral sides. The bipinnate branchial plume in H. hervei sp. nov. is two-toned with the upper one-third of leaves and rachises translucent white with a thin dark dorsal stripe, while the lower-two thirds are translucent yellow with ventral dark spots. The gill plume in H. mesophotica sp. nov. is similar; however, there are fewer large secondary branches, and the leaves and rachises are translucent white with a thick brown dorsal stripe and no spots. In H. jennyae the gill is also semi-translucent white with a dorsal black stripe, but there are also numerous small random black spots along the rachises and leaves.
Internally, the radula of H. hervei sp. nov. is more similar to H. jennyae, while the reproductive system is more similar to H. mesophotica sp. nov. In H. hervei sp. nov., there are small light brown spots around the buccal opening and no pigment on the buccal mass, while in H. jennyae the buccal mass is pigmented. In contrast, there is no pigmentation on the buccal mass or around the buccal opening in H. mesophotica sp. nov. In H. hervei sp. nov., the oral tube is much smaller than the buccal bulb while in H. mesophotica sp. nov., they are much more similar in size with the buccal bulb being 1.5 × the length of the oral tube. The radular teeth in H. hervei sp. nov. include 19 small inner lateral teeth with thick, sharply curved cusps; while in H. mesophotica sp. nov., there are 31 small inner lateral teeth with elongated stems and sharply curved cusps. In H. jennyae there are 16-18 small inner lateral teeth with thick cusps, that are shorter than those in H. hervei sp. nov. (Tibiriçá et al. 2018, Fig. 15a). The outermost radular teeth are different in all three species. In H. hervei sp. nov., the outermost three teeth are reduced with the outermost two teeth small, paddle-shaped, and semi-fimbriate. In H. mesophotica sp. nov., the three outermost teeth are reduced versions of the other outer laterals with more pointed tips and in H. jennyae the three to five outermost teeth are also reduced and similar to the other outer teeth, but less elongated and pointed than those in H. mesophotica sp. nov. (Tibiriçá et al. 2018, Fig. 15d). The reproductive systems of H. hervei sp. nov. and H. mesophotica sp. nov. share a large spherical bursa copulatrix partially covered by the granular portion of the prostate, a large vagina and penis, and a much smaller semirounded receptaculum seminis. However, in H. hervei sp. nov., the ejaculatory portion of the vas deferens is longer than in H. mesophotica sp. nov. with a distinct loop. The vagina of H. hervei sp. nov. gradually narrow and has a narrow distal portion that enters the bursa copulatrix, whereas in H. mesophotica sp. nov., the bulbous vagina abruptly narrows and is uniformly narrow to its junction with the bursa. Additionally, the duct connecting the vagina to the bursa and the bursa to the receptaculum/uterine duct split is much shorter and thicker in H. hervei sp. nov. In contrast, the smaller bursa is completely covered by the granular prostate in H. jennyae, the penis and vagina smaller, the ejaculatory portion shorter, and the receptaculum is also slightly larger (Tibiriçá et al. 2018, Fig. 14b). Based on the unique internal and external characteristics including dorsum and tubercle patterning, the translucent yellow gill, a short oral tube, athe paddle-shaped semi-fimbriate outer radular teeth, H. hervei sp. nov. is a distinct species with Halgerda despite the lack of molecular data for the species. Ecologically, H. hervei sp. nov. and H. jennyae are found in moderately shallow water 10-40 m and 20 m, respectively, whereas H. mesophotica sp. nov. is found at mesophotic depths from 61-70 m. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ 5DF42 2FE-F830-46F0-B1F0-8BEA3 9CD5A C0 (Fig. 17f,19f,24a Type locality: Aliwal Shoal, KwaZulu-Natal Province, South Africa Etymology: This species is named in honor of Maaike, the owner of the dive center that helps facilitate specimen collections with Brian Sellick in South Africa.
External morphology: Living animals 29-41 mm length (Fig. 24a). Body oval, semi-rigid, and gelatinous. Dorsum color semi-translucent white with low unconnected irregularly patterned orange ridging. Reddish purple viscera semivisible through mantle. Thin white marginal band along mantle edge. Mantle underside semi-translucent white. Gill surround slightly elevated anus with seven bipinnate branchial leaves. Gill coloration semi-translucent white with dorsal dark brown/black stripe. Gill pocket smooth with similar dorsum coloration. Opaque white glands present in gill branches. Rhinophores tapered, perfoliate with semi-translucent white stalks and 14-16 dark brown/black lamellae. Rhinophoral sheath smooth with similar dorsum coloration. Foot is broad, anteriorly notched, semi-translucent white, and extends slightly beyond mantle. Oral tentacles digitiform.
