Stirring up the muck: the systematics of soft-sediment Fionidae (Nudibranchia: Aeolidina) from the tropical Indo-Pacific

Molecular study

All specimens processed for molecular work were preserved in 95% EtOH. A total of 150 specimens, six newly sequenced and 144 with two or more genes previously published and available on GenBank from Faucci, Toonen & Hadfield (2007), Pola & Gosliner (2010), Moore & Gosliner (2011), Carmona et al. (2013), Cella et al. (2016), Korshunova et al. (2017, 2018b, 2019, 2020, 2023a), Ekimova, Deart & Schepetov (2019), Ekimova, Grishina & Nikitenko (2024), and Mehrotra et al. (2020, 2024) were used in the phylogenetic analyses. Seventeen new sequences were deposited on GenBank with the following accession numbers: partial fragments of the 16S ribosomal RNA gene (16S rRNA; PP759731–PP759736, PP751617), cytochrome C oxidase subunit I gene (COI; PP747810–PP747814, PP751619), histone H3 gene (H3; PP750950–PP750955, PP768732). Sampled specimens with previous and present species identifications, voucher numbers, locality information, GenBank accession numbers, and references are listed in Table 1. Members of Tritoniidae, Aeolididae, Babakinidae, Facelinidae, and Flabellinidae were used for outgroup comparisons based on molecular phylogenetic analysis by Pola & Gosliner (2010) and Cella et al. (2016). Vouchers of newly sequenced specimens and holotypes are deposited at the California Academy of Sciences (CASIZ) and the National Museum of Philippines (NMP).

DNA extraction, amplification, and Sanger sequencing

Genomic DNA from two mitochondrial genes, cytochrome oxidase I (COI) and 16S rRNA, and one nuclear gene, histone 3 (H3), were extracted, amplified using polymerase chain reaction (PCR) and universal primers, and sequenced on an ABI3130 Genetic Analyzer following protocols by Kristen Cella and Terrence M. Gosliner outlined in Cella et al. (2016) (Supplemental Material). Briefly, each PCR reaction used universal gene-specific primers: 16S rRNA (16S arL, 16S R; Palumbi et al., 1991); COI (HCO 2198, LCO 1490; Folmer et al., 1994); and H3 (H3 AF, H3 AR; Colgan et al., 1998) and gene-specific protocols which were run on a BioRad MyCycler ThermocyclerB (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplification occurred in 25 μl reactions containing: 1 μl of genomic DNA template, 2.5 μl of 10× PCR buffer, 0.5 μl dNTPs (10 μM stock), 1.5 μl MgCl2 (25 μM stock), 1 μl of bovine serum albumin, 0.2 μl each primer (25 μM stock), 0.5 μl Taq-Apex (1.25 units/mL) and 17.6 μl of de-ionized water (ddH20). PCR protocols are as follows: for COI and 16S rRNA, 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 50–56 °C, and extension for 1 min at 72 °C with a final extension period of 5 min at 72 °C and for H3, an initial denaturing for 3 min at 94 °C, followed by 35 cycles of denaturing for 35 s at 94 °C, annealing for 1 min at 50–55 °C, and extension for 2 min at 72 °C with a final extension period of 5 min at 72 °C. Successful PCR products were visualized using electrophoresis on 1% agarose gel and then cleaned following the standard protocol for ExoSap-IT™ (USB, Affymetrix, Fremont, CA). The clean PCR products were fluorescently labeled with dye-terminators (Big Dye 3.1; Applied Biosystems, Foster City, CA, USA) during cycle sequencing following SteP protocol (Platt, Woodhall & George, 2007). Each reaction contained: 1.5 μL of 5× reaction buffer, 0.3 μL of primer (10 mM stock), 0.75 μL of Big Dye, 4.45–5.45 μL of Millipore-H2O, and 2–3 μL of cleaned PCR product. The newly labeled single-stranded DNA was precipitated using EDTA and then sequentially washed in 100% and 70% ethanol. HiDi formamide (10 μL, Applied BioSystems, Foster City, CA, USA) was added to each DNA pellet, denatured 95 °C for 2 min, and then immediately cooled on ice for 5 min. Both directions of the DNA fragments were sequenced on an ABI3130 Genetic Analyzer in the Center for Comparative Genomics at the California Academy of Sciences.

Sequence alignment and analyses

For each gene fragment, both strands sequenced were assembled, trimmed to remove primers, and manually edited using Geneious v9.0 (Kearse et al., 2012). Single gene datasets were aligned with MAFFT (Katoh, Asimenos & Toh, 2009) using the algorithm E-INS-I with additional editing by hand for the 16S rRNA alignment. Each gene was analyzed independently using Bayesian inference (BI) and maximum likelihood (ML) and then concatenated into a three-gene alignment (16S rRNA+COI+H3). For the BI analysis, the best-fit evolution model and partitions were determined using ModelTest-NG (Darriba et al., 2020). Bayesian inference analysis was performed in MrBayes v3.2.6 (Ronquist & Huelsenbeck, 2003) with the concatenated dataset partitioned by gene and codon position. The following evolution models were used: GTR+GAMMA+I (16S rRNA, COI codon position 1, and H3 codon position 2), GTR+GAMMA (COI codon position 3 and H3 codon position 1), HKY+GAMMA+I (COI codon position 2), and K80 (H3 codon position 3). The BI analysis was run for 5.0 × 10⁷ generations with Markov chains sampled every 1,000 generations. Convergence of the two chains was checked using TRACER (Drummond & Rambaut, 2007) and then the initial 25% of estimated trees were removed as burn-in. The remaining tree estimates were used to create a 50% majority rule consensus tree with calculated posterior probabilities (pp). Non-parametric bootstrap values (bs) were estimated using randomized accelerated maximum likelihood (RAxML) v8.2.7 (Stamatakis, 2014). Each gene and codon position in the ML analysis used the evolution model GTR+GAMMA+I and was run for 5 × 10⁴ bootstraps runs. Branches with posterior probabilities ≥0.95 and bootstrap values ≥70 were considered strongly supported, while posterior probabilities ≤0.95 and bootstrap values ≤70 were considered weakly supported (Alfaro, Zoller & Lutzoni, 2003).

Species delimitation analyses

Species were only delimited in the three clades with the newly described species from this study to provide genetic comparisons with sister species and those with similar morphology. Three different approaches were implemented (1) assemble species by automatic partitioning (ASAP) method by Puillandre, Brouillet & Achaz (2021), (2) the Bayesian Poisson tree process (bPTP) by Zhang et al. (2013), and (3) the general mixed Yule coalescent (GMYC) model approach (Pons et al., 2006, Fujisawa & Barraclough, 2013). The ASAP method detects intraspecific and interspecific variation using genetic pairwise distances and then creates a scoring system to determine the best partition without relying on a priori species hypotheses. An ingroup COI alignment and an ingroup 16S rRNA alignment created in Geneious were uploaded to the ASAP Web-based interface (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html). Bayesian PTP models the number of substitutions between branches of previously inputted phylogenetic tree branches and identifies groups descended from a single ancestor using Bayesian MCMC methods. This test was performed separately on each of the three clades using a pruned 16S rRNA+COI+H3 concatenated BI tree on the bPTP server (https://species.h-its.org/) with the following parameters: 500,000 generations, 100 thinning, 0.1 burn-in, and 123 seeds. The GMYC approach assumes that different branching rates due to species diversification and independent evolution, which can then be delimited using a Yule Model (Fujisawa & Barraclough, 2013). Individual COI and 16S rRNA alignments were used as inputs to estimate ultrametric trees in BEAST v1.10.4 using the following priors previously assigned in BEAUTI v1.10.4: uncorrelated lognormal relaxed clock, yule speciation process, GTR+GAMMA+I, and 10 million generations MCMC chain length sampled every 1,000 steps. The first 10% of trees were removed for burn-in and a maximum clade credibility tree was estimated from the remaining tree estimates using TreeAnnotator v1.10.4. The GMYC modeling approach was implemented using the “single threshold” model (Pons et al., 2006) in the package Species Limits by Threshold Statistics (SPLITS, v1.0-20, Fujisawa & Barraclough, 2013) in R v4.2.1. Uncorrected pairwise genetic distances (p-distances) for COI and 16S rRNA were generated with Jukes-Cantor (JC69) settings in MEGA 11 v11.0.13 (Tamura, Stecher & Kumar, 2021).

Morphological study

Specimens of new species described in this study were collected from the Philippines and Papua New Guinea via scuba diving. All the specimens from the Philippines were collected under our Gratuitous Permits (GP-0077-14, GP-0085-15) from the shallow waters of the municipalities of Mabini, Tingloy, Calatagan and Puerto Galera. Features of living animals were recorded in the field and from photographs. All the specimens were preserved in either Bouin’s fixative, 75% or 95% EtOH. Morphological analyses were undertaken using a Nikon SMZ-U dissecting scope to determine the overall morphology of external structures, radula, jaws and details of the arrangement of reproductive organs. The penis was also examined for each specimen. In all instances the buccal mass was dissolved in a 10% solution of sodium hydroxide (NaOH) for approximately 24 h and then the elements of the buccal armature were rinsed in deionized water and prepared on glass coverslips for microscopic examination. Elements of the digestive and details of the reproductive systems were drawn using a camera lucida drawing attachment on dissecting scope. The penis was dissected from the reproductive system and mounted on glass coverslips for imaging. The SEM samples were mounted onto SEM stubs and then covered with gold/palladium using a Cressington 108 Auto vacuum sputter coater. Imaging of the jaws, radular teeth, and penis was done on a Hitachi SU3500 scanning electron microscope. Cerata were examined for the presence of nematocysts in the cnidosacs for some species by placing the tip of a ceras on a microscope slide and examining it using a compound light microscope. External features were examined directly, by photographs, or by literature review depending on the availability of specimens. Specimens and dissected structures were deposited at the California Academy of Sciences Department of Invertebrate Zoology collection (CASIZ) and the National Museum of the Philippines (NMP).

Nomenclature acts

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is urn:lsid:zoobank.org:pub:1757F835-C668-4300-961A-FC1E8B409CE7. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.

Phylogenetic relationships

The final concatenated dataset was 1,448 bp in length including gaps; however, not all three genes were successfully sequenced for all specimens used in the analyses (Table 1). The BI and ML analyses (Fig. 1, Figs. S1, S2) of the three-gene concatenated dataset resulted in a similarly supported topology as Cella et al. (2016). In the genus Abronica (highlighted in light blue in Fig. 1), Abronica abronia (MacFarland, 1966) is basal in a well-supported (pp = 1.00, bs = 99) clade that includes the newly described Abronica payaso sp. nov. and Abronica turon sp. nov. as well as Abronica purpureoanulata (Baba, 1961). The genus Tenellia (highlighted in yellow in Fig. 1) is mostly well-supported (pp = 100, bs = 96) and includes species previously assigned to Zelentia, Trinchesia, Diaphoreolis, Catriona, and Phestilla.

In Tenellia the lowest nodes of the clade are well-supported (pp = 1, bs = 100) and composed of species previously identified as “Zelentia” and an undescribed species of Tenellia. The rest of Tenellia is composed of a polytomy between three large clades. The first clade is a well-supported clade (pp = 1, bs = 100) of species previously identified as “Diaphoreolis”. The second clade is a well-supported clade (pp = 1, bs = 95) composed of mostly undescribed species from the Central Pacific. It includes a specimen of Tenellia foliata (Forbes & Goodsir, 1839) and T. yamasui, which were previously assigned to “Trinchesia”, the newly described Tenellia puti sp. nov. and its sister species Tenellia bughaw sp. nov., and the undescribed Tenellia sp. 29, Tenellia sp. 39, Tenellia sp. 46, and Tenellia sp. 52.

The third clade of Tenellia is un-supported (pp = 0.52, bs = NA) and includes a weakly-supported subclade (pp = 0.87, bs = NA) of the rest of “Trinchesia” and a specimen of Tenellia ornata (Baba, 1937) which was also included in “Trinchesia”. The second subclade is well-supported (pp = 1, bs = 100) and is composed of Tenellia sensu stricto, Tenellia sp. 13, and “Catriona” gymnota, and “Catriona” aurantia, and a clade of the rest of “Catriona”. The third subclade is well-supported (pp = 0.98, bs = 95) and has a polytomy with undescribed species of Tenellia and species previously assigned to “Phestilla”. This polytomy is composed of a single specimen of the undescribed Tenellia sp. 58, a well-supported clade (pp = 0.95, bs = 91) of a single specimen of Tenellia chaetopterana with the newly described T. nakapila sp. nov., and a larger less supported (pp = 0.81, bs = 90) clade of Tenellia lugubris (Bergh, 1870), Tenellia melanobranchia (Bergh, 1874), Tenellia poritophages (Rudman, 1979), Tenellia subodiosa (Wang et al., 2020); Tenellia arnoldi (Mehrotra & Caballer, 2024), Tenellia fuscostriata (Hu et al., 2020), Tenellia viei (Mehrotra et al., 2020), and the undescribed Tenellia sp. A from Palau and Tenellia sp. B from Guam.

Species delimitation

The COI ASAP analysis (Table 2, Fig. S3) and 16S rRNA ASAP analysis (Table 3, Fig. S4) using Jukes-Cantor (JC69) both recovered four partitions within the genus Abronica (ASAP scores of 2.00 and 1.00, respectively) and ten partitions within the Tenellia clade including T. foliata, T. yamasui and the newly described T. puti sp. nov. and T. bughaw sp. nov. (ASAP score 1.50 for both analyses). The COI ASAP analysis recovered eleven partitions and the 16S rRNA ASAP analysis recovered fourteen partitions within the Tenellia clade including T. poritophages, T. melanobranchia, T. subodiosa, T. arnoldi, T. fuscostriata, T. viei, T. chaetopterana and the newly described T. nakapila sp. nov. (ASAP scores of 3.00 and 2.00, respectively). The COI ASAP analysis with eleven partitions fails to split T. arnoldi from T. subodiosa and T. fuscostriata from T. viei. All four species are morphologically distinct, therefore the partition with thirteen groups is congruent with our molecular and morphological analyses (ASAP score of 6.00). The 16S rRNA ASAP analysis with fourteen partitions over splits one specimen of T. nakapila sp. nov.; however, all three specimens are from the Philippines and are morphologically identical. Therefore, the partition with thirteen groups (ASAP score = 8.00) is congruent with our other analyses.

The three-gene bPTP analysis recovered the same four partitions within Abronica and the same ten partitions in the first Tenellia subclade; however, the second Tenellia subclade was over-partitioned. In the second subclade, fourteen partitions were recovered, but one specimen of T. nakapila sp. nov. (NMP 041347) was split from the other two specimens potentially due to the lack of successful COI sequencing or perhaps due to high intraspecific divergence of the 16S rRNA alignment. The GMYC analysis for COI recovered the same number of partitions as the COI and 16S rRNA ASAP analyses; since, the 16S rRNA GMYC analysis also over-partitioned the T. nakapila sp. nov. clade into fourteen partitions similar to the bPTP analysis.