Internal anatomy (Figs. 17f,: Buccal mass (Fig. 17f) unpigmented. Oral tube slightly longer than buccal bulb and narrowed anteriorly. Radular sac very elongate, rounded tip, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth without denticles (Fig. 25a). Radular formula 35 × 35.0.35 in the holotype SAMC-A094639. Inner 14 lateral teeth (Fig. 25b) small, broad-based with short, thick, highly curved cusps form a steep V in the radula center. Middle lateral teeth (Fig. 25c) larger and thicker than middle laterals with more elongated cusps and rounded tips. Outer lateral teeth (Fig. 25d) larger and more elongate than middle laterals. Outermost two teeth greatly reduced versions of other outer laterals.
Reproductive system (Fig. 19f): Triaulic. Thin preampullary duct continues into thin ampulla which quickly expands, loops, and then gradually narrows into postampullary duct which splits into short vas deferens and short, thin oviduct. Vas deferens gradually expands into similarly wide, elongate granular prostate which widens, loops, and then abruptly narrows into thin, elongate, looped ejaculatory portion. Ejaculatory portion quickly expands into large, wide penis which shares common genital atrium with slightly smaller muscular vagina. Large, wide vagina gradually narrows proximally into thinner elongate duct which enters large, bursa copulatrix slightly covered by vagina. Thin, semi-elongate duct connects bursa to small oval-shaped receptaculum seminis. Short, thin uterine duct connects near receptaculum base and enters large, oblong-shaped female gland mass.
Geographical distribution: Known from South Africa. Ecology: Found on deeper reef faces below 25 m (Sellick et al. 2020).
Remarks: In our molecular phylogeny Halgerda maaikeae sp. nov. from South Africa is part of an unresolved clade including H. mozambiquensis, H. indotessellata, and H. tessellata. Within this clade, H. maaikeae sp. nov. is separated from H. mozambiquensis genetically by a minimum divergence of 5.2% in the COI gene and morphologically by the lack of large, dark brown spots found between the orange ridging seen in H. mozambiquensis (Tibiriçá et al. 2018, Fig. 11b). In comparison to H. indotessellata and H. tessellata there is a minimum COI genetic divergence of 5.8% and 8.6%, respectively. Externally, H. maaikeae sp. nov. also lacks the brown pigmentation with white spots between the pronounced ridging seen in both species and the rhinophores are a solid black in H. maaikeae sp. nov. rather than spotted as seen in H. indotessellata and H. tessellata (Tibiriçá et al. 2018, Fig. 11a;Bergh 1880b, Tafel F, Figs. 22 and 23;respectively).
Morphologically, H. maaikeae sp. nov. is most similar to H. diaphana (Fig. 24b) from Okinawa and H. dichromis (Fig. 24c) from South Africa, so further morphological comparisons are made below and summarized in Table 5. The four species delimitation analyses support H. maaikeae sp. nov. as a distinct species and there is a strong minimum COI divergence of 13.1% between H. maaikeae sp. nov. and H. diaphana and 11.8% between H. maaikeae sp. nov. and H. dichromis. Externally, H. maaikeae sp. nov. is semi-translucent white with low, unconnected orange ridging, no tubercles, and a thin white marginal band near the mantle edge. The rhinophores and seven bipinnate branchial leaves are also semi-translucent white with black lamellae and a dark brown/black dorsal stripe, respectively. The dorsal color of H. diaphana is more translucent white with moderately pronounced and connected orange ridging, secondary orange lines, low tubercles, and a thick orange marginal band offset from a thinner, white marginal band near the mantle edge. The rhinophores and four pinnate (sometimes bipinnate) branchial leaves are translucent white with numerous black speckles (Fahey and Gosliner 1999b, Fig. 1c). Halgerda dichromis is more grayish white in coloration with moderately pronounced, unconnected orange and black ridging, secondary black spots, no tubercles, and a thin white marginal band (Fahey and Gosliner 1999a, Fig. 4b). The rhinophores and branchial leaves are similarly patterned to H. maaikeae sp. nov., but the rhinophores also have a white apical tip and there are fewer, more highly pinnate branchial leaves.