The maximum genetic distance for COI within the Abronica clade (highlighted in blue in Fig. 1) was 23.6% between A. abronia and A. purpureoanulata, while the maximum distance for 16S rRNA was 16.1% between A. payaso sp. nov. and A. abronia. Intraspecific variation within two species of Abronica ranged between 0.0–0.8% within COI and 0.0–1.1% within 16S rRNA; however, no intraspecific variation was calculated for A. turon sp. nov. due to a lack of additional sequenced specimens. Within the first subclade of Tenellia the maximum genetic distance for COI was 22.6% between Tenellia sp. 39 and a specimen of Tenellia sp. 46 (CASIZ 177722), while the maximum distance for 16S rRNA was similar (20.1%) between Tenellia sp. 52 and T. yamasui. Intraspecific variation for two species within this clade (T. bughaw sp. nov., and T. puti sp. nov.) ranged between 0.0–0.9% for COI and 0.0–0.5% for 16S rRNA. In the second subclade of Tenellia the maximum genetic distance for COI was 23.4% between T. arnoldi and T. sp. 58, while the maximum distance for 16S rRNA was only 14.4% between T. viei and Tenellia sp. A. Within this clade, intraspecific variation within one species, T. nakapila sp. nov., was 1.4% for 16S rRNA.

Systematics

Tenellia yamasui (Hamatani, 1993)

Figures 2–4A

Cuthona yamasui Hamatani, 1993: 127, figs. 1-3.

Cuthona sp. 36 Gosliner, Behrens & Valdés, 2008: 370, misidentification

Material examined

CASIZ 182828, one specimen sequenced and dissected, ST 24, Bethlehem, 13.672800°N, 120.841339°E, Maricaban Island, Tingloy, Batangas Province, Luzon Philippines, 1 m depth, 20 May 2010, Alicia Hermosillo McKowen, collector.

Distribution

Originally described from Okinawa (Hamatani, 1993) and also recorded from Indonesia (Gosliner, Behrens & Valdés, 2008), and the Philippines (Gosliner, Valdés & Behrens, 2015).

Natural history

Found on shallow coral rubble habitats where it feeds on the thecate hydroid Pachyrhynchia cupressina (Kirchenpauer, 1872).

External morphology

The overall body color (Fig. 2) is an opaque white suffused with orange pigment. The concentration of orange pigment is particularly intense on the head and anterior portion of the body. Most of the body surface is covered with cerata. There is a thick honey-orange colored stripe traveling along the dorsal side of the body. The oral tentacles are opaque white in the basal half and bright orange in the outer half. Anterior to the oral tentacles is a bright orange spot on the front of the head. At the base of each oral tentacle, an opaque white line runs diagonally and unites into a medial longitudinal line that extends between the rhinophores and continues posteriorly a short distance behind the rhinophores. The rhinophore color is orange basally, followed by an opaque white band, another orange band, and a short opaque white apex. The thin, conical rhinophores are slightly longer than the oral tentacles. The texture is smooth and they are narrowly tapered, resembling a perfect icicle.

The cerata are very specialized to camouflage on their prey hydroid. The overall color of the cerata is a rusty reddish-brown color. Numerous small opaque white spots are present on the cerata with the exception of the apical portion. The tips are a burnt orange color with a white color at the distal end where the cnidosacs are situated. The widest part of the cerata is in the middle. They are folded in a chevron pattern along the dorsal side of the body. The cerata are arranged in numerous linear rows. There are four rows in the precardiac ceratal rows. In the one specimen (CASIZ 182828) examined here, the precardiac rows beginning with the most anterior row contain 4, 5, 4, 5 cerata per row. After the interhepatic space, there are 7–8 postcardiac ceratal rows, each of which contains 1–4 cerata. The anus is dorsal and acleioproctic and is located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

On either side of the buccal mass are large, dendritic oral glands that extend posteriorly two-thirds of the body length. In the one specimen examined (CASIZ 182828), the jaw resembles a clam shell shape. The texture has grooves like an undulating wave pattern. The jaws (Fig. 3A) are dark brown in color and relatively thickly cuticularized with a thickened area on the anterior side of the jaw. The masticatory margin is elongated and contains numerous irregular denticles (Fig. 3B) along its edge. The radula contains a single row of 54 teeth (Fig. 3C). The radula is elongated and relatively narrow with a few wider teeth near the middle of the ribbon. There is a prominent central denticle and the other denticles are positioned more posteriorly in an evenly graded fashion. There are 5–6 primary lateral cusps on either side of the central cusp (Fig. 3D). There are no smaller secondary denticles between the large denticles. The lateral cusps are acutely pointed and slightly curved inwardly.

Reproductive system

The reproductive system is androdiaulic (Fig. 4A). The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penial gland is pyriform whereas the penial papilla is conical. The penial stylet is straight and has a conical shape (Fig. 4A). The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via an elongated duct.

Remarks

The present specimen (CASIZ 182828) closely matches the original description of Cuthona yamasui by Hamatani (1993) from Okinawa, Japan, in its external morphology, color pattern, detail of the jaws and radula, and reproductive anatomy. The most distinguishing external features of this species are the presence of orange and white pigment on the head, rhinophores and oral tentacles and the rusty brown cerata with opaque white spots over their surface. The jaws and radular teeth closely resemble those described by Hamatani. Scanning electron microscopy reveals the presence of fine secondary denticles on the denticles of the masticatory margin of the present specimen. The radular teeth are characterized by having 5–6 evenly graduated lateral denticles on either side of the wider, triangular central cusp. The reproductive system also closely resembles that illustrated by Hamatani, but the bursa copulatrix of the present specimens has a more elongated duct. The penial stylet of our specimen was visible during dissection but was not evident in the scanning electron microscopic preparation.

Confusion around this species has arisen from considering species with very different color patterns as being color variants of the same species. Most of these variants have blue or green body pigment and lack the orange-white pigment on the head. Rudman (2002) and later postings on the Sea Slug Forum were identified as C. yamasui, but most of these variants represent the two species described subsequently as T. puti sp. nov. and T. bughaw sp. nov. This confusion was further exacerbated by Gosliner, Behrens & Valdés (2008), where some of these color variants were identified as C. yamasui and the true C. yamasui was identified as Cuthona sp. 36. This was corrected in Gosliner, Valdés & Behrens (2015) where C. yamasui was correctly identified. In Cella et al. (2016) numerous species of Cuthona, Catriona, Trinchesia, and Phestilla were moved to the genus Tenellia including C. yamasui. Two additional similar-looking species depicted in Gosliner, Valdés & Behrens (2015) as Cuthona sp. 13 and Cuthona sp. 14, which correspond to Tenellia sp. E and Tenellia sp. F, respectively, in Cella et al. (2016), are described as T. puti sp. nov. and T. bughaw sp. nov. in the present work. In the present study, we have shown that T. yamasui is a distinct species based on our phylogenetic analyses and is further corroborated by our morphological examination.

Tenellia puti Kim & Gosliner sp. nov.

LSID:urn:lsid:zoobank.org:act:65A96380-78E1-48DC-B344-6D0FC8549A19

Figures 4B, 5A–5D, 6

Cuthona yamasui Hamatani, 1993: Gosliner, Behrens & Valdés, 2008: 360, first row, second row, middle and right photos, misidentification.

Cuthona sp. 13: Gosliner, Valdés & Behrens, 2015: 345, left photo.

Tenellia sp. E: Cella et al., 2016

Tenellia sp. 15: Gosliner, Valdés & Behrens, 2018: 287, left photo.

Holotype

NMP 041346 (formerly CASIZ 217192) one specimen, sequenced, and dissected, ST DAU 06, 9.2007°N, 123.2789°E, San Miguel, Dauin, Negros Oriental, Philippines, 7 April, 2016, T. M. Gosliner.

Paratypes

CASIZ 177469, one specimen, sequenced and dissected, St 14, 13.6864°N, 120.8954°E, Mainit Point, Mabini Batangas, Luzon, Philippines, 20 March, 2008, T. Gosliner. CASIZ 177553, one specimen, sequenced and dissected, St 23, 13.6864°N, 120.8954°E, Mainit Point, Mabini Batangas, Luzon, Philippines, 21 March, 2008, T. Gosliner. CASIZ 177554, one specimen, sequenced and dissected, St 23, 13.6864°N, 120.8954°E, Mainit Point, Mabini Batangas, Luzon, Philippines, 21 March, 2008, T. Gosliner. CASIZ 186220, one specimen, St HEP 62, sequenced and dissected, St 14, 13.68627°N, 120.89544°E, Mainit Bubbles, Mabini Batangas, Luzon, Philippines, 13 May, 2011, T. Gosliner.

Distribution

This species is known from the Philippines (Gosliner, Valdés & Behrens, 2015, 2018) and Indonesia (Ianniello, 2003; Sozzani, 2006), and possibly Queensland, Australia (Rudman, 2002), Thailand (Panyarachun, 2007) and Tanzania (Picton, 2002).

Natural history

Found on soft bottom habitats of silty and sandy substrate where it feeds on the thecate hydroid Macrorhynchia balei (Nutting, 1906).

Etymology

Puti is the Filipino word for white, reflecting the characteristic opaque white patches on the notum of this species. The white triangular patch on the Philippine flag stands for equality.

External morphology

The living animals (Figs. 5A–5D) reach 25 mm in length. The body color is generally translucent white with a solid darker patch of opaque white occupying most of the dorsal surface and extending onto the head region in front of the rhinophores. The pattern of white pigment is irregular and does not extend to the lateral margins of the body, leaving areas of translucence laterally. The opaque patches often contain wrinkles. The digestive gland is a grayish brown, appearing like oxidized milk chocolate. The base of the cerata is translucent white, where the gray digestive gland is readily visible, whereas the medial portion is the most extensive and is an opaque white to light gray band. This band is variable in width and at its apical end has another area of transparency through which the digestive gland is visible. This band is followed by another smaller band of diffuse dull to medium blue, and the most distal part is a creamy yellow at the tip of the cerata. Between these three areas on the cerata, there are translucent areas through which the thin black digestive gland is visible. The rhinophores are thick and dark brown with red color tones that bear a resemblance to kelp leaves. The oral tentacles are the same color as the rhinophores as is the anterior end of the head at the base of the oral tentacles. The rhinophores are smooth, thick, and conical, slightly longer than the narrower oral tentacles. The anterior end of the foot is simply rounded to angular. The cerata project outwards randomly and cover most of the notum.

The cerata are arranged in numerous linear rows. There are 5–6 rows in the precardiac ceratal rows. In one specimen (NMP 041346), the precardiac rows, beginning with the most anterior row, contain 1, 2, 5, 3, 4, 1 cerata per row. After the interhepatic space, there are six to 13 postcardiac ceratal rows, each of which contains 1–4 cerata. The anus is dorsal and acleioproctic and it is located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

The jaws (Fig. 6A) are dark brown in color and relatively thickly cuticularized with a thickened area on the anterior side of the jaw. The masticatory margin is elongated and contains numerous irregular triangular denticles (Fig. 6B) along its edge. The denticles have fine granular edges. The radula is elongated and tapers considerably with the newest teeth becoming three times as wide as the oldest teeth. In one specimen (NMP 041346) the radula contains a single row of 64 teeth. The central cusp is somewhat shorter than the adjacent lateral denticles and in the oldest teeth (Fig. 6E) is markedly shorter than in the newest teeth (Fig. 6C). There are 4–5 primary lateral cusps on either side of the central cusp. Usually, there are 1–4 secondary denticles between the primary lateral cusps. The posterior end of the tooth is narrow with long spurs extending away from the cutting edge of the tooth (Figs. 6D–6E).

Reproductive system

The reproductive system is androdiaulic (Fig. 4B). The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penial gland is pyriform whereas the penial papilla is conical with a short, straight, cuticular penial stylet (Fig. 6F). The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via an elongate duct.

Remarks

Our phylogenetic and species delimitation analyses clearly indicate that T. puti sp. nov. is distinct from T. yamasui. There is a minimum divergence of 18.1% in the COI between T. puti sp. nov. and T. yamasui and a 12.5% divergence in the 16S rRNA gene. The two species appear to be considered the same species by Rudman (2002), without explanation but are readily distinguishable by the external color pattern and by aspects of their internal anatomy. Externally, the color pattern of T. puti sp. nov. is very different from that of T. yamasui. In T. puti sp. nov., the cerata have opaque white pigment basally, with yellow, black, and blue bands located more apically (Fig. 2), in contrast to the orange cerata with white speckling found in T. yamasui (Figs. 5A–5D). Also, T. yamasui has bright orange and white pigment on the head that is not present in T. puti sp. nov. Additionally, T. puti sp. nov. has broad areas of opaque white on the notum that is absent in T. yamasui. Internally, the masticatory border of the jaw has denticles with jagged secondary denticles that are not observed in T. puti sp. nov. (Fig. 3B). The radular teeth of T. yamasui also have a central cusp that is longer than the adjacent denticles (Fig. 3D), whereas the central cusp is shorter than the adjacent denticles in T. puti sp. nov. (Figs. 6C–6E). Furthermore, the teeth of T. yamasui lack shorter denticles between the large denticles (Fig. 3D) whereas, T. puti sp. nov. has smaller denticles between the larger ones (Figs. 6C–6E). The reproductive system of T. yamasui (Fig. 4A) has a relatively short vas deferens that is only slightly convoluted in contrast to the much longer, highly convoluted vas deferens of T. puti sp. nov. (Fig. 4B). Similarly, the penial gland of T. yamasui has a relatively short duct of the penial gland (Hamatani, 1993; present study: Fig. 4A) in contrast to T. puti sp. nov. elongate convoluted duct. Ecologically, the two species are found in different habitats. Tenellia yamasui is characteristic of living reefs and coral rubble habitats while T. puti sp. nov. is found on isolated bits of rock and rubble in soft sediment habitats and feeds on different species of aglaopheniid hydroids.

Tenellia bughaw Kim & Gosliner sp. nov.

LSID:urn:lsid:zoobank.org:act:04C4F439-5D0C-46AD-8245-CDD49BF15C0F

Figures 4C, 5E–5H, 7

Cuthona yamasui Hamatani, 1993: Gosliner, Behrens & Valdés, 2008: 226, second row left photo, misidentification.

Cuthona sp. 13: Gosliner, Valdés & Behrens, 2015: 345, right photo.

Cuthona sp. 14: Gosliner, Valdés & Behrens, 2015: 345, left and right photos.

Tenellia sp. F: Cella et al., 2016.

Tenellia sp. 15: Gosliner, Valdés & Behrens, 2018: 287, right photo.