Internally, the radular teeth and reproductive systems vary noticeably between the three species. In H. maaikeae sp. nov., there are 14 small inner lateral teeth with short, thick, highly curved cusps that form a steep V shape in the middle of the radula. In H. diaphana there are only 10 small inner lateral teeth with very short and pointed cusps (Fahey and Gosliner 1999b, Fig. 5b) and in H. dichromis there are 20 small inner lateral teeth with less curved and more pointed cusps (Fahey and Gosliner 1999a, Fig. 7b). The middle and outer teeth are larger with more elongate cusps in all three species, but the outermost teeth vary drastically. In H. maaikeae sp. nov., the two outermost teeth are greatly reduced versions of the other outer laterals, while in H. diaphana the three outermost teeth are similarly reduced with blunt tips and denticulation (Fahey and Gosliner 1999b, Fig. 5a), and in H. dichromis the three outermost teeth are either fimbriate or pointed depending on the row (Fahey and Gosliner 1999a, Fig. 7a). The reproductive system of H. maaikeae sp. nov. includes a small receptaculum seminis; a large, spherical bursa copulatrix uncovered by the prostate; a large, wide penis connecting to an elongate, looped ejaculatory portion of the prostate; a wide, looped, elongated granular portion of the prostate; and a large, wide, muscular vagina (slightly smaller than the penis) that narrows proximally. In contrast, H. diaphana has a large, spherical bursa copulatrix covered by the granular portion of the prostate and the ejaculatory portion of the prostate is much shorter and connects into a wide, elongated penis that is slightly smaller than the large and glandular vagina (Fahey and Gosliner 1999b, Fig. 2c). In H. dichromis, the large and spherical bursa copulatrix is also covered by the granular portion of the prostate and connects to a thin, elongated ejaculatory portion of the prostate, which expands into a large, wide penis (Fahey and Gosliner 1999a, Fig. 3c). The vagina is smaller and composed of a large, glandular portion that narrows into a short, muscular portion, before narrowing and connecting to the bursa. Despite the morphological similarities, there are clear internal and external differences and strong genetic divergences between H. maaikeae sp. nov., H. diaphana, and H. dichromis, which supports each of them as distinct species within Halgerda. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ E4F5C ACA-4254-452F-ACA3-B3BFD BEDA6 49 (Fig. 17g,19g,24d and 26) Halgerda toliara Poddubetskaia (2003). Halgerda cf. toliara Poddubetskaia (2004).
Material examined: Holotype: One specimen, UF 422800, dissected and sequenced, 10 mm preserved, type locality: Mayotte Island (12° 51′ 18.5″ S 45° 16′ 06.8″ E), Comoros Archipelago, 15-17 m depth, 02 June 2008, collected by Arthur Anker and François Michonneau.
Type locality: Mayotte Island, Comoros Archipelago. Etymology: This species is named in honor of Patti Ann Kerscher, the first author's very supportive and beloved mother.
External morphology: Preserved animals 10 mm in length (Fig. 24d, Supplementary Figure S6e). Body oval, semirigid, and gelatinous. Dorsum color translucent white with low irregular ridging formed by series of low conical tubercles Anteriorly forked wide yellow longitudinal line along central ridge begins before rhinophores and rings the gill pocket. Central lateral ridges maybe also be light yellow or translucent white with translucent white or vibrant yellow small tubercles which gradually lose color towards mantle edge. (Supplementary Figure S6f). Numerous small semiopaque white conical tubercles present along lateral ridges and mantle edge. Thin white marginal band along mantle edge. Viscera with reddish purple coloration visible through mantle. Mantle underside translucent white. Gill surrounds slightly elevated anus with five unipinnate branchial leaves. Gill coloration white with translucent white rachis and dorsal dark brown stripe. Gill pocket smooth. Opaque white glands semi-visible within branchial leaves. Rhinophores tapered, perfoliate with translucent white stalks and 15 dark brown lamellae forming the rhinophoral club. Rhinophoral sheath smooth with similar dorsum coloration. Foot is broad, anteriorly notched, and translucent white. Oral tentacles digitiform.
Internal anatomy (Figs. 17g,: Buccal mass (Fig. 17g) unpigmented. Oral tube 1.5 × longer than buccal bulb. Radular sac elongate, curved, slightly expanded at tip, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 26a). Radular formula 38 × 35.0.35 in the holotype UF 422800. Inner 16 lateral teeth (Fig. 26b) small, broad-based with slightly elongated, gently curved, thin cusps and pointed tips form a V in the center. Middle lateral teeth (Fig. 26c) larger and thicker than inner laterals with more elongated cusps and rounded tips. Outer lateral teeth (Fig. 26d) slightly larger than middle laterals with more elongated tips. Outermost two-three teeth greatly reduced and fimbriate.
Reproductive system (Fig. 19g): Triaulic. Thin preampullary duct rapidly expands into thick ampulla, then gradually narrows into postampullary duct which splits into short vas deferens and short oviduct. Vas deferens gradually expands into similarly wide, elongate granular prostate which widens, loops, and then abruptly narrows into thin, elongate, curved ejaculatory portion. Ejaculatory portion quickly expands into large, wide, elongate penis which shares common genital atrium with similarly sized muscular vagina. Large, wide muscular vagina abruptly narrows proximally and enters medium-sized, irregularly rounded bursa copulatrix. Very thin, elongate duct connects bursa to small, oblong receptaculum seminis. Short, slightly thin uterine duct connects near receptaculum base and enters medium sized irregularly rounded female gland mass.
Geographical distribution: Known from Mayotte Island in the Comoros Archipelago.
Ecology: Found on exposed coral and rocks between 15-17 m.
Remarks: Our molecular phylogeny shows that Halgerda pattiae sp. nov. from Mayotte Island in the Comoros Archipelago is sister to the similarly colored H. meringuecitrea (Fig. 24e) from Mozambique. All four species delimitation analyses support H. pattiae sp. nov. as a distinct species from H. meringuecitrea and reveal a minimum divergence of 6.0% in the COI gene. Halgerda pattiae sp. nov. is also similarly colored to H. toliara from Madagascar; however, there is a stronger genetic divergence of 11.3% between these two species. Furthermore, these two species are found within separate well-supported clades in our molecular phylogeny. Due to their morphological similarities, further comparisons between H. pattiae sp. nov., H. meringuecitrea, and H. toliara are made below and summarized in Table 5.
Externally, H. pattiae sp. nov. is translucent white with low yellow ridging that gradually loses color; a wide, forked, yellow longitudinal line along the central ridge; and numerous small yellow and white tubercles along the ridge edges and offset from the thin, white mantle band along the mantle margin. The rhinophores are translucent white with dark brown lamellae and there are five, unipinnate white branchial leaves with translucent white rachis and a dorsal dark brown stripe. The dorsum of H. meringuecitrea is also translucent white; however, the low yellow-orange ridging is less pronounced and more uniform in color and there are no tubercles present along the ridging or offset from the thin, white mantle band along the mantle margin (Tibiriçá et al. 2018, Fig. 11g). Also, the central yellow line of H. pattiae sp. nov. extends around the gill pocket, whereas it terminates anteriorly of the gill pocket in H. meringuecitrea. The rhinophores of H. meringuecitrea are similar in coloration to H. pattiae sp. nov. There are five to six unipinnate (occasionally bipinnate) translucent white branchial leaves with dark brown coloration along the top two-thirds of the rachis. In H. toliara, the dorsum is whiter in coloration with more extensive low orange ridging (Fahey and Gosliner 1999a, Fig. 4c). There are no tubercles present along the ridging; however, there are small yellow/orange tubercles offset from the thin, white mantle band along the mantle margin. The rhinophores are similar to both H. pattiae sp. nov. and H. meringuecitrea; however, the lamellae are darker and blacker in coloration. The gill is composed of only four pinnate leaves which are translucent white with approximately one-half to one-third black in coloration.
Internally, the radular teeth and reproductive systems also noticeably vary between these species. In H. pattiae sp. nov., there are 16 small inner lateral teeth with slightly elongated, gently curved, thin cusps, while in H. meringuecitrea, there are 13-14 small inner lateral teeth with shorter, thicker, more abruptly curved cusps (Tibiriçá et al. 2018, Fig. 16b), and in H. toliara, there are only four small inner lateral teeth which are more similar in size and shape to H. pattiae sp. nov. (Fahey and Gosliner 1999a, Fig. 8a). The middle and outer teeth are all larger and more elongate in each species, but the outermost teeth vary slightly. In H. pattiae sp. nov. and H. toliara, the outermost two to three lateral teeth are reduced and fimbriate (Fahey and Gosliner 1999a, Fig. 8b), while in H. meringuecitrea, the two to three outermost teeth are also reduced with a slightly pointed hook (Tibiriçá et al. 2018, Fig. 16c,d). The reproductive system of H. pattiae sp. nov. is composed of a thick ampulla; a large, wide, elongate penis and a similarly sized muscular vagina; a medium-sized irregularly rounded bursa copulatrix; and a small, oblong receptaculum seminis. In contrast, H. meringuecitrea has a narrower ampulla and elongated penis; a much smaller bursa copulatrix and a minute, rounded receptaculum seminis. The vagina is similarly muscular to the one found in H. pattiae sp. nov.; however, it gradually narrows in the proximal portion and is overall smaller in size and width (Tibiriçá et al. 2018, Fig. 14c). In H. toliara, the penis is short and bulbous and the vagina is narrow and extremely elongated, while the rounded bursa copulatrix is very large and the rounded receptaculum seminis is approximately one-sixth the size of the bursa (Fahey and Gosliner 1999a, Fig. 3d).