Tenellia sp. 16: Gosliner, Valdés & Behrens, 2018: 287, left and right photos.

Holotype

CASIZ 176737, one specimen, sequenced, St. M24, 2.720101°N. 104.194977°E, Waterfall Bay (S end of the island), near Cahaya Asah Resort, Pulau Tioman, Malaysia, South China Sea, 15m depth, 4 October, 2007, T. Gosliner.

Paratypes

CASIZ 176739, one specimen, sequenced, ST. M 09, 3.25569°N, 103.76038°E, Pulau Varella, Malaysia, South China Sea, 15 m depth, 1 October, 2007, T. Gosliner. CASIZ 177552, one specimen, sequenced and dissected, St 23, 13.6864°N, 120.8954°E, Mainit Point, Mabini Batangas, Luzon, Philippines, 21 March, 2008, T. Gosliner. CASIZ 181298, one specimen, St. 14, 3.6859167°N 120.8952167°E, Mainit Bubbles dive site, Mabini, Batangas, Luzon, Philippines, 18 May 2009, Charles Delbeek. CASIZ 200553, one specimen, sequenced and dissected, St. MAB64, 13.65676°N, 20.89682°E, 64, Coconut Point, black Coral Forest, Tingloy, Batangas Province, Luzon, Philippines, 5 May 2014, Alexis Principe.

Distribution

Known from eastern Malaysia and the Philippines (present study).

Natural history

Found on soft bottom habitats of silty substrate where it is found on small bits of rock and rubble where it feeds on the thecate hydroid Macrorhynchia balei (Nutting, 1906).

Etymology

Bughaw is the Filipino word for blue, referring to the bright blue pigment that is dominant in this species.

External morphology

The living animals (Figs. 5E–5H) reach 25 mm in length. The overall body color is a translucent white with a light blue or purple ombre shade on the dorsal area of the head. The rhinophores are black or deep plum, but the tip is light blue to white in color. The oral tentacles are very dark plum to black with a small white apex. The cerata are mostly translucent bluish-white, like the body color, but at the tip, there is a grayish-blue shading. Below the tip of each ceras is a medium yellow (with orange tones) band that has a crisp black band above and below it. Also, the cerata are the widest in the medial portion making them appear plump. The rhinophores are slightly shorter than the oral tentacles. They are a little thicker at the base. The tips of the rhinophores are smooth. The anterior end of the foot is simply rounded to angular. The cerata are arranged in numerous linear rows. There are 6–7 rows in the precardiac ceratal rows. In one specimen (CASIZ 200553), the precardiac rows beginning with the most anterior row contain 2, 3, 3, 4, 3, 5, 4 cerata per row. After the interhepatic space, there are seven to 10 postcardiac ceratal rows, each of which contains 1–4 cerata. The anus is dorsal and acleioproctic and it’s located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

The concave jaws (Fig. 7A) are brownish and roughly ovate in shape. The masticatory border (Fig. 7B) bears numerous well-spaced triangular denticles. In one specimen (CASIZ 200553) the radula contains a single row of 54 teeth. Each tooth (Figs. 7C–7E) is relatively narrow with a well-elevated central cusp. The central cusp is shorter than the adjacent lateral denticles. The lateral denticles in the middle are longer than the adjacent lateral ones. There are 0–4 secondary denticles located between the primary denticles. They are considerably shorter and narrower than the primary ones. The primary denticles are relatively straight and are acutely pointed to slightly rounded. There are 4–6 primary lateral cusps on either side of the central cusp.

Reproductive system

The reproductive system is androdiaulic (Fig. 4C). The overall appearance is lobate and sharply curved. The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penial gland is pyriform, whereas the penial papilla is conical. The penial stylet has a tip shape that resembles a floss pick (Fig. 7F), while the base of the stylet has a curved shape. The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via an elongated duct.

Remarks

In our phylogenetic analysis, Tenellia bughaw sp. nov. is most closely related to T. puti sp. nov.; however, there is a strong genetic divergence between the two species. The minimum genetic divergence between the two species is 14.1% in the COI gene and 9.5% in the 16S rRNA gene. This exceeds the amount of genetic divergence that Korshunova et al. (2019) found for closely related species of Tenellia (as Trinchesia) where divergence for COI ranged from 2.98–12.99%. Externally, the two are similar in appearance and sometimes difficult to distinguish. The two species are both present sympatrically in the Philippines and have been collected from the same dive site at the same time. Specimens from the Philippines have a more bluish color (Figs. 5F, 5H), whereas Malaysian specimens have a more greenish-white color (Figs. 5E, 5G). Unlike T. puti sp. nov., specimens of T. bughaw sp. nov. have a uniform frosting of white or blue pigment. Specimens of T. puti sp. nov. have patches of white pigment that do not extend to the notal margins. The coloration of the cerata is also distinctive between the two species. In T. puti sp. nov., the basal two-thirds of the cerata are covered with opaque white followed by blue subapical band. Above the blue band, near the apex is a succession of diffuse black followed by a band of yellow and another diffuse black area at the very tip. In contrast, in T. bughaw sp. nov., the basal two-thirds of the cerata is white, occasionally with a gradual transition to blue. Above the white and blue, there is a sharply defined black band, followed by the yellow-orange band and a second well-defined black band with a white ceratal apex. Internally, the radula of T. bughaw sp. nov. (Figs. 7D, 7E) appears to have narrower teeth with more abundant secondary denticles between the primary ones than is found in T. puti sp. nov. (Figs. 6C–6E). The reproductive systems of the two species also have several differences (Figs. 4B, 4C). In T. bughaw sp. nov., the vas deferens is longer and more highly convoluted than in T. puti sp. nov. and the penial gland of T. bughaw sp. nov. is proportionately larger than in T. puti sp. nov.

Tenellia nakapila Kim & Gosliner sp. nov.

LSID:urn:lsid:zoobank.org:act:C6697544-2DBD-41E3-B00E-9F47C39BB411

Figures 4D, 8A–8D, 9A–9F

Cuthona sp. 14: Gosliner, Behrens & Valdés, 2008, 364.

Cuthona sp. 18: Gosliner, Valdés & Behrens, 2015, 346.

Tenellia sp. 20: Gosliner, Valdés & Behrens, 2018: 288.

Holotype

NMP 041347 (formerly CASIZ 202110), one specimen, sequenced, St. CAL03, 13.91422°N 120.60643°E, Lago de Oro house reef, Calatagan, Batangas, Luzon, Philippines, 9 May 2014, T. Gosliner.