The slight variation in external morphology, particularly the extent of the dorsal yellow pigment, and the noticeable differences in the radula and reproductive systems of H. pattiae sp. nov., H. meringuecitrea, and H. toliara are supported by strong genetic divergences, which help distinguish these three distinct, but similar species within Halgerda. The presence of ridges formed by lines of small tubercles, the presence of yellow pigment encircling the gill and the details of the reproductive system noted above clearly differentiate H. pattiae sp. nov. from other described species of Halgerda. Donohoo & Gosliner sp. nov. https:// zooba nk. org/ 9879B 020-0D6B-40F4-A120-FFBF3 21919 A0 (Fig. 17h,19h,24f and 27) Halgerda toliara? Gaggeri (2006) External anatomy: Preserved animals 12-16 mm in length (Fig. 24f). Body oval, semi-rigid, and gelatinous. Dorsum color translucent white with moderate irregular ridging devoid of tubercles. Small semi-white to translucent white tubercles present offset from mantle edge. Forked wide yellow longitudinal line with thick short offshoots along central ridge begins before rhinophores and terminates anterior to gill sheath. Viscera with purple coloration visible through mantle. Thin white marginal band present along mantle edge. Mantle underside translucent white. Gill surrounds elevated anus with six unipinnate branchial leaves. Gill coloration translucent white with rachis and top two-thirds of branchial leaves black. Gill pocket smooth with similar dorsum coloration. Opaque white glands present within branchial leaves. Rhinophores tapered, perfoliate, black pigment present e club and with black pigment extending downward on top of rhinophore stalk. lub base and 14-16 black lamellae. Rhinophoral sheath smooth, translucent white. Foot is broad, anteriorly notched, translucent white and extends beyond mantle. Oral tentacles digitiform.
Internal anatomy (Figs. 17h,: Buccal mass (Fig. 17h) unpigmented. Buccal bulb 1.5 × longer than short, anteriorly narrowed oral tube. Radular sac thin, elongated, and unpigmented. Labial cuticle smooth. Radula composed of smooth hamate teeth with no denticles (Fig. 27a). Radular formula 32 × 29.0.29 in the holotype CASIZ 173456. Inner 15 lateral teeth (Fig. 27b) small, broad-based with short stalks and quickly curved, elongated thick cusps. Middle lateral teeth (Fig. 27c) larger, thicker, and more elongated than inner laterals with rounded tips. Outer lateral teeth (Fig. 27d) are slightly larger and thicker than middle laterals. Outermost four teeth gradually reduce in size with outer three teeth fimbriate.
Reproductive system (Fig. 19h): Triaulic. Thin, elongate preampullary duct expands into thicker ampulla, then gradually narrows into postampullary duct which splits into vas deferens and oviduct. Vas deferens gradually expands into wide, elongate, and looped granular prostate which narrows abruptly into thin, elongate ejaculatory portion. Ejaculatory portion abruptly narrows into large, wide, curved penis which shares common genital atrium with smaller, similarly elongate vagina. Narrow, elongate vagina gradually narrows and enters medium-sized irregularly shaped bursa copulatrix. Semi-thin, elongate duct connects bursa to small irregularly oblong shaped receptaculum seminis. Very thin, short uterine duct connects near receptaculum base and enters medium sized, irregularly rounded female gland mass.
Geographical distribution: Known from Iles Radama, Madagascar (present study) and Nosy Be, Madagascar (Gaggeri 2006). Specimens attributed from Mayotte Island, Comoros Archipelago (Gosliner et al. 2008(Gosliner et al. , 2015(Gosliner et al. , 2018) ) represent H. pattiae sp. nov.
Ecology: Found on reef faces between 6-19 m. Remarks: Molecular sequencing of both the holotype (CASIZ 173456) and paratype (CASIZ 234753) of Halgerda radamaensis sp. nov. was unsuccessful due to prior fixation in formalin; however, the morphological characteristics are distinct enough to distinguish H. radamaensis sp. nov. from similar species of Halgerda. Halgerda radamaensis sp. nov. is morphologically similar to H. pattiae sp. nov. (Fig. 24d) and H. meringuecitrea (Fig. 24e) and morphological comparisons between the three are made below. Externally, the dorsum coloration of all three species is translucent white with a thin, white marginal band; however, there are distinct patterns and colorations in the ridging and tubercless within each species. In H. radamaensis sp. nov., the moderately pronounced ridging is colorless except for a forked, wide, bright yellow longitudinal line with short offshoots present down the central ridging. There are small semi-white to translucent white submarginal tubercles alongside the ridging and offset from the marginal band along the mantle edge. In contrast, the ridging in H. meringuecitrea is less pronounced and more irregular with yellow-orange coloration and there are no tubercles present along the ridging or the edge of the mantle. Halgerda pattiae sp. nov. also has less pronounced ridging with vibrant, yellow or white tubercles and a wide, yellow longitudinal line down the central ridging. The rest of the ridging may have yellow coloration that gradually lightens towards the edge of the mantle, but there are always numerous small semi-opaque white tubercles along the lighter parts of the ridging and the edge of the mantle. In H. radamaensis sp. nov., the yellow terminates anteriorly to the gill cavity while in H. pattiae sp. nov., the central yellow line encircles the gill. The rhinophores are similar between the three species (translucent white with dark brown/black lamellae) but in H. radamaensis sp. nov., the black extends below the rhinophore club with some black extending onto the apical portion of the stalk, whereas in H. meringuecitrea and H. pattiae sp. nov., the black pigment is confined to the rhinophoral club and does not extend on to the stalk. The gill plume varies slightly between species. In H. radamaensis sp. nov., the branchial leaves are unipinnate and translucent white with the rachis and top two-thirds of the branchial leaves black. The branchial leaves in H. meringuecitrea are similarly patterned, but with dark brown coloration and occasionally bipinnate rachises. In H. pattiae sp. nov., the branchial leaves are also unipinnate, but with white coloration and translucent white rachis with a dark brown dorsal stripe.