Paratypes

CASIZ 071139, one specimen, St. 90, Pig (Tab) Island, Madang Papua New Guinea, 10 m depth, January, 1988, T. Gosliner. CASIZ 110386, one specimen, St. 28, Club Ocellaris, Mabini, Batangas Province, Luzon, Philippines, 24 April, 1997, Clay Carlson. CASIZ 208184, one specimen, Airport Beach, Maui, Hawaiian Islands, 8 May 2015. Cory Pittman. CASIZ 208511, one specimen, St. GAL 81, 13.51664°N. 120.95917°E, Schoolhouse Beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 13 April, 2015, T. Gosliner. CASIZ 208513, St. GAL91, 13.51688°N, 120.95983°E, Schoolhouse beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 16 April, 2015, T. Gosliner. CASIZ 208648, one specimen, St. GAL52, 13.51688°N, 120.95983°E, Schoolhouse beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 8 April, 2015, T. Gosliner. CASIZ 217318, CASIZ 208579, one specimen, sequenced, St. GAL15, 13.52482°N, 120.97139°E, La Laguna Pt., Puerto Galera, Mindoro Occidental, Philippines, mixed coral and sand, 20 m depth, 27 March, 2015, T. Gosliner. CASIZ 217292, one specimen, ST DAU 02, 9.1864°N, 123.2685°E, Ceres dive site, Dauin, Negros Oriental, Philippines, 15 m depth, 02 April 2016, T. Gosliner. CASIZ 217295, one specimen, St. DAU05, 9.1798°N, 123.259°E, Ginama-An dive site, Dauin, Negros Oriental, Philippines, 2 April 2016, T, Gosliner. CASIZ 217299, one specimen, St. DAU06, 9.2007°N, 123.2789°E, San Miguel, Dauin, Negros Oriental, Philippines, 2 April, 2016, T. Gosliner. CASIZ 217303, one specimen, sequenced and dissected, St. DAU07, 9.2007°N, 123.2789°E, San Miguel, Dauin, Negros Oriental, Philippines, 3 April, 2016, T. Gosliner. CASIZ 217310, St. DAU13,9.2007°N, 123.2789°E. San Miguel, Dauin, Negros Oriental, Philippines, 9 April, 2016, T. Gosliner. CASIZ 217311, one specimen, St. DAU01, 9.1975°N, 23.2754°E, house reef, Atlantis Resort, Dauin, Negros Oriental, Philippines, 1 April, 2016, T. Gosliner. CASIZ 217313, St. DAU08, 9.1864°N, 123.2685°E, Ceres dive site, Dauin, Negros Oriental, Philippines, 15 m depth, 04 April 2016, M. Burke. CASIZ 217318, five specimens, with eggs_, St. DAU16, 9.1968°N, 23.2752°E, VIP resort house reef, Dauin, Negros Oriental, Philippines, 9 April 2016, T, Gosliner & M. Burke.

Distribution

Known from the Hawaiian Islands Japan, the Philippines and Papua New Guinea (Gosliner, Behrens & Valdés, 2008).

Natural history

Living animals are found on soft sandy to silty substrate where they feed on specimens of the solitary athecate hydroid, Corymorpha sp.

Etymology

The name is derived from the Filipino word nakapila meaning, in a straight line, referring to the very distinctly separated rows of cerata.

External morphology

The living animals have a narrow, elongated body and reach a length of 7–8 mm. The body color of Tenellia nakapila sp. nov. is opaque white on the dorsal side of the anterior end. A lighter purple color in a “V” shape stretches on the dorsal side of the rest of the body. The oral tentacles are a fiery orange-red color with darker yellow speckles. The color perfectly camouflages with the substrate color. The rhinophores are a lighter shade compared to the oral tentacles and have a prominent yellow band near the tip. The anterior end of the foot is simply rounded and somewhat thickened. The cerata base has a dusty light brown color. The tip color is a very bright fiery red. The overall color of the cerata is a light sand color. The cerata color most proximal to the body varies throughout; some are sandy or creamy white. When resting, the animal holds its cerata folded flat against the body. The rhinophores are slightly shorter than the oral tentacles. They are a little thicker at the base. The tips of the rhinophores are sharply pointed to bluntly rounded. The cerata are arranged in numerous linear rows. There are three rows in the precardiac ceratal rows. In one specimen (CASIZ 217303), the precardiac rows beginning with the most anterior row contain 4, 6, 5 cerata per row. After the interhepatic space, there are four postcardiac ceratal rows, each of which contains 1–5 cerata. The anus is dorsal and acleioproctic and it’s located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

On either side of the buccal mass are large, dendritic oral glands that extend posteriorly two-thirds of the body length. There is also a pair of short salivary glands. In one specimen (CASIZ 217318), the surface of the jaw has a smooth texture. The jaws (Fig. 9A) are dark brown in color and relatively thickly cuticularized with a thickened area on the anterior side of the jaw. The masticatory margin is elongated and contains numerous irregular denticles (Fig. 9B) along its edge. The radula is elongated and the central cusp is about four times as large as any other denticle. In one specimen (CASIZ 217318), the radula contains a single row of 27 teeth. There are a couple of secondary denticles toward the middle of each tooth (Figs. 9C–9E). There are 5–8 primary lateral cusps on either side of the wider central cusp. Usually, there are 1–4 secondary denticles between the primary lateral cusps. The lateral cusps look like imperfect icicles.

Reproductive system

The reproductive system is androdiaulic (Fig. 4D). The overall shape is irregularly ovoid. The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penial stylet has a very prominent curved shape, resembling a hook. The base of the stylet is considerably thicker than the tip and looks like a tree trunk (Fig. 9F). The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via an elongated duct.

Remarks

Tenellia nakapila sp. nov. is easily distinguishable by its pale body color and distinctly separated ceratal rows that are held close to the notum. This species is a member of the clade that included coralivorous species formerly placed in the distinct genus Phestilla. Our molecular analyses recovered T. nakapila sp. nov. as sister to Tenellia chaetopterana, which was previously described by Ekimova, Deart & Schepetov (2019) from polychaete tubes in Vietnam. This species, T. chaetopterana, was recovered as a member of Phestilla but with some unusual morphological features. Fritts-Penniman et al. (2020) found that T. chaetopterana was nested in clade D with other corallivorous species, despite the ecological divergence of this species from its other close relatives. Tenellia chaetopterana, like its close corallivorous relatives, has thin, elongated denticles on its radular teeth, but unlike its corallivorous relatives, T. chaetopterana, may have cnidosacs that contain nematocysts. In contrast, T. chaetopterana sister species, the newly described Tenellia nakapila sp. nov., and the undescribed Tenellia sp. 58 both feed on hydroids but are also members of the largely corallivorous clade. Tenellia nakapila and T. sp. 58 are also divergent in that adult have cnidosacs that contain fully functional nematocysts, whereas only some adult individuals of T. chaetopterana store nematocysts and only some juvenile specimens of T. lugubris have functional nematocysts (Putz, König & Wägele, 2010). Tenellia nakapila has radular teeth with lateral cusps that are more typical of other species of Tenellia represented in other clades of the phylogenetic tree, whereas T. chaetopterana has elongate lateral denitcles more typical of other “coral eating” members of the clade (Rudman, 1981). Additionally, Tenellia nakapila sp. nov. differs markedly in color from T. chaetopterana and Tenellia sp. 58 (Gosliner, Valdés & Behrens, 2018: 295, middle left image). It also clearly has demarcated rows of cerata that are held close to the body, rather than being held upright as seen in Tenellia sp. 58 or T. chaetopterana. The three species also differ genetically since there is a strong genetic divergence in the COI/16S rRNA genes of 17.5%/10.3% and 20.3%/9.3% between T. nakapila sp. nov. and Tenallia sp. 58 and T. chaetopterana, respectively.

Abronica payaso Kim and Gosliner, sp. nov.

LSID:urn:lsid:zoobank.org:act:8A25A6A9-9921-4709-B92B-48CAF67E57CE

Figures 4E, 8E–8G, 10A–10F

Cuthona sp. 6: Gosliner, Behrens & Valdés, 2008: 362.

Cuthona sp. 39: Gosliner, Valdés & Behrens, 2015: 350.

Abronica sp. 3: Gosliner, Valdés & Behrens, 2018: 282.

Holotype

NMP 041348 (formerly CASIZ 177417), one specimen, sequenced, St. 25, 13.673313°N, 120.841566°E, Bethlehem, Tingloy, Batangas, Luzon, Philippines, 19 March 2008, T. Gosliner.