Internally, the radular teeth and reproductive systems noticeably vary between the three species. In H. radamaensis sp. nov., there are 15 small inner lateral teeth with short stalks and quickly curved, elongated thick cusps. The middle and outer lateral teeth are thicker and more elongated, but the outermost four teeth are reduced with outer three teeth also fimbriate. In contrast, H. meringuecitrea has 13-14 inner lateral teeth with a similar thickness, but less elongated and with more abruptly curved cusps; while the outermost two to three teeth are reduced in size with a slightly pointed hook (Tibiriçá et al. 2018, Fig. 16b-d). In H. pattiae sp. nov., the 16 inner small lateral teeth are also elongated, but thinner with a gentle curve and narrow, pointed cusps; while the outermost two to three teeth are similarly reduced and fimbriate. The reproductive system of H. radamaensis sp. nov. is composed of an elongate ampulla; a large, wide, curved penis and a smaller, more elongate vagina; a medium-sized irregularly rounded bursa copulatrix; and a smaller, irregularly oblong receptaculum seminis. Halgerda meringuecitrea has a similarly elongate ampulla, but the penis is more elongate and narrower, the vagina is muscular, and the bursa copulatrix and the receptaculum seminis are also much smaller and more rounded (Tibiriçá et al. 2018, Fig. 14c). Halgerda pattiae sp. nov. has a similarly large penis, medium-sized bursa copulatrix, and small receptaculum seminis; however, the ampulla is much shorter and wider, the receptaculum more oblong shaped, and the muscular vagina wider than the non-muscular one seen in H. radamaensis sp. nov. Based on the unique internal and external characteristics including dorsum morphology and coloration and the presence of an elongated, non-muscular vagina, H. radamaensis sp. nov. is a distinct species from H. meringuecitrea and H. pattiae sp. nov., despite the lack of molecular data for the species. Halgerda radamaensis sp. nov. is likely a member of the same clade as H. meringuecitrea and H. pattiae sp. nov., based on the morphology described. This clade already demonstrates strong regional endemism in the western Indian Ocean with corresponding morphological and molecular divergence. It is highly likely that H. radamaensis sp. nov. shows a similar pattern of divergence and that the morphological differences noted above are significant and support the distinctness of this species. We have been trying to obtain additional material from Madagascar since this material was collected in 2005, but have been unsuccessful and feel it is necessary to describe this species based on its unique morphological features.
This study produced an updated phylogenetic analysis of the genus Halgerda and revealed unexpected diversity in the central and western Pacific Ocean and the western Indian Ocean, including the Red Sea. By combining morphological data with molecular data from two mitochondrial and two nuclear genes, we have uncovered two new species in the Red Sea, four new species in the western Indian Ocean, one new species in Japan, and seven new species in the Indo-Pacific including New Caledonia, French Polynesia, and the Verde Island Passage. We also synonymized H. elegans with H. willeyi, provided a juvenile description for the previously described H. dalanghita, and established a new mesophotic clade. This study brings the total number of described Halgerda species from 42 to 55, making Halgerda one of the most diverse genera in Discodorididae.
Despite the numerous morphological studies on Halgerda, the position and sister taxon of Halgerda within Discodorididae varies from study to study. Halgerda has been previously suggested as the sister taxon of Asteronotus after a morphological study of caryophyllidia-bearing dorids, and a morphological review of cryptobranch dorids showed that they shared several characteristics including a rigid body, dorsum ridging, and a well-differentiated prostate (Valdés and Gosliner 2001;Valdés 2002). This relationship was later supported in two morphological phylogenies focused on Halgerda (Fahey andGosliner 1999b, 2001a) as well as the first molecular phylogeny (Fahey 2003). It should be noted that in the first molecular phylogeny, Fahey (2003) sequenced only one mitochondrial gene (COI), utilized only one additional species of Discodorididae for outgroup comparisons, and was focused predominately on species from the Pacific. In 2017, Discodoris, rather than Asteronotus, was suggested as the sister taxon of Halgerda during a four gene molecular phylogenetic study of Doridina; however, that study used limited representation from each family (including one specimen of Halgerda and one specimen of Asteronotus) to evaluate an entire suborder (Hallas et al. 2017). Tibiriçá et al. (2018) expanded upon the first Halgerda molecular phylogeny by sequencing COI and two additional genes (16S and H3) from specimens of Halgerda across the Pacific Ocean and the western Indian Ocean. Their phylogeny suggests that Paradoris Bergh, 1884 or Peltodoris Bergh, 1880b is the sister taxon of Halgerda; however, it is unclear which genus is more closely related (Tibiriçá et al. 2018). In our study, we expanded the outgroup to include 16 different genera from Discodorididae including the previously suggested genera Asteronotus, Discodoris, Paradoris, and Peltodoris; however, we are still unable to establish a clear sister taxon for Halgerda. We suggest a more comprehensive review of the entire family is needed to better understand the relationships between Discodorid genera and establish a sister taxon for Halgerda.
Species delimitation Specimens thought to be previously undescribed species of Halgerda (i.e., Halgerda sp. 3 and Halgerda sp. 4; Gosliner et al. 2018) are shown in our molecular phylogeny to be juveniles of the newly described H. mango sp. nov. and the previously described H. dalanghita, respectively. Externally, these juvenile specimens share only a few obvious morphological characteristics with their adult counterparts; however, closer inspection of the radula and reproductive systems reveals limited differences in the internal juvenile and adult anatomy. This noticeable difference in coloration and patterning between juveniles and adults can also be found in other species of nudibranchs. For example, in Jorunna rubescens (Bergh, 1876), a second species of Discodorididae, the adults are white with brown lines and yellow spots; however, the juveniles are more translucent white with opaque white lines (Gosliner et al. 2015(Gosliner et al. , 2018)). In the chromodorid Goniobranchus charlottae (Schrödl, 1999), the adults and juveniles share a similar reddish-brown coloration; however, the juveniles have large areas of cream-white coloration and a light blue submarginal band. In the adults, the white areas are much smaller and more scattered; there are also small red spots scattered along the dorsum, and the light blue submarginal band is also broken apart into small light blue spots (Schrödl 1999;Gosliner et al. 2015Gosliner et al. , 2018)). Some nudibranch juveniles (e.g., Phestilla melanobrachia Bergh, 1874) may change their coloration depending on available food or host regardless of the parent's original color (Yiu et al. 2021).
Occasionally, juveniles may be described as a new species. For example, our molecular phylogeny reveals that specimens matching Bergh's original description of H. elegans from 1905 are in fact juveniles of the adult species H. willeyi described by Eliot in 1904. Genetic differences in the mitochondrial COI gene between specimens representing these two morphologies and a second adult morphology described in this study ranges between 0.3 and 1.8% (Supplementary Table S3); however, all three morphologies share a suite of internal and external characteristics (see remarks section of H. willeyi). We have also determined that specimens formerly identified as H. willeyi from the Red Sea and Okinawa, Japan, are actually two new species of Halgerda. Both H. willeyi and the new species, H. paulayi sp. nov. and H. labyrinthus sp. nov., are found in separate subclades within our molecular phylogeny and have a strong genetic divergence of 9.5-11.4% between all three species. The morphological differences between these three species are detailed in their respective remark sections. Furthermore, we suggest that H. willeyi may very well suffer from the "catch-all problem" given that H. willeyi, H. paulayi sp. nov., and H. labyrinthus sp. nov. share a wide morphological description built around the dorsum coloration and patterning of the pronounced tubercles and lined ridging. All these species highlight the importance of utilizing molecular data in coordination with morphological data to prevent either the over-description of new species solely based on a "novel" or "unique" morphological characteristic (i.e., the novelty problem/splitting) or the lack of new descriptions due to over-grouping based on a universal morphological characteristic or wide morphological description (i.e., the catch-all problem/grouping) (Rudman 1984(Rudman , 1987;;Dayrat and Gosliner 2005;Johnson and Gosliner 2012).
Mesophotic species A new mesophotic clade has been identified within Halgerda and includes the following species: H. scripta sp. nov., H. mesophotica sp. nov., H. profunda sp. nov., H. takipsilim sp. nov., and H. okinawa. Within this clade, H. mesophotica sp. nov., H. profunda sp. nov., and H. takipsilim sp. nov. form a species complex with low genetic diversity (0.9-1.5%); however, the morphological internal and external differences detailed in the remarks section of H. takipsilim sp. nov. support the distinctness of each species within Halgerda. This new clade is predominantly found in the Philippines and the temperate Central Pacific including Japan; however, all five species within this clade have a similar depth profile (50-90 m). They also have shared morphological characteristics such as large secondary branchial leaves, a thin colored mantle band, numerous inner radular teeth, and a partially covered bursa copulatrix that set them apart from species found in the deep sea. Similarly patterned species of Halgerda found in the deep sea between 90 and 400 + m in depth including Halgerda abyssicola Fahey & Gosliner, 2000;Halgerda azteca Fahey & Gosliner, 2000;Halgerda fibra Fahey & Gosliner, 2000;and Halgerda orstomi Fahey & Gosliner, 2000 generally have fewer small inner radular teeth (apart from H. orstomi) and occasional denticulation along the outermost radular teeth. The reproductive system of these species includes a large, bulbous vagina that may be glandular or have a proximal sphincter connecting to the bursa copulatrix and a very small, rounded receptaculum seminis. The dorsum coloration and patterning of the deep-sea species noticeably vary from the mesophotic species. In the deep-sea species, there is thick, dark ridging with dark spots, perpendicular dark lines along the dorsum edge, pronounced tubercles, and a similar gill pattern (Fahey and Gosliner 2000). To date, there is no molecular sequencing for any of the previously mentioned deep-sea species due to a lack of collecting and suitable preservation for sequencing; however, we have sequenced other species of Halgerda found at similar depths including H. brunneomaculata, H. carlsoni, and H. dalanghita. These three species are found in different clades within our molecular phylogeny and exhibit numerous morphological differences between each other, the previously mentioned deep-sea species, and the newly established mesophotic species. Further collection and sequencing of H. abyssicola, H. azteca, H. fibra, and H. orstomi may expand the mesophotic clade identified here, or it may reveal an entirely new and separate clade within Halgerda.