Paratypes

CASIZ 070430, one specimen, St. 13, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 13 January 1988, T. Gosliner. CASIZ 070454, one specimen, St. 80, −4.836487°S, 145.783869°E, the Quarry, near Bunn Village, Madang, Papua New Guinea, 11 February 1988, T. Gosliner. CASIZ 071148, one specimen, St. 35, −5.1719232°S, 145.822717°E, Cement Mixer Reef, Madang Papua New Guinea, 19 January 1998, T. Gosliner. CASIZ 071218, one specimen, St. 56, −5.212653°S, 145.815276°E, off of Madang lighthouse, Madang Papua New Guinea, 1 February 1988. T. Gosliner. CASIZ 072769, one specimen, −5.1719232°S, 145.822717°E, Cement Mixer Reef, Madang Papua New Guinea, 20 October, 1986, T. Gosliner. CASIZ 072771, one specimen, St. 60, −5.1719232°S, 145.822717°E, Cement Mixer Reef, Madang Papua New Guinea, 18 October 1986, T. Gosliner. CASIZ 072981, one specimen, St. 33, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 27 July, 1989, T. Gosliner. CASIZ 072985, 6 specimens, St. 88, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 27 August 1989, T. Gosliner. CASIZ 072986, four specimens, St. 94, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 7 m depth, 31 August 1989, T. Gosliner. CASIZ 072991, one specimen, St. 39, −5.164299°S, 145.838340°E, off of the north end of Pig (Tab) Island, Madang, Papua New Guinea, 30 July 1989, T. Gosliner. CASIZ 073009, St. 33, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 27, July 1989, T. Gosliner. CASIZ 073010, two specimens, St. 64, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 11 August, 1989, T. Gosliner. CASIZ 073401, two specimens, −5.1719232°S, 145.822717°E, Cement Mixer Reef, Madang Papua New Guinea, 2 m. depth, 22 October 1986. CASIZ 075223, one specimen, St. 29, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 14 November 1990, T. Gosliner. CASIZ 083850, St. 32, 13.650515°N, 120.841552°E, Devil’s Pt., Maricaban Island, Tingloy, Luzon, Philippines, 26 February, 1992, T. Gosliner. CASIZ 086311, one specimen, St. 33, −5.267982°S, 145.830621°E, Planet Rock, Madang Papua New Guinea, 16 June 1992, T. Gosliner. CASIZ 086449, three specimens, one dissected, St. 21, −5.155241°N, 145.830305°E, south side Rasch Passage, Madang, Papua New Guinea, 11 June 1992, T. Gosliner. CASIZ 088083, one specimen, St. 14, 13.720163°N, 120.873558°E, Koala dive site, Mabini, Batangas Luzon, Philippines, 25 March, 1993, T. Gosliner. CASIZ 106592, one specimen, St. 20, 13.690831°N, 120.889058°E, Twin Rocks, Mabini, Luzon, Philippines, 19 April 1996, T. Gosliner. CASIZ 113672, St. 31, −10.203500, 150.937833, Taodovu Reef, off East Cape, SW end of Goschen Strait) Milne Bay Province, Papua New Guinea, 6 June 1998, T. Gosliner. CASIZ 177350, one specimen, sequenced, St. 8, 13.673313°N, 120.841566°E, Bethlehem, Tingloy, Batangas, Luzon, Philippines, 18 March 2008, T. Gosliner. CASIZ 177353, one specimen, sequenced and dissected, St. 8, 13.673313°N, 120.841566°E, Bethlehem, Tingloy, Batangas, Luzon, Philippines, 18 March 2008, T. Gosliner. CASIZ 191475, one specimen, Pig (Tab) Island, Madang, Papua New Guinea, 30 November 2012, V. Knutson. CASIZ 191609, one specimen, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 8 December 2012, Jessica Goodheart. CASIZ 208447, one specimen, School Beach Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 16 April 2015, T. Gosliner. CASIZ 208635, one specimen, St. GAL 52, 13.51688°N, 120.95983°E, Schoolhouse beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 8 April, 2015, T. Gosliner. CASIZ 208690, one specimen, ST. GAL 72, 13.51688°N, 120.95983°E, Schoolhouse beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 12 April 2015, P. J. Aristorenas. CASIZ 208709, one specimen, St. GAL 132, 13.51688°N, 120.95983°E, Schoolhouse beach, Batangas Channel, Puerto Galera, Mindoro Oriental, Philippines, 29 April 2015, T, Gosliner.

Distribution

Midway Atoll, Japan, Papua New Guinea and the Philippines (Gosliner, Behrens & Valdés, 2008).

Natural history

Found on the undersides of coral rubble where this species feeds on thecate hydroids.

Etymology

The name comes from the Filipino word payaso, meaning clown owing to the colorful ornamentation on the body of this species.

External morphology

The body color is complex. The body is a translucent peach color covered with many small opaque white speckles. There is a denser concentration of white speckles posterior to the head, making it appear like a white clump. Near the head is a darker peach color. The oral tentacles are the same color as the head with dark red and white spots that are in the shape of sprinkles. The rhinophores follow the same pattern as the oral tentacles but have a yellow band near the tip. The anterior end of the foot is simply rounded. There are no speckles on the cerata. The tip of the cerata have a white, yellow, and dark red band in order from most proximal to the body. The cerata located farther away from the head have a wider opaque white color band. The cerata point in random directions from the body. The body and cerata blend in very well with the environment. The rhinophores are about the same length as the oral tentacles and are smooth in texture. The cerata are arranged in numerous linear rows. There are three precardiac ceratal rows. In one specimen (CASIZ 086449), the precardiac rows beginning with the most anterior row, contain 1, 3, 3 cerata per row. After the interhepatic space, there are five postcardiac ceratal rows, each of which contains 1–3 cerata. The anus is dorsal and acleioproctic and is located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

In one specimen (CASIZ 086449), the jaw is thin and folded when it was air-dried (Fig. 10A). The jaw texture is smooth with undulating grooves over its surface. The jaws are dark brown in color and relatively thickly cuticularized with a thickened area on the anterior side of the jaw. The masticatory margin is elongated and contains numerous irregular denticles (Fig. 10B) along its edge. In one specimen (CASIZ 086449) the radula contains a single row of 28 teeth (Fig. 10C). The individual teeth are broadly arched and elongated (Figs. 10D, 10F). There are 5–6 primary lateral cusps on either side of the equally wide central cusp. The lateral denticles may be longer than the central cusp. There is a pair of secondary denticles flanking the central cusp.

Reproductive system

The reproductive system is androdiaulic (Fig. 4F). The texture of the surface of the reproductive system is lobate and irregular in outline. The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penis is readily visible. The penial gland is pyriform whereas the penial papilla is conical. The penial stylet (Fig. 10F) is curved like a rainbow shape. The penis is very similar to Tenellia nakapila sp. nov., but it is slightly larger in width and length (Fig. 4E). The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via an elongated duct.

Remarks

Abronica payaso sp. nov. is clearly distinct from the other described species of Abronica: A. abronia and A. purpureoannulata. Both of these species have purple bands on the oral tentacles and rhinophores, whereas A. payaso sp. nov. has red and white spots and an additional yellowish subapical band on the rhinophores. The most distinctive features of A. payaso sp. nov. are the peach body color with numerous opaque white speckles and the opaque white, yellow and red bands on the cerata. In our phylogenetic analysis, A. payaso sp. nov. is the sister species to both A. purpureoannulata and another species described here, A. turon sp. nov. Abronica abronia is sister to the remaining species of the genus. Genetically, A. payaso sp. nov. is 21.6% different from A. abronia in its COI p-distance and 20.5% different from A. purpureoanulata.