In 2018, Tibiriçá et al. described two species complexes within Halgerda: the H. carlsoni complex predominately from the central and western Pacific and the H. wasinensis complex from the western Indian Ocean.
Here, we provide clarification on the lack of genetic differences between two species in the H. carlsoni clade (H. okinawa and H. malesso) previously identified in Tibiriçá et al. 2018 and provide additional insight into the H. wasinensis clade. During their three gene phylogenetic analysis, Tibiriçá et al. used the original 599 bp mitochondrial COI sequences from Fahey (2003), which included a specimen of H. okinawa from Japan and a specimen of H. malesso from Guam. The results of their molecular phylogeny and species delimitation analyses revealed no genetic differences between these two specimens (p = 0%). As such, Tibiriçá et al. (2018) then question whether H. malesso and H. okinawa are one extremely variable species or if there was a lack of genetic diversity between the two species. For our study, we successfully resequenced several of the original Fahey (2003) samples including the specimens of H. okinawa and H. malesso. Our COI sequences were longer (~ 658 bp); however, our H. malesso resequence was 100% identical to Fahey's original sequence and exhibited very little genetic difference with an additional specimen of H. malesso (UF 289547) also from Guam. The resequencing of the H. okinawa specimen reveals a strong genetic difference of 12.6% in the COI gene between H. okinawa and H. malesso suggesting that H. okinawa is no longer a part of the H. carlsoni species complex. Instead, H. okinawa is found nested within a new mesophotic clade described in this study. It is likely that Fahey's original H. okinawa sequence was an exact duplication of the H. malesso sequence and possibly a duplication error during the initial PCR, sequencing, or the submission to Genbank. Sequencing of three additional genes for both H. okinawa and H. malesso also supports the separation between these two species. Therefore, it is unlikely that H. malesso and H. okinawa are a single variable species given the strong genetic separation and lack of morphological similarities.
In our species delimitation analyses, the H. wasinensis complex identified in Tibiriçá et al. 2018 is partitioned into either four distinct groups (COI ABGD), partially split into two groups (16S ABGD, GYMC), or part of one large taxonomic unit (COI ASAP, 16S ASAP, bPTP) with other species including H. anosy sp. nov., H. cf. formosa, and H. dichromis (Fig. 2). The COI ABGD and 16S ABGD analyses successfully recover H. aff. wasinensis separately from H. wasinensis. Futhermore, in the COI ABGD, H. wasinensis is partitioned into three taxonomic units, while the COI and 16S ASAP and bPTP analyses group H. wasinensis with other members of the clade. The COI ABGD partitions do exhibit some geographical signal as the largest partition is composed of eight specimens from Mozambique, followed by two specimens from Zanzibar and a lone specimen (MB28-004972) from Zavora, Mozambique. The COI intraspecific variation within the H. wasinensis studied here ranges between 0.0 and 1.7%, which overlaps slightly with the 1.5-2.8% divergence between H. wasinensis and H. aff. wasinensis (Supplementary Table S4). The over-partitioning of H. wasinensis may be attributed to the low genetic diversity within the entire subclade. For instance, the maximum divergence within this subclade is only 3.3% between H. anosy sp. nov. and H. cf. formosa (Table 3). Overlap between intraspecific and interspecific variation may impact DNA barcoding in recently diverged species (Meier et al. 2008;Hubert and Hanner 2015;Tibiriçá et al. 2018); however, hidden diversity may cause an under or overestimation in species richness and may lead the "barcode gap" to be an artifact of limited sampling (Jörger et al. 2012). Further geographical sampling for both H. wasinensis and H. aff. wasinensis in coordination with next-generation sequencing and extensive morphological study may provide clarification for both the H. wasinensis and the H. carlsoni species complexes.
Continued exploration and discovery of other members of Halgerda not already sequenced may help to provide additional deep node support and potentially provide better resolution for the entire genus of Halgerda within Discodorididae.