Abronica turon Kim & Gosliner sp. nov.

LSID:urn:lsid:zoobank.org:act:B3B9E430-20FA-4197-82DD-A0F483D4D240

Figures 4F, 8H, 8I, 11A–11D

Cuthona sp. 7: Gosliner, Behrens & Valdés, 2008: 362.

Cuthona sp. 15: Gosliner, Valdés & Behrens, 2015: 346.

Abronica sp. 2: Gosliner, Valdés & Behrens, 2018: 282.

Holotype

CASIZ 179946, one specimen, sequenced, Airport Beach (Kahekili Beach Park), 20.936931°N, −156.694314°W, Maui, Hawai’i, 25 May 2008, C. Pittman.

Paratypes

CASIZ 070453, one specimen, S. side Sek Passage, −5.123433°S, 145.823733°E, Madang, Papua New Guinea, 21 February 1988, G. Williamson. CASIZ 071142, one specimen, St. 80, −4.836487°S, 145.783869°E, the Quarry, near Bunn Village, Madang, Papua New Guinea, 11 February 1988, T. Gosliner. 071225, one specimen, St. 41, s. e. side Pig (Tab) Island, Madang, Papua New Guinea, 24 January 1988, R. C. Willan. CASIZ 073001, one specimen, St. 88, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 31 August 1989, T. Gosliner. CASIZ 073002, St. 94, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 27 August 1989, M. Ghiselin. CASIZ 073003, St.64, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 11 August 1989, T, Gosliner. PNG, 073008 St. 14, −5.1715212°S, 145.842062°E, Barracuda Pt., Madang Papua New Guinea, 19 July 1989, T. Gosliner. CASIZ 073514, one specimen, St. 61, −5.1719232°S, 145.822717°E, Cement Mixer Reef, Madang Papua New Guinea, 19 October 1988, T. Gosliner. CASIZ 086397, one specimen, St. 36, −5.155241°N, 145.830305°E, south side Rasch Passage, Madang, Papua New Guinea, 17 June 1992, T. Gosliner. CASIZ 086452, one specimen, St. 12, −5.155241°N, 145.830305°E, south side Rasch Passage, Madang, Papua New Guinea, 8 June 1992, T. Gosliner. CASIZ 086397, one specimen, St. 36, −5.155241°N, 145.830305°E, south side Rasch Passage, Madang, Papua New Guinea, 17 June 1992, T. Gosliner. CASIZ 191474, one specimen, St. PR 135, −5.212653°S, 145.815276°E, off of Madang lighthouse, Madang Papua New Guinea, 29 November, 2012, A. Berberian. CASIZ 241493, one specimen (dissected), St. 28 Daphne’s Reef, Madang, Papua New Guinea, 25 July 1989, T. Gosliner. CASIZ 241494, two specimens (one dissected), St. 32, near Madang Lighthouse, Madang, 27 July 1986, T. Gosliner.

Distribution

Known from the Hawai’ian Islands and Papua New Guinea

Etymology

The name turon comes from the delicious Filipino dessert that is made from fried banana and can be combined with ube (sweet purple yam).

External morphology

The overall body color of this species is a dark reddish purple (Figs. 8H, 8I). Their oral tentacles are comparably very long; about one-third of their body length. The main color of the oral tentacles is a very dark purplish blue, almost black color. The tips are bright and greenish-white, like glow-in-the-dark stars. The rhinophores are mostly a dark cherry color with bright greenish-white dots on each one that may be clustered to form a patch. The tips are the same color as the tips on the oral tentacles. On the head are two larger bright greenish-white masses, which are more abundant and widespread in some Hawai’ian specimens (Fig. 8H). More posteriorly from the head, the body color has an ombre effect that transitions to a dark cherry color. The cerata color medially near the base is a very dark charcoal gray, followed by a thickened area of opaque white which has a honey-yellow band at its apical end. The tips, which abruptly narrow above the opaque white and yellow bands, are a translucent white, fluorescent color. The coloring of this animal makes it appear more fluorescent against their substrate. The direction of the cerata lay somewhat flat against the body or may be slightly elevated. The rhinophores are about half the length of the oral tentacles. The shape is evenly tapered with a somewhat rounded apex. The anterior end of the foot is simply rounded. The cerata are arranged in numerous linear rows. There are two rows in the precardiac ceratal rows. In one specimen the precardiac rows beginning with the most anterior row contain 3, 3 cerata per row. After the interhepatic space, there are 6–7 postcardiac ceratal rows, each of which contains 1–3 cerata. The anus is dorsal and acleioproctic and is located anterior to the first ceratal row of the postcardiac cerata. The genital opening is ventral to the third and fourth precardiac ceratal rows.

Buccal mass and radula

In one specimen, the jaw is translucent and flat. The texture has undulating grooves. The jaws (Fig. 11A) are dark brown in color and relatively thickly cuticularized with a thickened area on the anterior side of the jaw. The masticatory margin is elongated and contains numerous regular triangular denticles (Fig. 11B) along its edge. In one specimen (CASIZ 241493), the radula contains a single row of 24 teeth (Fig. 11C). The individual teeth (Figs. 11D, 11E) have a very characteristic shape with three primary denticles on either side of the prominent, acutely pointed central cusp. There are numerous smaller secondary denticles prominently on both sides of the central cusp located in the middle. There are up to nine secondary denticles flanking either side of the primary cusp. Additional secondary denticles are found between the primary denticles. The central cusps are longer than the adjacent denticles.

Reproductive system

The reproductive system is androdiaulic (Fig. 4F). The texture of the surface is lobate in the fully mature specimen. The ovotestis follicles contain a large female acinus surrounded by a series of smaller male acini. The large, saccate ampulla divides distally into the short oviduct and vas deferens. The vas deferens begins with a thick prostatic portion of and narrows into a short, convoluted ejaculatory duct, entering the penis near the junction of the penial gland with the penial papilla. The penial gland is pyriform whereas the penial papilla is conical. The penial stylet (Fig. 11F) is very curved and smooth. The base is very thick and tapers outwardly. There are three prominent tubercles on the inner side of the anterior portion of the penial stylet. The female glands are well-developed and small albumen and membrane glands are clearly visible, as is the larger mucous gland. A spherical bursa copulatrix is present at the distal end of the reproductive system and connects to the gonopore via a very short duct.

Remarks

In our phylogenetic analysis, Abronica turon sp. nov. is sister to A. purpureoanulata but has a COI divergence of 20.7%. The most characteristic external features of this species are the purple body color with cerata having an abrupt narrowing just apically from the bright yellow band. However, there are also several other internal morphological features that distinguish A. turon sp. nov. from the three other known members of the genus. The radular teeth of A. turon sp. nov. have numerous secondary denticles whereas A. abronia, A purpureoanulata, and A. payaso sp. nov. lack secondary denticles that are smaller than the primary denticles (MacFarland, 1966: pl. 68, figs. 20, 21; Baba, 1961: pl. 15, fig. 5; present study: Figs. 10D, 10F). Additionally, A. turon sp. nov. is the only species that has a curved penial stylet with three tubercles on its inner edge (fig. 11F). The remaining species have a smooth, curved stylet (MacFarland, 1966: pl. 70, fig. 2; Baba, 1961: pl. 15, fig. 8; present study: Fig. 10F).